Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 198282 12 pageshttpdxdoiorg1011552013198282

Research ArticleMoxibustion Inhibits the ERK Signaling Pathway and IntestinalFibrosis in Rats with Crohnrsquos Disease

Xiaomei Wang1 Yuan Lu2 Luyi Wu2 Chen Zhao1 Chunbin Song2

Shuguang Yu3 Baixiao Zhao4 Tianping Zhao2 Huirong Liu2 Chuanzi Dou2

Yingying Zhang2 and Huangan Wu12

1 Yueyang Hospital of Integrated Traditional Chinese and Western Medicine Shanghai University of Traditional Chinese MedicineShanghai 200437 China

2 Key Laboratory of Acupuncture-Moxibustion and Immunological Effects Shanghai University of Traditional Chinese MedicineShanghai 200030 China

3 Laboratory of Experimental Acupuncture of College of Acumox and Tuina Chengdu University of Traditional Chinese MedicineChengdu 610075 China

4 School of Acupuncture-Moxibustion and Tuina Beijing University of Chinese Medicine Beijing 100029 China

Correspondence should be addressed to Huangan Wu wuhuangan126com

Received 27 May 2013 Accepted 2 July 2013

Academic Editor Yong-Qing Yang

Copyright copy 2013 Xiaomei Wang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Intestinal fibrosis is the main pathological process in Crohnrsquos disease (CD) acupuncture andmoxibustion can inhibit the process offibrosis in CD rats but the regulatory mechanism remains unknown The present study observed the effect of moxibustion on theextracellular signal-regulated kinase (ERK) signaling pathway in the CD rat The result shows that the phosphorylation of the RasRaf-1MEK-1 and ERK-12 proteins and the expression of the correspondingmRNAs in the colon tissue of CD rat were significantlyhigher than the normal control group Both treatments with mild moxibustion and with herb-separated moxibustion significantlyreduced the expression of the Ras Raf-1 MEK-1 and ERK-12 proteins and Ras and Raf-1 mRNA MEK-1 and ERK-12 mRNAexpression in each treatment group showed a downward trend and the ERK-12 mRNA levels were significantly lower in the mildmoxibustion group It indicates that Ras Raf-1 MEK-1 and ERK-12 are involved in the process of intestinal fibrosis in CD ratsMoxibustion can downregulate the abnormal expression of colonic Ras Raf-1 MEK-1 and ERK-12 protein and mRNA levels inCD intestinal fibrosis in rats Moxibustion may play a role in the treatment of CD intestinal fibrosis by regulating ERK signalingpathway

1 Introduction

Crohnrsquos disease (CD) is an agnogenic chronic and nonspe-cific granulomatous inflammatory disease of the intestine[1] The histological features of CD are characterized bytransmural inflammation lymphangiectasia and lymphaticand fibrous tissue hyperplasia CD is more common inEurope and the USA than in other countries The annualincidence in Europe and theUSA is 5ndash10 per 100000 personsand the prevalence is 50ndash100 per 100000 persons [2] Theincidence of CD in Asia is lower than in Western countriesalthough the incidence in China has noticeably increased inrecent years [3 4]

Under the pathological conditions of the chronic andrecurring inflammation of CD collagen decomposition islimited and the continuously increasing deposition of col-lagen fibers results in excessive scar tissue which promotesthe hardening of the intestinal wall and luminal stenosisTherefore local intestinal function is impaired [5] Theresults of impaired intestinal function range from the loss ofintestinal wall compliance severe abdominal pain anorexiaand diarrhea to priming fistula formation and even increasingthe possibility of malignant transformation in which diseaseduration is persistent and refractory [6] Studies have shown[7 8] that early mild intestinal fibrosis can be reversedwhich has great significance for slowing disease At present

2 Evidence-Based Complementary and Alternative Medicine

the basis of therapy for CD intestinal fibrosis is long-termanti-inflammatory treatment However although they mayreduce inflammation anti-inflammatory treatments do notreduce fibrous stenosis Therefore an understanding of thepathogenesis of CD intestinal fibrosis and the discovery ofeffective prophylactic measures with fewer side effects areurgently needed in this field

Intestinal fibrosis is a complicated pathological process inwhichmany cell types cytokines and signaling pathways par-ticipate including the extracellular signal-regulated kinase(ERK) signaling pathway which has become a popular topicof current study ERK including ERK1 and ERK2 is oneof the mitogen-activated protein kinase (MAPK) signalingpathwaysThe ERK12-MAPK signaling pathway is a cascadethat is related to the pathological fibrosis of multiple diseases[9] An activated Ras-Raf-MEK-ERK pathway promotesfibrosis and plays an important role inTGF-120573-mediated type Icollagen expression extracellular matrix (ECM) productionand myofibroblast formation [10 11]

Preliminary studies have shown that medicine-separatedmoxibustion can significantly inhibit the process of fibrosisin CD rats [12 13] but the regulatory mechanism underlyingthis process remains to be further explored In this study aCD intestinal fibrosis rat model was prepared by the Morrismethod [14] EnVision immunohistochemistry and qPCRwere used to measure the levels of Ras Raf MEK and ERKproteins and mRNA expression in colon tissues to explorethe key signaling molecules involved in the regulation of CDintestinal fibrosis in rats and reveal the mechanisms underly-ing the effects of moxibustion on CD intestinal fibrosis

2 Materials and Methods

21 Experimental Animals A total of 60 male Sprague-Dawley (SD) rats with a body weight of 180 plusmn 20 g wereprovided by the Experimental Animal Center at the ShanghaiUniversity of Traditional Chinese Medicine All of the ratswere kept for one week at room temperature after receiptTherats that behaved normally with respect to feeding drinkingand other behaviors were included in the experiment Thisstudy received permission from the Ethics Committee inYueyang Hospital which is affiliated with Shanghai Univer-sity of Traditional Chinese Medicine China All protocolswere performed in strict accordance with the GuidanceSuggestions for the Care and Use of Laboratory Animalsformulated by the Ministry of Science and Technology of thePeoplersquos Republic of China [15]

22 Grouping and Model Establishment A total of 60 SD ratswere randomly divided into a normal group of 12 rats anda model group of 48 rats A 246-trinitrobenzene sulfonicacid (TNBS) (Sigma Co St Louis USA) enema of 5 TNBSand 50 ethanol was prepared at a 2 1 ratio The rats in eachgroup were fasted but not deprived of water for 48 hThe ratsin the model group were anesthetized by the intraperitonealinjection of 2 sodium pentobarbital (China Langchem IncShanghai China) at a dose of 20mgkg and were verticallyinverted to introduce 100mgkg TNBS by rectal infusiononce per 7 days for a total of four treatments whereas the

normal group was not treated To validate the model thedistal colons from one rat in the normal group and two ratsin the model group were examined by visual observation andHampE staining After the success of the model was confirmedthe 44 rats were randomly assigned to a model group a mildmoxibustion group a herb-separatedmoxibustion group andan EA group with 11 rats per group Two rats died during themodeling process and one rat from themodel group and onerat from the mild moxibustion group died during the courseof treatment

23 Treatment

NC Group No treatment

MC Group TNBS enema only no treatment

MM Group A TNBS enema was administered and theST25 (bilateral) and RN6 acupuncture points were located Amoxa stick (Nanyang Hanyi Moxa Co Ltd China) 5mm indiameter was lit and placed at points at a distance of 1-2 cmfrom the ST25 and RN6 acupuncture points Moxibustionwas performed for 10min per point per treatment once a dayfor a total of seven treatments

HSP Group A TNBS enema was administered and theST25 (bilateral) and RN6 acupuncture points were locatedAconite cinnamon salvia and other drugs were milled intoa powder andmixed with rice wine to form a pasteThe pastewas then pressed into amedicinal cake 5mm in diameter and3mm in thickness A moxa cone was formed from 90mg ofrefined moxa Two moxa cones were used for moxibustionfor each point per day and a total of seven moxibustiontreatments were performed for 7 days

EA Group A TNBS enema was administered and the ST25(bilateral) and RN6 acupuncture points were located Nee-dles (Suzhou Huatuo Co Ltd Suzhou China) 025mm indiameter and 25mm in length were inserted to a depth of 3-4mm and connected to an LD202HHan electroacupunctureapparatus (Beijing Huawei Co Ltd Beijing China) TheRN6 and bilateral ST25 acupuncture points were stimulatedalternately by a continuouswave of 03ms pulsewidth 100Hzfrequency and 1mA current once a day for 10min withcontinuous treatment for 7 days

24 Specimen Collection Following treatment the rats werefasted for 24 hwith access towater andwere then anesthetizedby intraperitoneal injection of 2 sodium pentobarbital at adose of 50mgkg The anesthetized rats were then surgicallysectioned to remove approximately 6 cm of the distal colonThemesenteriumwas then cut longitudinally and rinsedwith4∘C saline The mucosal injury was visually examined andevaluated using a gross injury score [16 17] Two pieces oftissue with obvious lesions were removed of which one wastransferred to liquid nitrogen until testing and the other wasfixed in a 10 neutral formaldehyde solution

25 Detection of Collagen Fibers in the Rat Colon Massontrichrome staining was used to detect collagen fibers in

Evidence-Based Complementary and Alternative Medicine 3

Table 1 The sequence of the forward primer and reverse primer used for fluorescent quantitative PCR assays

Name Primer sense Sequence (51015840rarr31015840) Amplification product (bp)

GAPDH Forward 51015840-CCGAGGGCCCACTAAAGG-31015840 116 bpReverse 51015840-GCTGTTGAAGTCACAGGAGACAA-31015840

Ras Forward 51015840-TGGTGGGCAACAAGTGTGAC-31015840 181 bpReverse 51015840-GGGTTCAGTTTCCGCAGTTTATG-31015840

Raf-1 Forward 51015840-CAGGAGCACAAAGGTAAGAAAGC-31015840 201 bpReverse 51015840-GCCACAAGTCTGACATCGAAATC-31015840

MEK-1 Forward 51015840-AATATGTCAGGTTCAAGGAAAGGAC-31015840 169 bpReverse 51015840-AGGTAAGGACGAAGCCACAAG-31015840

ERK-12 Forward 51015840-TATATACATTCAGCTAATGTTCTGC-31015840 169 bpReverse 51015840-TCTACCTGCAGCTGGAACCTCTC-31015840

the rat colon Green staining was interpreted as collagenmucus and cartilage red staining denotes muscle and nervefibers and black indicates nuclei

26 EnVision Plus Immunohistochemical Detection of RasRaf MEK and ERK-12 in Colonic Tissue Fixed tissues wereconventionally deparaffinized in water and washed threetimes for 3min in 001M PBS pH 74 The tissue sampleswere heated and allowed to equilibrate to room temperaturefor 20min and then washed three times in PBS for 3min Toinhibit endogenous peroxidase the samples were treatedwith03 H

2O2at room temperature for 20min and then rinsed

three times with PBS for 3min The tissue samples wereincubated in 20normal goat serum at room temperature for30min and then with one drop of the appropriately dilutedprimary antibody (Santa Cruz Bio Inc Santa Cruz USA) at4∘C for 18 h The tissues were washed three times with PBSfor 3min mixed with one drop of Envision reagent (HRP-M) and incubated in a humidified chamber for 30min at37∘C After washing three times with PBS for 3min the assaywas developed in 004 DAB and 03 H

2O2for 8ndash12min

then washed in water stained with hematoxylin for 1minand bathed in hot water for 3 sec to allow the blue color todevelop Finally the tissue sections were dried and mountedwith a neutral resin All the samples were analyzed by aMoticMed 60 image analysis system (MoticGroupCo Ltd)Threefields were randomly selected the positive target value ofthe integral optical density was calculated under an opticalmicroscope (Olympus Co Ltd) at 200x magnification

27 qPCR Detection of Ras Raf MEK and ERK-12 mRNALevels in Colonic Tissues The total RNA was extracted fromcolon tissue samples The cDNA was synthesized by reversetranscription (RT) according to the qPCR kit (ShanghaiDaweike Bio Inc Shanghai China) instructions and ampli-fied by PCR according to the kit instructions (Table 1) Thefollowing cycling conditions were used RT at 37∘C for 1 h95∘C for 5min to inactivate the MMLV RT enzyme 50∘C for2min 95∘C for 5min and 40 cycles of 95∘C for 15 sec and60∘C for 45 sec

28 Statistical Analysis SPSS 180 statistical software (SPSSInc Chicago USA) was used for statistical analysis The

012345678

NC MC MM EA HSP

Gro

ss in

jury

scor

es

lowast

Figure 1 The gross injury scores of the colon tissue in eachrat group NC normal control group MC model control groupMM mild moxibustion EA electroacupuncture and HSM herb-separated moxibustion lowast119875 lt 001 versus NC 119875 lt 001 versus MC

measurement data are presented as the mean plusmn SD Fornormally distributed data one-way analysis of variance(ANOVA) was applied The least significant differencemethod (LSD) was used for the homogeneity of variance andGames-Howell was used to compare the differences betweengroups for the heterogeneity of variance A value of 119875 lt 005was considered statistically significant The nonparametricMann-Whitney U-test was used for abnormal distributions

3 Results

31 Gross Injury in the Colon Tissue of Rat When the colonictissues were observed by visual inspection the rats in thenormal group exhibited a uniformity of colon bowel andintestinal wall a smooth colonic mucosa surface clear vascu-lar texture no bleeder and no erosion or ulcers The modelgroup exhibited partial intestinal canal stenosis or intestinalwall thicken and harden fuzzy vascular texture edematouscolonic mucosa serious congestion erosion and differentsize shape and depth ulcer formation After treatmentsthere were lacked smooth colonicmucosa surface edematouscolonic mucosa and erosion in both MM and EA groupand lacked smooth colonic mucosa surface with no obviouserosion and ulcer in HPM group

As shown in Figure 1 the gross injury score of the colonictissue in the NC group was significantly higher than the MCgroup (119875 lt 001) After treatment the scores were lower in

4 Evidence-Based Complementary and Alternative Medicine

(a) (b)

(c) (d)

(e)

Figure 2 Expression of collagen fibers in the colon tissue of rat Masson trichrome staining method times100 (a) Normal (b) Crohnrsquos disease(c) mild moxibustion (d) electroacupuncture (e) herb-separated moxibustion

both the HPM group MM group and the EA group (119875 lt001) It suggests that these treatments are able to improve thetissue damage of CD

32 Expression of Collagen Fibers in the Colon Tissue of RatAs shown in Figure 2 Masson trichrome staining revealedthat in the MC group a large number of collagen fibers werepresent in the mucosa muscularis mucosa submucosa andmuscularis of the rat colon with a disordered arrangementIn the MM group and the HSM group there were smallnumbers of collagen fibers in the colonic lamina propria andsubmucosa at the original ulcer-healing location which wasless severe compared with that of the model group In the EAgroup there were a small number of collagen fibers in the

mucosa submucosa muscularis and serosa of the rat colonand the effect was less severe than in the MC group

33 Ras Expression in the Colonic Mucosa of Rat As shownin Figures 3 and 4 the phosphorylation of Ras protein inthe rat colon was significantly higher in the MC group thanin the NC group (119875 lt 001) Ras protein expression wassignificantly reduced (119875 lt 001) to normal levels after acu-puncture treatment

Figure 5 indicates that Ras mRNA expression in the MCgroup was higher than that in the NC group (119875 lt 001) andthat HSM effectively reduced the Ras mRNA levels in the ratcolon (119875 lt 005) whereas there was no significant differencebetween the EA group and the NC group

Evidence-Based Complementary and Alternative Medicine 5

0

100

200

300

400

500

600

700

NC MC MM EA HSPRa

s pho

spho

ryla

tion

prot

ein

(IO

D)

lowast

Figure 3The integral optical density (IOD) of Ras phosphorylation in each rat group NC normal control group MC model control groupMM mild moxibustion EA electroacupuncture and HSM herb-separated moxibustion lowast119875 lt 001 versus NC 119875 lt 001 versus MC

(a) (b)

(c) (d)

(e)

Figure 4 Expression of phosphorylated Ras protein in the colonic mucosa of rat The phosphorylation of Ras protein was expressedin epithelial cells and lymphocyte in the colonic mucosa of rats EnVision Plus method times200 (a) Normal (b) Crohnrsquos disease (c) mildmoxibustion (d) electroacupuncture (e) herb-separated moxibustion

6 Evidence-Based Complementary and Alternative Medicine

0

2

4

Ras m

RNA

leve

l (

)

6

8

10

12 17

25

21

NC MC MM EA HSP

lowast

lowast

Figure 5 The Ras mRNA levels in the rat colon NC normalcontrol group MC model control group MM mild moxibustionEA electroacupuncture and HSM herb-separated moxibustionlowast119875 lt 001 versus NC 119875 lt 005 versus MC

0100200300400500600700800900

NC MC MM EA HSP

Raf-1

pro

tein

(IO

D)

lowast

998771998771 998771

Figure 6 The IOD of Raf-1 protein expression in rat colonNC normal control group MC model control group MM mildmoxibustion EA electroacupuncture and HSM herb-separatedmoxibustion lowast119875 lt 001 versus NC 119875 lt 005 119875 lt 001 versusMC 998771998771119875 lt 005 998771119875 lt 001 versus EA

34 The Raf-1 Expression in the Colonic Mucosa of Rat Asshown in Figures 6 and 7 the Raf-1 protein expression inthe colonic tissue of the MC group was significantly higherthan in the NC group (119875 lt 001) and that the Raf-1 proteinexpression in the colon was reduced to varying degrees afteracupuncture treatment (119875 lt 005 or 119875 lt 005)

The results in Figure 8 show that the Raf-1 mRNAlevels in the MC group were significantly higher than in theNC group (119875 lt 001) and that MM and HSM treatmentssignificantly reduced rat colon Raf-1 mRNA levels (119875 lt 005)whereas EA treatment did not significantly affect rat colonRaf-1 mRNA levels

35 The MEK-1 Expression in the Colonic Mucosa of Rat Asshown in Figures 9 and 10 the MEK-1 protein expression inthe colonic tissue of the MC group was significantly higherthan in NC rats (119875 lt 001) and MEK-1 protein expressionwas significantly reduced in each group after acupuncturetreatment (119875 lt 001) although the levels were still higherthan normal levels (119875 lt 001)

The results in Figure 11 show that theMEK-1mRNA levelsin the model group were significantly higher than those inthe NC group (119875 lt 001) and that MM HSM and EAtreatment reduced MEK-1 mRNA levels although there was

no statistically significant difference between the treatmentand no treatment groups or among the treatment groups

36 The ERK-12 Expression in the Colonic Mucosa of Rat Asshown in Figures 12 and 13 the phosphorylation of ERK-12significantly increased in the MC group compared with theNC group (119875 lt 001) and that the levels of phosphorylatedERK-12 proteins were significantly reduced after the MMand HSM treatments (119875 lt 001)

The results in Figure 14 show that the ERK-12 mRNAexpression in the MC group was significantly higher thanthat in the NC group (119875 lt 001) and that ERK-12 mRNAexpression was significantly lower in the MM group afteracupuncture treatment compared with the MC group (119875 lt005)

4 Discussion

Extracellular signal-regulated kinase (ERK) a member ofthe mitogen-activated protein kinase (MAPK) family playsan important biological role in transmitting signals fromsurface receptors to the nucleus The ERK12 pathway is themost thoroughly researched pathway in the ERK family andplays a role in a wide range of cellular functions includ-ing regulating meiosis mitosis and anaphase processes indifferent cells A variety of stimulating factors such as growthfactors cytokines viruses G protein-coupled receptor lig-ands and oncogenes can activate the ERK12 pathway [18]The ERK signaling pathway is a Ras-Raf-MEK-ERK path-way Phosphorylation-activated ERK12 translocates fromthe cytoplasm to the nucleus and thereby mediates thetranscriptional activation of Elk-1 ATF NF-120581B AP-1 and c-fos which in turn regulate cell proliferation and differentia-tion the maintenance of cell shape the construction of thecytoskeleton apoptosis and the malignant transformation ofcells among other biological functions

One of the features of CD is the transformation of smoothmuscle cells into fibroblasts andor myofibroblasts [8] Thisabundance of fibroblasts and myofibroblasts increases theexpression of type I collagen mRNA and produces collagentissue within the muscle [19] There are a large number offibroblast cells and type I III IV and V collagen transcriptsin the small intestines of CD patients [20] which producemore ECM including collagen elastin and glycoproteinsthat are deposited intramurally to produce a thickening of theintestinal wall [10] Based on a large number of experimentalreports the ERK-12 signaling pathway appears to participatein myofibroblast proliferation differentiation and collagenproduction [10 11 21ndash25] extracellular matrix synthesis [2627] smooth muscle cell proliferation and apoptosis [28]and the migration of vascular smooth muscle cells [29]Inhibiting the expression and activity of ERK can inhibit theproliferation of vascular smooth muscle cells [30] to reducethe accumulation of ECM [31] Therefore it is inferred thatthe ERK-12 signaling pathway may induce the activationand proliferation of CD intestinal myofibroblasts and theirtransformation intomuscle fiber cells Additionally the ERK-12 signaling pathway may regulate the proliferation andapoptosis of smooth muscle cells

Evidence-Based Complementary and Alternative Medicine 7

(a) (b)

(c) (d)

(e)

Figure 7 Expression of Raf-1 protein in the colonic mucosa of rat The Raf-1 protein was expressed in epithelial cells in the colonic mucosaof rats EnVision Plus method times200 (a) Normal (b) Crohnrsquos disease (c) mild moxibustion (d) electroacupuncture and (e) herb-separatedmoxibustion

005

115

225

335

4

NC MC MM EA HSP

Raf-1

mRN

A le

vels

()

lowast

Figure 8 The Raf-1 mRNA levels in the rat colon NC normalcontrol group MC model control group MM mild moxibustionEA electroacupuncture and HSM herb-separated moxibustionlowast119875 lt 001 versus NC 119875 lt 005 versus MC

050

100150200250300350400450500

NC MC MM EA HSP

MEK

-1 p

rote

in (I

OD

)

lowast

Figure 9 The IOD of MEK-1 protein expression in the rat colonNC normal control group MC model control group MM mildmoxibustion EA electroacupuncture and HSM herb-separatedmoxibustion lowast119875 lt 001 versus NC 119875 lt 001 versus MC

8 Evidence-Based Complementary and Alternative Medicine

(a) (b)

(c) (d)

(e)

Figure 10 Expression of MEK-1 protein in the colonic mucosa of rat The MEK-1 protein was expressed in epithelial cells in the colonicmucosa of rats EnVision Plus method times200 (a) Normal (b) Crohnrsquos disease (c) mild moxibustion (d) electroacupuncture and (e) herb-separated moxibustion

As an upstream protein in the Raf-MEK-ERK pathwayRas is the product of the proto-oncogene c-ras A variety ofstimulating factors particularly growth factors (eg PDGFEGF TNF and PKC activators as well as Src family members)can activate Ras which further activates Raf-1 ActivatedRaf-1 then activates MEK and ERK downstream [32 33] toinitiate the MAPK pathway Raf-1 protein kinase is widelyexpressed in various types of cells [34] Ras and Raf-1 oftenexist in the form of complexes Ras the upstream activatorprotein can translocate Raf from the cytoplasm to theplasma membrane where the latter is activated Howeverthe mechanism by which Raf is activated remains unclearalthough it may include phosphorylation [35] All that isknown is that Raf activation is related to its phosphorylation

on SerThr residues [33] Recent studies have shown thatRaf-1 protein kinase may be a point of crosstalk with othersignaling pathways This protein may participate in a varietyof biological processes in signal transduction including itsown regulation by interactingwith other signalingmoleculesor via reciprocal regulation

The MAPK kinases include MEK1 (MKK1) and MEK2(MKK2)MEK1 andMEK2 are the central links in theMAPKpathway The activated upstream Raf-1 protein kinase cancombine with MEK1 and 2 and doubly phosphorylate Tyrand Thr its two regulatory sites to activate ERK [36] MEKsare not only ERK activators but may also relocalize theactivated ERKs to the nucleus or other active sites to furtherphosphorylate downstream substrates [37]

Evidence-Based Complementary and Alternative Medicine 9

0

2

1

3

5

4

6

19

NC MC MM EA HSP

MEK

-1 m

RNA

leve

ls (

)

lowast

Figure 11 The MEK-1 mRNA levels in the rat colon NC normalcontrol group MC model control group MM mild moxibustionEA electroacupuncture and HSM herb-separated moxibustionlowast119875 lt 001 versus NC

0200400600800

100012001400

NC MC MM EA HSPPhos

phor

ylat

ed E

RK-1

2 (I

OD

)

lowast

Figure 12 The IOD of phosphorylated ERK-12 levels in therat colon NC normal control group MC model control groupMM mild moxibustion EA electroacupuncture and HSM herb-separated moxibustion lowast119875 lt 001 versus NC 119875 lt 005 119875 lt 001versus MC

The upstream kinase MEK1 is a dual-specificity enzymeActivatedMEK1 can phosphorylate ERK1 and ERK2There isevidence that the Tyr residues of ERK1 and 2 are phosphory-lated before the Thr residues both intracellularly and extra-cellularly [38] ERK translocates into the nucleus by forminga homodimer of phosphorylated ERK and phosphorylatedor unphosphorylated ERK [39] ERK is evenly distributedin quiescent cells but when the cells are stimulated byserum persistently activated ERK can partially translocateinto the nucleus whereas transiently activated ERK cannotrelocalize Activated ERKs accumulate in the nucleus andthen phosphorylate the corresponding transcription factorswhich drive expression of specific gene products Becauseof the difference in nuclear localization transiently activated(or low-activity) ERK and persistently activated (or high-activity) ERK cause cells to express genes at a different qualityor quantity resulting in different biological effects [40ndash43]

The ERK1-ERK2-MAPK pathway incorporates a mech-anism for inactivation after activation which can preventexcessive activation-induced adverse consequences At thesame time the inactivated kinase is prepared for the nextactivation The mechanisms of inhibition of this pathwayinclude the following (1) the activation of ERK12-MAPK is

the result of the phosphorylation cascade and can be inac-tivated by dephosphorylation by the corresponding phos-phatases (2) Raf-1 is the direct substrate of PKA which canphosphorylate Ser43 of c-Raf an inhibitory phosphorylationsite in the N-terminus of Raf that interacts with Ras andthe phosphorylation of this site can inhibit the bindingbetween RasGTP and the N-terminus of Raf which blocksthe activation of ERK (3) the phosphatase PP2 can inactivateMEK and ERK via dephosphorylation (4) activated ERKphosphorylates Sos and Raf while ERK dephosphorylationinactivates them thereby generates negative feedback regula-tion of the pathwayTherefore the study of themechanism bywhich acupuncture regulates the expression of ERKupstreamreceptors the phosphorylation of ERK cascade members thetargets of downstream signal transduction and the potentialimpact of other signaling pathways on this pathway is neces-sary to confirm acupuncture as an antifibrotic treatment

Our experiments studied the regulation of Ras RafMEKand ERK protein and gene expression by acupuncture inCD intestinal fibrosis in the rat colon Our results showthat the mRNA levels of Ras Raf-1 MEK-1 and ERK-12 were significantly increased in CD rat intestinal tissueand that the expression of p-Ras Raf-1 MEK-1 and p-ERK-12 proteins in intestinal epithelial cells were also sig-nificantly increased These results suggest that the activityof the ERK pathway in CD intestinal fibrosis process wasaltered Medicine-separated moxibustion mild moxibustionand electroacupuncture treatments could effectively down-regulate the expression of p-Ras Raf-1 MEK-1 and p-ERK-12 but different inhibitory effects on Ras Raf-1 MEK-1and ERK-12 mRNA levels were shown in ERK pathwayactivity changes In this discussion we propose the following(1) medicine-separated moxibustion mild moxibustion andelectroacupuncture can inhibit CD intestinal fibrosis byregulating ERK pathway activity and (2) the acupuncture-induced inhibition of p-Ras Raf-1 MEK-1 and p-ERK-12 expression may be the key elements in the treatmentrsquosinhibition of CD intestinal fibrosis At present most reportsregarding the role of acupuncture in the regulation of theERK signaling pathway have focused on EA such as EAmodel rats After EA on the Dazhui (DU14) and Baihui(DU20) acupuncture points the number of ERK-positivestriatal cells increased significantly in the cerebral cortex andERK protein expression was increased in rats with damagedneurons after cerebral ischemia and reperfusion thereforeEA can increase ERK pathway activity [44 45] After the EAtreatment of gastric mucosa-injured rats the phosphoryla-tion levels of ERK in gastric mucosal cells from extractedserum were significantly increased and were significantlydifferent between the serum of the normal rats and that ofthe model rats These observations suggest that ERK maybe involved in signal transduction in the EA stimulation atacupoints of stomach meridian of foot yangming repairinggastric mucosal injury [46] and that EA had an analgesiceffect by downregulating p-ERK expression levels in thedorsal horn of the spinal cord of rats with adjuvant arthritis[47] The previous reports have demonstrated that EA canregulate the ERK signal transduction pathway The results ofour experiments show that medicine-separated moxibustion

10 Evidence-Based Complementary and Alternative Medicine

(a) (b)

(c) (d)

(e)

Figure 13 Expression of phosphorylated ERK-12 protein in the colonicmucosa of ratThe phosphorylation of ERK-12 proteinwas expressedin epithelial cells and lymphocyte in the colonic mucosa of rats EnVision Plus method times200 (a) Normal (b) Crohnrsquos disease (c) mildmoxibustion (d) electroacupuncture and (e) herb-separated moxibustion

005

115

225

335

4

NC MC MM EA HSP

ERK-

12

mRN

A le

vels

()

lowast

Figure 14 The ERK-12 mRNA levels in the rat colon NC normalcontrol group MC model control group MM mild moxibustionEA electroacupuncture and HSM herb-separated moxibustionlowast119875 lt 001 versus NC 119875 lt 005 versus MC

andmildmoxibustion can also regulate the ERK signal trans-duction pathway and inhibit Ras and Raf-1 better than EAThe functional mechanisms underlying these observationsneed to be explored further

Abbreviations

CD Crohnrsquos diseaseNC Normal control groupMC Model control groupEA ElectroacupunctureMM Mild moxibustionHSM Herb-separated moxibustionST25 Tianshu acupoint

Evidence-Based Complementary and Alternative Medicine 11

RN6 Qihai acupointERK Extracellular signal-regulated kinaseMAPK Mitogen-activated protein kinaseTNBS 246-Trinitrobenzene sulfonic acid

Acknowledgments

The authors thank Professor Shuhui Jiang for technical assis-tance and the support of the National Natural Science Foun-dation of China (no 81173331) and National Basic ResearchProgram of China (973 program no 2009CB522900)Xiaomei Wang and Yuan Lu have the same right

References

[1] G Manning D B Whyte R Martinez T Hunter and SSudarsanam ldquoThe protein kinase complement of the humangenomerdquo Science vol 298 no 5600 pp 1912ndash1934 2002

[2] C Mowat A Cole A Windsor et al ldquoGuidelines for themanagement of inflammatory bowel disease in adultsrdquoGut vol60 no 5 pp 571ndash607 2011

[3] Inflammatory Bowel Disease Co-operation Group ChineseSociety of Gastroenterology ldquoChinese consensus on diagnosisand treatment standard of inflammatory bowel diseaserdquoChineseJournal of Gastroenterology vol 12 no 8 pp 490ndash494 2007

[4] J J Zheng X S Zhu Z Huangfu Z X Gao Z R Guo andZ Wang ldquoCrohnrsquos disease in mainland China a systematicanalysis of 50 years of researchrdquo Chinese Journal of DigestiveDiseases vol 6 no 4 pp 175ndash181 2005

[5] M Dai and B M Li ldquoResearch progress in mechanisms ofintestinal fibrosis in Crohnrsquos diseaserdquo Shijie Huaren XiaohuaZazhi vol 17 no 11 pp 1117ndash1121 2009

[6] O Grip J Malm B Veress A Bjartell S Lindgren and AEgesten ldquoIncreased presence of cells containing transforminggrowth factor alpha (TGF-120572) in ulcerative colitis both duringactive inflammation and in remissionrdquo European Journal ofGastroenterology and Hepatology vol 12 no 7 pp 761ndash7662000

[7] I E Koutroubakis E Petinaki P Dimoulios et al ldquoSerumlaminin and collagen IV in inflammatory bowel diseaserdquoJournal of Clinical Pathology vol 56 no 11 pp 817ndash820 2003

[8] S Speca I Giusti F Rieder and G Latella ldquoCellular andmolecular mechanisms of intestinal fibrosisrdquo World Journal ofGastroenterology vol 18 no 28 pp 3635ndash3661 2012

[9] Y F Song J H Hong and L M Xu ldquoTGF-120573 1ERK pathwaysand its inhibitors and fibrosisrdquo Chinese Journal of ClinicalHepatology vol 21 no 2 pp 125ndash127 2005

[10] H C Huang M Yang J Z Li and H Y Wang ldquoConnectivetissue growth factor promotes the proliferation ofmyofibroblastthrough Erk-12 signaling pathwayrdquoNational Medical Journal ofChina vol 85 no 19 pp 1322ndash1326 2005

[11] L P Guo H C Huang and J Z Li ldquoHypoxia inducesmyofibroblast formation and stimulates production of collagenI in myofibroblasts through ERK12 pathwayrdquo Chinese Journalof Pathophysiology vol 24 no 12 pp 2428ndash2432 2008

[12] H R Liu X P Ma T P Zhao and H G Wu ldquoThe adjustmentof acupuncture on colon collagen synthesis and TGF-120573mRNAexpression in Crohnrsquos disease ratrdquo Journal of Anhui TraditionalChinese Medical College vol 24 no 4 pp 25ndash30 2005

[13] X P Ma C P An H G Wu et al ldquoHerbs-partitionedmoxibustion and electro-acupuncture effects on transforming

growth factor-beta 1 connective tissue growth factor collagentype I and fibronectin expression in the colon of Crohnrsquo sdisease ratsrdquo Journal of Clinical Rehabilitative Tissue EngineeringResearch vol 12 no 20 pp 3853ndash3858 2008

[14] G P Morris P L Beck M S Herridge W T Depew M RSzewczuk and J LWallace ldquoHapten-inducedmodel of chronicinflammation and ulceration in the rat colonrdquoGastroenterologyvol 96 no 3 pp 795ndash803 1989

[15] TheMinistry of Science andTechnology of the Peoplersquos Republicof China Guidance Suggestions for the Care and Use ofLaboratory Animals 2006

[16] J D Butzner R Parmar C J Bell andVDalal ldquoButyrate enematherapy stimulates mucosal repair in experimental colitis in theratrdquo Gut vol 38 no 4 pp 568ndash573 1996

[17] X M Wang Y Lu L Y Wu et al ldquoMoxibustion inhibitsinterleukin-12 and tumor necrosis factor alpha and modulatesintestinal flora in rat with ulcerative colitisrdquo World Journal ofGastroenterology vol 18 no 46 pp 6826ndash6835 2012

[18] J B Pucilowska K K McNaughton N K Mohapatra et alldquoIGF-I and procollagen 1205721(I) are coexpressed in a subset ofmesenchymal cells in active Crohnrsquos diseaserdquo American Journalof Physiology vol 279 no 6 pp G1307ndashG1322 2000

[19] M Gharaee-Kermani B Hu S H Phan and M R GyetkoldquoRecent advances in molecular targets and treatment of idio-pathic pulmonary fibrosis focus on TGF120573 signaling and themyofibroblastrdquo Current Medicinal Chemistry vol 16 no 11 pp1400ndash1417 2009

[20] E M Zimmermann L Li Y T Hou M Cannon G MChristman andKN Bitar ldquoIGF-I induces collagen and IGFBP-5 mRNA in rat intestinal smooth musclerdquo American Journal ofPhysiology vol 273 no 4 pp G875ndashG882 1997

[21] L J ZhangQ Li P Chen et al ldquoMediating effect of extracellularsignal-regulated kinase 12 in PDGF-induced proliferation andcollagen synthesis of cultured rat cardiac fibroblastsrdquo ChineseJournal of Anatomy vol 13 no 4 p 223 2007

[22] M Tang W Zhang H Lin H Jiang H Dai and Y ZhangldquoHigh glucose promotes the production of collagen types Iand III by cardiac fibroblasts through a pathway dependenton extracellular-signal-regulated kinase 12rdquo Molecular andCellular Biochemistry vol 301 no 1-2 pp 109ndash114 2007

[23] H Y Dai Z M Ge and Y H Li ldquoEffect of urotensin II on pro-liferative potential and phosphorylation of extracellular signal-regulated kinase 12 adventitial fibroblasts from spontaneouslyhypertensive ratrdquoActaAcademiaeMedicinae Sinicae vol 28 no6 pp 776ndash780 2006

[24] I Fuentes-Calvo A M Blazquez-Medela N Eleno E SantosJ M Lopez-Novoa and C Martınez-Salgado ldquoH-Ras isoformmodulates extracellular matrix synthesis proliferation andmigration in fibroblastsrdquo American Journal of Physiology vol302 no 4 pp C686ndashC697 2012

[25] Y W Ma G T Bai and M X Zhang ldquoStudies on signal trans-duction pathway induced by fibronectin in human embryoniclung fibroblastsrdquo Shanxi Medical Journal vol 38 no 7 pp 587ndash589 2009

[26] J H Jang and C P Chung ldquoFibronectin-mediated adhesionrescues cell cycle arrest induced by fibroblast growth factor-1 bydecreased expression of P21CIPWAF in human chondrocytesrdquoIn Vitro Cellular and Developmental Biology vol 41 no 5-6 pp126ndash129 2005

[27] B Zhu Y J Wang C F Zhu et al ldquoTriptolide inhibitsextracellularmatrix protein synthesis by suppressing the Smad2

12 Evidence-Based Complementary and Alternative Medicine

but not the MAPK pathway in TGF-1205731-stimulated NRK-49Fcellsrdquo Nephrology Dialysis Transplantation vol 25 no 10 pp3180ndash3191 2010

[28] J Bai X S Liu Y J Xu M Xie and W Ni ldquoEffects ofextracellular signal regulated kinase signaling pathway on cellcycle of airway smooth muscle cells in asthmatic ratsrdquo ChineseJournal of Respiratory and Critical Care Medicine vol 9 no 1pp 23ndash27 2010

[29] S Matsusaka and I Wakabayashi ldquoEnhancement of vascu-lar smooth muscle cell migration by urotensin IIrdquo Naunyn-Schmiedebergrsquos Archives of Pharmacology vol 373 no 5 pp 381ndash386 2006

[30] Y Chen Z Dai Y M Liu et al ldquoInhibitory effects ofCGRP on vascular smooth muscle cell proliferation role ofcaveolaecaveolin-1ERK12 signal pathwayrdquo Progress in Bio-chemistry and Biophysics vol 40 no 5 pp 445ndash453 2013

[31] W L Wu A Q Zhang Q Yin and W H Gan ldquoEffectsof ERK signal pathway on extracellular matrix synthesis ofrat mesangial cells treated with PDGF-BBrdquo ACTA UniversityMedicinalis Nanjing vol 27 no 10 pp 1126ndash1129 2007

[32] L S Steelman W H Chappell S L Abrams et al ldquoRoles ofthe RafMEKERK and PI3KPTENAktmTOR pathways incontrolling growth and sensitivity to therapy-implications forcancer and agingrdquo Aging vol 3 no 3 pp 192ndash222 2011

[33] W Kolch ldquoMeaningful relationships the regulation of theRasRafMEKERK pathway by protein interactionsrdquo Biochem-ical Journal vol 351 no 2 pp 289ndash305 2000

[34] D Matallanas M Birtwistle D Romano et al ldquoRaf familykinases old dogs have learned new tricksrdquo Genes and Cancervol 2 no 3 pp 232ndash260 2011

[35] C S Mason C J Springer R G Cooper G Superti-Furga C JMarshall and RMarais ldquoSerine and tyrosine phosphorylationscooperate in Raf-1 but not B-Raf activationrdquo The EMBOJournal vol 18 no 8 pp 2137ndash2148 1999

[36] R J Fiddes P W Janes S P Sivertsen R L Sutherland EA Musgrove and R J Daly ldquoInhibition of the MAP kinasecascade blocks heregulin-induced cell cycle progression in T-47D human breast cancer cellsrdquo Oncogene vol 16 no 21 pp2803ndash2813 1998

[37] S T Eblen J K SlackM JWeber andA D Catling ldquoRac-PAKsignaling stimulates extracellular signal-regulated kinase (ERK)activation by regulating formation of MEK1-ERK complexesrdquoMolecular and Cellular Biology vol 22 no 17 pp 6023ndash60332002

[38] M Cargnello and P P Roux ldquoActivation and function ofthe MAPKs and their substrates the MAPK-activated proteinkinasesrdquo Microbiology and Molecular Biology Reviews vol 75no 1 pp 50ndash83 2011

[39] A V Khokhlatchev B Canagarajah J Wilsbacher et al ldquoPhos-phorylation of theMAP kinase ERK2 promotes its homodimer-ization and nuclear translocationrdquo Cell vol 93 no 4 pp 605ndash615 1998

[40] R H Chen C Sarnecki and J Blenis ldquoNuclear localization andregulation of erk- and rsk-encoded protein kinasesrdquoMolecularand Cellular Biology vol 12 no 3 pp 915ndash927 1992

[41] S Traverse K Seedorf H Paterson C J Marshall P Cohenand A Ullrich ldquoEGF triggers neuronal differentiation of PC12cells that overexpress the EGF receptorrdquo Current Biology vol 4no 8 pp 694ndash701 1994

[42] T T Nguyen J C Scimeca C Filloux P Peraldi J L Car-pentier and E Van Obberghen ldquoCo-regulation of the mitogen-activated protein kinase extracellular signal-regulated kinase 1

and the 90-kDa ribosomal S6 kinase in PC12 cells Distincteffects of the neurotrophic factor nerve growth factor and themitogenic factor epidermal growth factorrdquo Journal of BiologicalChemistry vol 268 no 13 pp 9803ndash9810 1993

[43] I Dikic J Schlessinger and I Lax ldquoPC12 cells overexpressingthe insulin receptor undergo insulin-dependent neuronal dif-ferentiationrdquo Current Biology vol 4 no 8 pp 702ndash708 1994

[44] M H Shen Z R Li X R Xiang W M Niu and C B ZhangldquoEffect of electro-acupuncture on ERK protein expression ofMCAO ratsrdquo Journal of Clinical Acupuncture and Moxibustionvol 23 no 7 pp 62ndash63 2007

[45] M H Shen Z R Li X R Xiang W M Niu and C BZhang ldquoInvolvement of extracellular regulated protein kinasesignal transduction pathway in electroacupuncture-inducedimprovement of ischemic cerebral injury in rats with cerebralischemia reperfusionrdquo Acupuncture Research vol 32 no 6 pp368ndash372 2007

[46] Z B Yang J Yan and S X Yi ldquoUp-regulation of the ERK phos-phorylation in gastric mucosal cell by the serum of rats treatedwith electroacupuncture at stomach meridian acupointsrdquo Basicamp Clinical Medicine vol 29 no 2 pp 135ndash138 2009

[47] C L Wang S X Wang and W Gong ldquoChanges in phos-phatized ERK in the spinal dorsal horn in adjuvant arthritisrats during electroacupuncture of Jiaji points for analgesiardquoShanghai Journal of Acupuncture and Moxibustion vol 24 no12 pp 33ndash35 2005

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Behavioural Neurology

EndocrinologyInternational Journal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

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Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Evidence-Based Complementary and Alternative Medicine

the basis of therapy for CD intestinal fibrosis is long-termanti-inflammatory treatment However although they mayreduce inflammation anti-inflammatory treatments do notreduce fibrous stenosis Therefore an understanding of thepathogenesis of CD intestinal fibrosis and the discovery ofeffective prophylactic measures with fewer side effects areurgently needed in this field

Intestinal fibrosis is a complicated pathological process inwhichmany cell types cytokines and signaling pathways par-ticipate including the extracellular signal-regulated kinase(ERK) signaling pathway which has become a popular topicof current study ERK including ERK1 and ERK2 is oneof the mitogen-activated protein kinase (MAPK) signalingpathwaysThe ERK12-MAPK signaling pathway is a cascadethat is related to the pathological fibrosis of multiple diseases[9] An activated Ras-Raf-MEK-ERK pathway promotesfibrosis and plays an important role inTGF-120573-mediated type Icollagen expression extracellular matrix (ECM) productionand myofibroblast formation [10 11]

Preliminary studies have shown that medicine-separatedmoxibustion can significantly inhibit the process of fibrosisin CD rats [12 13] but the regulatory mechanism underlyingthis process remains to be further explored In this study aCD intestinal fibrosis rat model was prepared by the Morrismethod [14] EnVision immunohistochemistry and qPCRwere used to measure the levels of Ras Raf MEK and ERKproteins and mRNA expression in colon tissues to explorethe key signaling molecules involved in the regulation of CDintestinal fibrosis in rats and reveal the mechanisms underly-ing the effects of moxibustion on CD intestinal fibrosis

2 Materials and Methods

21 Experimental Animals A total of 60 male Sprague-Dawley (SD) rats with a body weight of 180 plusmn 20 g wereprovided by the Experimental Animal Center at the ShanghaiUniversity of Traditional Chinese Medicine All of the ratswere kept for one week at room temperature after receiptTherats that behaved normally with respect to feeding drinkingand other behaviors were included in the experiment Thisstudy received permission from the Ethics Committee inYueyang Hospital which is affiliated with Shanghai Univer-sity of Traditional Chinese Medicine China All protocolswere performed in strict accordance with the GuidanceSuggestions for the Care and Use of Laboratory Animalsformulated by the Ministry of Science and Technology of thePeoplersquos Republic of China [15]

22 Grouping and Model Establishment A total of 60 SD ratswere randomly divided into a normal group of 12 rats anda model group of 48 rats A 246-trinitrobenzene sulfonicacid (TNBS) (Sigma Co St Louis USA) enema of 5 TNBSand 50 ethanol was prepared at a 2 1 ratio The rats in eachgroup were fasted but not deprived of water for 48 hThe ratsin the model group were anesthetized by the intraperitonealinjection of 2 sodium pentobarbital (China Langchem IncShanghai China) at a dose of 20mgkg and were verticallyinverted to introduce 100mgkg TNBS by rectal infusiononce per 7 days for a total of four treatments whereas the

normal group was not treated To validate the model thedistal colons from one rat in the normal group and two ratsin the model group were examined by visual observation andHampE staining After the success of the model was confirmedthe 44 rats were randomly assigned to a model group a mildmoxibustion group a herb-separatedmoxibustion group andan EA group with 11 rats per group Two rats died during themodeling process and one rat from themodel group and onerat from the mild moxibustion group died during the courseof treatment

23 Treatment

NC Group No treatment

MC Group TNBS enema only no treatment

MM Group A TNBS enema was administered and theST25 (bilateral) and RN6 acupuncture points were located Amoxa stick (Nanyang Hanyi Moxa Co Ltd China) 5mm indiameter was lit and placed at points at a distance of 1-2 cmfrom the ST25 and RN6 acupuncture points Moxibustionwas performed for 10min per point per treatment once a dayfor a total of seven treatments

HSP Group A TNBS enema was administered and theST25 (bilateral) and RN6 acupuncture points were locatedAconite cinnamon salvia and other drugs were milled intoa powder andmixed with rice wine to form a pasteThe pastewas then pressed into amedicinal cake 5mm in diameter and3mm in thickness A moxa cone was formed from 90mg ofrefined moxa Two moxa cones were used for moxibustionfor each point per day and a total of seven moxibustiontreatments were performed for 7 days

EA Group A TNBS enema was administered and the ST25(bilateral) and RN6 acupuncture points were located Nee-dles (Suzhou Huatuo Co Ltd Suzhou China) 025mm indiameter and 25mm in length were inserted to a depth of 3-4mm and connected to an LD202HHan electroacupunctureapparatus (Beijing Huawei Co Ltd Beijing China) TheRN6 and bilateral ST25 acupuncture points were stimulatedalternately by a continuouswave of 03ms pulsewidth 100Hzfrequency and 1mA current once a day for 10min withcontinuous treatment for 7 days

24 Specimen Collection Following treatment the rats werefasted for 24 hwith access towater andwere then anesthetizedby intraperitoneal injection of 2 sodium pentobarbital at adose of 50mgkg The anesthetized rats were then surgicallysectioned to remove approximately 6 cm of the distal colonThemesenteriumwas then cut longitudinally and rinsedwith4∘C saline The mucosal injury was visually examined andevaluated using a gross injury score [16 17] Two pieces oftissue with obvious lesions were removed of which one wastransferred to liquid nitrogen until testing and the other wasfixed in a 10 neutral formaldehyde solution

25 Detection of Collagen Fibers in the Rat Colon Massontrichrome staining was used to detect collagen fibers in

Evidence-Based Complementary and Alternative Medicine 3

Table 1 The sequence of the forward primer and reverse primer used for fluorescent quantitative PCR assays

Name Primer sense Sequence (51015840rarr31015840) Amplification product (bp)

GAPDH Forward 51015840-CCGAGGGCCCACTAAAGG-31015840 116 bpReverse 51015840-GCTGTTGAAGTCACAGGAGACAA-31015840

Ras Forward 51015840-TGGTGGGCAACAAGTGTGAC-31015840 181 bpReverse 51015840-GGGTTCAGTTTCCGCAGTTTATG-31015840

Raf-1 Forward 51015840-CAGGAGCACAAAGGTAAGAAAGC-31015840 201 bpReverse 51015840-GCCACAAGTCTGACATCGAAATC-31015840

MEK-1 Forward 51015840-AATATGTCAGGTTCAAGGAAAGGAC-31015840 169 bpReverse 51015840-AGGTAAGGACGAAGCCACAAG-31015840

ERK-12 Forward 51015840-TATATACATTCAGCTAATGTTCTGC-31015840 169 bpReverse 51015840-TCTACCTGCAGCTGGAACCTCTC-31015840

the rat colon Green staining was interpreted as collagenmucus and cartilage red staining denotes muscle and nervefibers and black indicates nuclei

26 EnVision Plus Immunohistochemical Detection of RasRaf MEK and ERK-12 in Colonic Tissue Fixed tissues wereconventionally deparaffinized in water and washed threetimes for 3min in 001M PBS pH 74 The tissue sampleswere heated and allowed to equilibrate to room temperaturefor 20min and then washed three times in PBS for 3min Toinhibit endogenous peroxidase the samples were treatedwith03 H

2O2at room temperature for 20min and then rinsed

three times with PBS for 3min The tissue samples wereincubated in 20normal goat serum at room temperature for30min and then with one drop of the appropriately dilutedprimary antibody (Santa Cruz Bio Inc Santa Cruz USA) at4∘C for 18 h The tissues were washed three times with PBSfor 3min mixed with one drop of Envision reagent (HRP-M) and incubated in a humidified chamber for 30min at37∘C After washing three times with PBS for 3min the assaywas developed in 004 DAB and 03 H

2O2for 8ndash12min

then washed in water stained with hematoxylin for 1minand bathed in hot water for 3 sec to allow the blue color todevelop Finally the tissue sections were dried and mountedwith a neutral resin All the samples were analyzed by aMoticMed 60 image analysis system (MoticGroupCo Ltd)Threefields were randomly selected the positive target value ofthe integral optical density was calculated under an opticalmicroscope (Olympus Co Ltd) at 200x magnification

27 qPCR Detection of Ras Raf MEK and ERK-12 mRNALevels in Colonic Tissues The total RNA was extracted fromcolon tissue samples The cDNA was synthesized by reversetranscription (RT) according to the qPCR kit (ShanghaiDaweike Bio Inc Shanghai China) instructions and ampli-fied by PCR according to the kit instructions (Table 1) Thefollowing cycling conditions were used RT at 37∘C for 1 h95∘C for 5min to inactivate the MMLV RT enzyme 50∘C for2min 95∘C for 5min and 40 cycles of 95∘C for 15 sec and60∘C for 45 sec

28 Statistical Analysis SPSS 180 statistical software (SPSSInc Chicago USA) was used for statistical analysis The

012345678

NC MC MM EA HSP

Gro

ss in

jury

scor

es

lowast

Figure 1 The gross injury scores of the colon tissue in eachrat group NC normal control group MC model control groupMM mild moxibustion EA electroacupuncture and HSM herb-separated moxibustion lowast119875 lt 001 versus NC 119875 lt 001 versus MC

measurement data are presented as the mean plusmn SD Fornormally distributed data one-way analysis of variance(ANOVA) was applied The least significant differencemethod (LSD) was used for the homogeneity of variance andGames-Howell was used to compare the differences betweengroups for the heterogeneity of variance A value of 119875 lt 005was considered statistically significant The nonparametricMann-Whitney U-test was used for abnormal distributions

3 Results

31 Gross Injury in the Colon Tissue of Rat When the colonictissues were observed by visual inspection the rats in thenormal group exhibited a uniformity of colon bowel andintestinal wall a smooth colonic mucosa surface clear vascu-lar texture no bleeder and no erosion or ulcers The modelgroup exhibited partial intestinal canal stenosis or intestinalwall thicken and harden fuzzy vascular texture edematouscolonic mucosa serious congestion erosion and differentsize shape and depth ulcer formation After treatmentsthere were lacked smooth colonicmucosa surface edematouscolonic mucosa and erosion in both MM and EA groupand lacked smooth colonic mucosa surface with no obviouserosion and ulcer in HPM group

As shown in Figure 1 the gross injury score of the colonictissue in the NC group was significantly higher than the MCgroup (119875 lt 001) After treatment the scores were lower in

4 Evidence-Based Complementary and Alternative Medicine

(a) (b)

(c) (d)

(e)

Figure 2 Expression of collagen fibers in the colon tissue of rat Masson trichrome staining method times100 (a) Normal (b) Crohnrsquos disease(c) mild moxibustion (d) electroacupuncture (e) herb-separated moxibustion

both the HPM group MM group and the EA group (119875 lt001) It suggests that these treatments are able to improve thetissue damage of CD

32 Expression of Collagen Fibers in the Colon Tissue of RatAs shown in Figure 2 Masson trichrome staining revealedthat in the MC group a large number of collagen fibers werepresent in the mucosa muscularis mucosa submucosa andmuscularis of the rat colon with a disordered arrangementIn the MM group and the HSM group there were smallnumbers of collagen fibers in the colonic lamina propria andsubmucosa at the original ulcer-healing location which wasless severe compared with that of the model group In the EAgroup there were a small number of collagen fibers in the

mucosa submucosa muscularis and serosa of the rat colonand the effect was less severe than in the MC group

33 Ras Expression in the Colonic Mucosa of Rat As shownin Figures 3 and 4 the phosphorylation of Ras protein inthe rat colon was significantly higher in the MC group thanin the NC group (119875 lt 001) Ras protein expression wassignificantly reduced (119875 lt 001) to normal levels after acu-puncture treatment

Figure 5 indicates that Ras mRNA expression in the MCgroup was higher than that in the NC group (119875 lt 001) andthat HSM effectively reduced the Ras mRNA levels in the ratcolon (119875 lt 005) whereas there was no significant differencebetween the EA group and the NC group

Evidence-Based Complementary and Alternative Medicine 5

0

100

200

300

400

500

600

700

NC MC MM EA HSPRa

s pho

spho

ryla

tion

prot

ein

(IO

D)

lowast

Figure 3The integral optical density (IOD) of Ras phosphorylation in each rat group NC normal control group MC model control groupMM mild moxibustion EA electroacupuncture and HSM herb-separated moxibustion lowast119875 lt 001 versus NC 119875 lt 001 versus MC

(a) (b)

(c) (d)

(e)

Figure 4 Expression of phosphorylated Ras protein in the colonic mucosa of rat The phosphorylation of Ras protein was expressedin epithelial cells and lymphocyte in the colonic mucosa of rats EnVision Plus method times200 (a) Normal (b) Crohnrsquos disease (c) mildmoxibustion (d) electroacupuncture (e) herb-separated moxibustion

6 Evidence-Based Complementary and Alternative Medicine

0

2

4

Ras m

RNA

leve

l (

)

6

8

10

12 17

25

21

NC MC MM EA HSP

lowast

lowast

Figure 5 The Ras mRNA levels in the rat colon NC normalcontrol group MC model control group MM mild moxibustionEA electroacupuncture and HSM herb-separated moxibustionlowast119875 lt 001 versus NC 119875 lt 005 versus MC

0100200300400500600700800900

NC MC MM EA HSP

Raf-1

pro

tein

(IO

D)

lowast

998771998771 998771

Figure 6 The IOD of Raf-1 protein expression in rat colonNC normal control group MC model control group MM mildmoxibustion EA electroacupuncture and HSM herb-separatedmoxibustion lowast119875 lt 001 versus NC 119875 lt 005 119875 lt 001 versusMC 998771998771119875 lt 005 998771119875 lt 001 versus EA

34 The Raf-1 Expression in the Colonic Mucosa of Rat Asshown in Figures 6 and 7 the Raf-1 protein expression inthe colonic tissue of the MC group was significantly higherthan in the NC group (119875 lt 001) and that the Raf-1 proteinexpression in the colon was reduced to varying degrees afteracupuncture treatment (119875 lt 005 or 119875 lt 005)

The results in Figure 8 show that the Raf-1 mRNAlevels in the MC group were significantly higher than in theNC group (119875 lt 001) and that MM and HSM treatmentssignificantly reduced rat colon Raf-1 mRNA levels (119875 lt 005)whereas EA treatment did not significantly affect rat colonRaf-1 mRNA levels

35 The MEK-1 Expression in the Colonic Mucosa of Rat Asshown in Figures 9 and 10 the MEK-1 protein expression inthe colonic tissue of the MC group was significantly higherthan in NC rats (119875 lt 001) and MEK-1 protein expressionwas significantly reduced in each group after acupuncturetreatment (119875 lt 001) although the levels were still higherthan normal levels (119875 lt 001)

The results in Figure 11 show that theMEK-1mRNA levelsin the model group were significantly higher than those inthe NC group (119875 lt 001) and that MM HSM and EAtreatment reduced MEK-1 mRNA levels although there was

no statistically significant difference between the treatmentand no treatment groups or among the treatment groups

36 The ERK-12 Expression in the Colonic Mucosa of Rat Asshown in Figures 12 and 13 the phosphorylation of ERK-12significantly increased in the MC group compared with theNC group (119875 lt 001) and that the levels of phosphorylatedERK-12 proteins were significantly reduced after the MMand HSM treatments (119875 lt 001)

The results in Figure 14 show that the ERK-12 mRNAexpression in the MC group was significantly higher thanthat in the NC group (119875 lt 001) and that ERK-12 mRNAexpression was significantly lower in the MM group afteracupuncture treatment compared with the MC group (119875 lt005)

4 Discussion

Extracellular signal-regulated kinase (ERK) a member ofthe mitogen-activated protein kinase (MAPK) family playsan important biological role in transmitting signals fromsurface receptors to the nucleus The ERK12 pathway is themost thoroughly researched pathway in the ERK family andplays a role in a wide range of cellular functions includ-ing regulating meiosis mitosis and anaphase processes indifferent cells A variety of stimulating factors such as growthfactors cytokines viruses G protein-coupled receptor lig-ands and oncogenes can activate the ERK12 pathway [18]The ERK signaling pathway is a Ras-Raf-MEK-ERK path-way Phosphorylation-activated ERK12 translocates fromthe cytoplasm to the nucleus and thereby mediates thetranscriptional activation of Elk-1 ATF NF-120581B AP-1 and c-fos which in turn regulate cell proliferation and differentia-tion the maintenance of cell shape the construction of thecytoskeleton apoptosis and the malignant transformation ofcells among other biological functions

One of the features of CD is the transformation of smoothmuscle cells into fibroblasts andor myofibroblasts [8] Thisabundance of fibroblasts and myofibroblasts increases theexpression of type I collagen mRNA and produces collagentissue within the muscle [19] There are a large number offibroblast cells and type I III IV and V collagen transcriptsin the small intestines of CD patients [20] which producemore ECM including collagen elastin and glycoproteinsthat are deposited intramurally to produce a thickening of theintestinal wall [10] Based on a large number of experimentalreports the ERK-12 signaling pathway appears to participatein myofibroblast proliferation differentiation and collagenproduction [10 11 21ndash25] extracellular matrix synthesis [2627] smooth muscle cell proliferation and apoptosis [28]and the migration of vascular smooth muscle cells [29]Inhibiting the expression and activity of ERK can inhibit theproliferation of vascular smooth muscle cells [30] to reducethe accumulation of ECM [31] Therefore it is inferred thatthe ERK-12 signaling pathway may induce the activationand proliferation of CD intestinal myofibroblasts and theirtransformation intomuscle fiber cells Additionally the ERK-12 signaling pathway may regulate the proliferation andapoptosis of smooth muscle cells

Evidence-Based Complementary and Alternative Medicine 7

(a) (b)

(c) (d)

(e)

Figure 7 Expression of Raf-1 protein in the colonic mucosa of rat The Raf-1 protein was expressed in epithelial cells in the colonic mucosaof rats EnVision Plus method times200 (a) Normal (b) Crohnrsquos disease (c) mild moxibustion (d) electroacupuncture and (e) herb-separatedmoxibustion

005

115

225

335

4

NC MC MM EA HSP

Raf-1

mRN

A le

vels

()

lowast

Figure 8 The Raf-1 mRNA levels in the rat colon NC normalcontrol group MC model control group MM mild moxibustionEA electroacupuncture and HSM herb-separated moxibustionlowast119875 lt 001 versus NC 119875 lt 005 versus MC

050

100150200250300350400450500

NC MC MM EA HSP

MEK

-1 p

rote

in (I

OD

)

lowast

Figure 9 The IOD of MEK-1 protein expression in the rat colonNC normal control group MC model control group MM mildmoxibustion EA electroacupuncture and HSM herb-separatedmoxibustion lowast119875 lt 001 versus NC 119875 lt 001 versus MC

8 Evidence-Based Complementary and Alternative Medicine

(a) (b)

(c) (d)

(e)

Figure 10 Expression of MEK-1 protein in the colonic mucosa of rat The MEK-1 protein was expressed in epithelial cells in the colonicmucosa of rats EnVision Plus method times200 (a) Normal (b) Crohnrsquos disease (c) mild moxibustion (d) electroacupuncture and (e) herb-separated moxibustion

As an upstream protein in the Raf-MEK-ERK pathwayRas is the product of the proto-oncogene c-ras A variety ofstimulating factors particularly growth factors (eg PDGFEGF TNF and PKC activators as well as Src family members)can activate Ras which further activates Raf-1 ActivatedRaf-1 then activates MEK and ERK downstream [32 33] toinitiate the MAPK pathway Raf-1 protein kinase is widelyexpressed in various types of cells [34] Ras and Raf-1 oftenexist in the form of complexes Ras the upstream activatorprotein can translocate Raf from the cytoplasm to theplasma membrane where the latter is activated Howeverthe mechanism by which Raf is activated remains unclearalthough it may include phosphorylation [35] All that isknown is that Raf activation is related to its phosphorylation

on SerThr residues [33] Recent studies have shown thatRaf-1 protein kinase may be a point of crosstalk with othersignaling pathways This protein may participate in a varietyof biological processes in signal transduction including itsown regulation by interactingwith other signalingmoleculesor via reciprocal regulation

The MAPK kinases include MEK1 (MKK1) and MEK2(MKK2)MEK1 andMEK2 are the central links in theMAPKpathway The activated upstream Raf-1 protein kinase cancombine with MEK1 and 2 and doubly phosphorylate Tyrand Thr its two regulatory sites to activate ERK [36] MEKsare not only ERK activators but may also relocalize theactivated ERKs to the nucleus or other active sites to furtherphosphorylate downstream substrates [37]

Evidence-Based Complementary and Alternative Medicine 9

0

2

1

3

5

4

6

19

NC MC MM EA HSP

MEK

-1 m

RNA

leve

ls (

)

lowast

Figure 11 The MEK-1 mRNA levels in the rat colon NC normalcontrol group MC model control group MM mild moxibustionEA electroacupuncture and HSM herb-separated moxibustionlowast119875 lt 001 versus NC

0200400600800

100012001400

NC MC MM EA HSPPhos

phor

ylat

ed E

RK-1

2 (I

OD

)

lowast

Figure 12 The IOD of phosphorylated ERK-12 levels in therat colon NC normal control group MC model control groupMM mild moxibustion EA electroacupuncture and HSM herb-separated moxibustion lowast119875 lt 001 versus NC 119875 lt 005 119875 lt 001versus MC

The upstream kinase MEK1 is a dual-specificity enzymeActivatedMEK1 can phosphorylate ERK1 and ERK2There isevidence that the Tyr residues of ERK1 and 2 are phosphory-lated before the Thr residues both intracellularly and extra-cellularly [38] ERK translocates into the nucleus by forminga homodimer of phosphorylated ERK and phosphorylatedor unphosphorylated ERK [39] ERK is evenly distributedin quiescent cells but when the cells are stimulated byserum persistently activated ERK can partially translocateinto the nucleus whereas transiently activated ERK cannotrelocalize Activated ERKs accumulate in the nucleus andthen phosphorylate the corresponding transcription factorswhich drive expression of specific gene products Becauseof the difference in nuclear localization transiently activated(or low-activity) ERK and persistently activated (or high-activity) ERK cause cells to express genes at a different qualityor quantity resulting in different biological effects [40ndash43]

The ERK1-ERK2-MAPK pathway incorporates a mech-anism for inactivation after activation which can preventexcessive activation-induced adverse consequences At thesame time the inactivated kinase is prepared for the nextactivation The mechanisms of inhibition of this pathwayinclude the following (1) the activation of ERK12-MAPK is

the result of the phosphorylation cascade and can be inac-tivated by dephosphorylation by the corresponding phos-phatases (2) Raf-1 is the direct substrate of PKA which canphosphorylate Ser43 of c-Raf an inhibitory phosphorylationsite in the N-terminus of Raf that interacts with Ras andthe phosphorylation of this site can inhibit the bindingbetween RasGTP and the N-terminus of Raf which blocksthe activation of ERK (3) the phosphatase PP2 can inactivateMEK and ERK via dephosphorylation (4) activated ERKphosphorylates Sos and Raf while ERK dephosphorylationinactivates them thereby generates negative feedback regula-tion of the pathwayTherefore the study of themechanism bywhich acupuncture regulates the expression of ERKupstreamreceptors the phosphorylation of ERK cascade members thetargets of downstream signal transduction and the potentialimpact of other signaling pathways on this pathway is neces-sary to confirm acupuncture as an antifibrotic treatment

Our experiments studied the regulation of Ras RafMEKand ERK protein and gene expression by acupuncture inCD intestinal fibrosis in the rat colon Our results showthat the mRNA levels of Ras Raf-1 MEK-1 and ERK-12 were significantly increased in CD rat intestinal tissueand that the expression of p-Ras Raf-1 MEK-1 and p-ERK-12 proteins in intestinal epithelial cells were also sig-nificantly increased These results suggest that the activityof the ERK pathway in CD intestinal fibrosis process wasaltered Medicine-separated moxibustion mild moxibustionand electroacupuncture treatments could effectively down-regulate the expression of p-Ras Raf-1 MEK-1 and p-ERK-12 but different inhibitory effects on Ras Raf-1 MEK-1and ERK-12 mRNA levels were shown in ERK pathwayactivity changes In this discussion we propose the following(1) medicine-separated moxibustion mild moxibustion andelectroacupuncture can inhibit CD intestinal fibrosis byregulating ERK pathway activity and (2) the acupuncture-induced inhibition of p-Ras Raf-1 MEK-1 and p-ERK-12 expression may be the key elements in the treatmentrsquosinhibition of CD intestinal fibrosis At present most reportsregarding the role of acupuncture in the regulation of theERK signaling pathway have focused on EA such as EAmodel rats After EA on the Dazhui (DU14) and Baihui(DU20) acupuncture points the number of ERK-positivestriatal cells increased significantly in the cerebral cortex andERK protein expression was increased in rats with damagedneurons after cerebral ischemia and reperfusion thereforeEA can increase ERK pathway activity [44 45] After the EAtreatment of gastric mucosa-injured rats the phosphoryla-tion levels of ERK in gastric mucosal cells from extractedserum were significantly increased and were significantlydifferent between the serum of the normal rats and that ofthe model rats These observations suggest that ERK maybe involved in signal transduction in the EA stimulation atacupoints of stomach meridian of foot yangming repairinggastric mucosal injury [46] and that EA had an analgesiceffect by downregulating p-ERK expression levels in thedorsal horn of the spinal cord of rats with adjuvant arthritis[47] The previous reports have demonstrated that EA canregulate the ERK signal transduction pathway The results ofour experiments show that medicine-separated moxibustion

10 Evidence-Based Complementary and Alternative Medicine

(a) (b)

(c) (d)

(e)

Figure 13 Expression of phosphorylated ERK-12 protein in the colonicmucosa of ratThe phosphorylation of ERK-12 proteinwas expressedin epithelial cells and lymphocyte in the colonic mucosa of rats EnVision Plus method times200 (a) Normal (b) Crohnrsquos disease (c) mildmoxibustion (d) electroacupuncture and (e) herb-separated moxibustion

005

115

225

335

4

NC MC MM EA HSP

ERK-

12

mRN

A le

vels

()

lowast

Figure 14 The ERK-12 mRNA levels in the rat colon NC normalcontrol group MC model control group MM mild moxibustionEA electroacupuncture and HSM herb-separated moxibustionlowast119875 lt 001 versus NC 119875 lt 005 versus MC

andmildmoxibustion can also regulate the ERK signal trans-duction pathway and inhibit Ras and Raf-1 better than EAThe functional mechanisms underlying these observationsneed to be explored further

Abbreviations

CD Crohnrsquos diseaseNC Normal control groupMC Model control groupEA ElectroacupunctureMM Mild moxibustionHSM Herb-separated moxibustionST25 Tianshu acupoint

Evidence-Based Complementary and Alternative Medicine 11

RN6 Qihai acupointERK Extracellular signal-regulated kinaseMAPK Mitogen-activated protein kinaseTNBS 246-Trinitrobenzene sulfonic acid

Acknowledgments

The authors thank Professor Shuhui Jiang for technical assis-tance and the support of the National Natural Science Foun-dation of China (no 81173331) and National Basic ResearchProgram of China (973 program no 2009CB522900)Xiaomei Wang and Yuan Lu have the same right

References

[1] G Manning D B Whyte R Martinez T Hunter and SSudarsanam ldquoThe protein kinase complement of the humangenomerdquo Science vol 298 no 5600 pp 1912ndash1934 2002

[2] C Mowat A Cole A Windsor et al ldquoGuidelines for themanagement of inflammatory bowel disease in adultsrdquoGut vol60 no 5 pp 571ndash607 2011

[3] Inflammatory Bowel Disease Co-operation Group ChineseSociety of Gastroenterology ldquoChinese consensus on diagnosisand treatment standard of inflammatory bowel diseaserdquoChineseJournal of Gastroenterology vol 12 no 8 pp 490ndash494 2007

[4] J J Zheng X S Zhu Z Huangfu Z X Gao Z R Guo andZ Wang ldquoCrohnrsquos disease in mainland China a systematicanalysis of 50 years of researchrdquo Chinese Journal of DigestiveDiseases vol 6 no 4 pp 175ndash181 2005

[5] M Dai and B M Li ldquoResearch progress in mechanisms ofintestinal fibrosis in Crohnrsquos diseaserdquo Shijie Huaren XiaohuaZazhi vol 17 no 11 pp 1117ndash1121 2009

[6] O Grip J Malm B Veress A Bjartell S Lindgren and AEgesten ldquoIncreased presence of cells containing transforminggrowth factor alpha (TGF-120572) in ulcerative colitis both duringactive inflammation and in remissionrdquo European Journal ofGastroenterology and Hepatology vol 12 no 7 pp 761ndash7662000

[7] I E Koutroubakis E Petinaki P Dimoulios et al ldquoSerumlaminin and collagen IV in inflammatory bowel diseaserdquoJournal of Clinical Pathology vol 56 no 11 pp 817ndash820 2003

[8] S Speca I Giusti F Rieder and G Latella ldquoCellular andmolecular mechanisms of intestinal fibrosisrdquo World Journal ofGastroenterology vol 18 no 28 pp 3635ndash3661 2012

[9] Y F Song J H Hong and L M Xu ldquoTGF-120573 1ERK pathwaysand its inhibitors and fibrosisrdquo Chinese Journal of ClinicalHepatology vol 21 no 2 pp 125ndash127 2005

[10] H C Huang M Yang J Z Li and H Y Wang ldquoConnectivetissue growth factor promotes the proliferation ofmyofibroblastthrough Erk-12 signaling pathwayrdquoNational Medical Journal ofChina vol 85 no 19 pp 1322ndash1326 2005

[11] L P Guo H C Huang and J Z Li ldquoHypoxia inducesmyofibroblast formation and stimulates production of collagenI in myofibroblasts through ERK12 pathwayrdquo Chinese Journalof Pathophysiology vol 24 no 12 pp 2428ndash2432 2008

[12] H R Liu X P Ma T P Zhao and H G Wu ldquoThe adjustmentof acupuncture on colon collagen synthesis and TGF-120573mRNAexpression in Crohnrsquos disease ratrdquo Journal of Anhui TraditionalChinese Medical College vol 24 no 4 pp 25ndash30 2005

[13] X P Ma C P An H G Wu et al ldquoHerbs-partitionedmoxibustion and electro-acupuncture effects on transforming

growth factor-beta 1 connective tissue growth factor collagentype I and fibronectin expression in the colon of Crohnrsquo sdisease ratsrdquo Journal of Clinical Rehabilitative Tissue EngineeringResearch vol 12 no 20 pp 3853ndash3858 2008

[14] G P Morris P L Beck M S Herridge W T Depew M RSzewczuk and J LWallace ldquoHapten-inducedmodel of chronicinflammation and ulceration in the rat colonrdquoGastroenterologyvol 96 no 3 pp 795ndash803 1989

[15] TheMinistry of Science andTechnology of the Peoplersquos Republicof China Guidance Suggestions for the Care and Use ofLaboratory Animals 2006

[16] J D Butzner R Parmar C J Bell andVDalal ldquoButyrate enematherapy stimulates mucosal repair in experimental colitis in theratrdquo Gut vol 38 no 4 pp 568ndash573 1996

[17] X M Wang Y Lu L Y Wu et al ldquoMoxibustion inhibitsinterleukin-12 and tumor necrosis factor alpha and modulatesintestinal flora in rat with ulcerative colitisrdquo World Journal ofGastroenterology vol 18 no 46 pp 6826ndash6835 2012

[18] J B Pucilowska K K McNaughton N K Mohapatra et alldquoIGF-I and procollagen 1205721(I) are coexpressed in a subset ofmesenchymal cells in active Crohnrsquos diseaserdquo American Journalof Physiology vol 279 no 6 pp G1307ndashG1322 2000

[19] M Gharaee-Kermani B Hu S H Phan and M R GyetkoldquoRecent advances in molecular targets and treatment of idio-pathic pulmonary fibrosis focus on TGF120573 signaling and themyofibroblastrdquo Current Medicinal Chemistry vol 16 no 11 pp1400ndash1417 2009

[20] E M Zimmermann L Li Y T Hou M Cannon G MChristman andKN Bitar ldquoIGF-I induces collagen and IGFBP-5 mRNA in rat intestinal smooth musclerdquo American Journal ofPhysiology vol 273 no 4 pp G875ndashG882 1997

[21] L J ZhangQ Li P Chen et al ldquoMediating effect of extracellularsignal-regulated kinase 12 in PDGF-induced proliferation andcollagen synthesis of cultured rat cardiac fibroblastsrdquo ChineseJournal of Anatomy vol 13 no 4 p 223 2007

[22] M Tang W Zhang H Lin H Jiang H Dai and Y ZhangldquoHigh glucose promotes the production of collagen types Iand III by cardiac fibroblasts through a pathway dependenton extracellular-signal-regulated kinase 12rdquo Molecular andCellular Biochemistry vol 301 no 1-2 pp 109ndash114 2007

[23] H Y Dai Z M Ge and Y H Li ldquoEffect of urotensin II on pro-liferative potential and phosphorylation of extracellular signal-regulated kinase 12 adventitial fibroblasts from spontaneouslyhypertensive ratrdquoActaAcademiaeMedicinae Sinicae vol 28 no6 pp 776ndash780 2006

[24] I Fuentes-Calvo A M Blazquez-Medela N Eleno E SantosJ M Lopez-Novoa and C Martınez-Salgado ldquoH-Ras isoformmodulates extracellular matrix synthesis proliferation andmigration in fibroblastsrdquo American Journal of Physiology vol302 no 4 pp C686ndashC697 2012

[25] Y W Ma G T Bai and M X Zhang ldquoStudies on signal trans-duction pathway induced by fibronectin in human embryoniclung fibroblastsrdquo Shanxi Medical Journal vol 38 no 7 pp 587ndash589 2009

[26] J H Jang and C P Chung ldquoFibronectin-mediated adhesionrescues cell cycle arrest induced by fibroblast growth factor-1 bydecreased expression of P21CIPWAF in human chondrocytesrdquoIn Vitro Cellular and Developmental Biology vol 41 no 5-6 pp126ndash129 2005

[27] B Zhu Y J Wang C F Zhu et al ldquoTriptolide inhibitsextracellularmatrix protein synthesis by suppressing the Smad2

12 Evidence-Based Complementary and Alternative Medicine

but not the MAPK pathway in TGF-1205731-stimulated NRK-49Fcellsrdquo Nephrology Dialysis Transplantation vol 25 no 10 pp3180ndash3191 2010

[28] J Bai X S Liu Y J Xu M Xie and W Ni ldquoEffects ofextracellular signal regulated kinase signaling pathway on cellcycle of airway smooth muscle cells in asthmatic ratsrdquo ChineseJournal of Respiratory and Critical Care Medicine vol 9 no 1pp 23ndash27 2010

[29] S Matsusaka and I Wakabayashi ldquoEnhancement of vascu-lar smooth muscle cell migration by urotensin IIrdquo Naunyn-Schmiedebergrsquos Archives of Pharmacology vol 373 no 5 pp 381ndash386 2006

[30] Y Chen Z Dai Y M Liu et al ldquoInhibitory effects ofCGRP on vascular smooth muscle cell proliferation role ofcaveolaecaveolin-1ERK12 signal pathwayrdquo Progress in Bio-chemistry and Biophysics vol 40 no 5 pp 445ndash453 2013

[31] W L Wu A Q Zhang Q Yin and W H Gan ldquoEffectsof ERK signal pathway on extracellular matrix synthesis ofrat mesangial cells treated with PDGF-BBrdquo ACTA UniversityMedicinalis Nanjing vol 27 no 10 pp 1126ndash1129 2007

[32] L S Steelman W H Chappell S L Abrams et al ldquoRoles ofthe RafMEKERK and PI3KPTENAktmTOR pathways incontrolling growth and sensitivity to therapy-implications forcancer and agingrdquo Aging vol 3 no 3 pp 192ndash222 2011

[33] W Kolch ldquoMeaningful relationships the regulation of theRasRafMEKERK pathway by protein interactionsrdquo Biochem-ical Journal vol 351 no 2 pp 289ndash305 2000

[34] D Matallanas M Birtwistle D Romano et al ldquoRaf familykinases old dogs have learned new tricksrdquo Genes and Cancervol 2 no 3 pp 232ndash260 2011

[35] C S Mason C J Springer R G Cooper G Superti-Furga C JMarshall and RMarais ldquoSerine and tyrosine phosphorylationscooperate in Raf-1 but not B-Raf activationrdquo The EMBOJournal vol 18 no 8 pp 2137ndash2148 1999

[36] R J Fiddes P W Janes S P Sivertsen R L Sutherland EA Musgrove and R J Daly ldquoInhibition of the MAP kinasecascade blocks heregulin-induced cell cycle progression in T-47D human breast cancer cellsrdquo Oncogene vol 16 no 21 pp2803ndash2813 1998

[37] S T Eblen J K SlackM JWeber andA D Catling ldquoRac-PAKsignaling stimulates extracellular signal-regulated kinase (ERK)activation by regulating formation of MEK1-ERK complexesrdquoMolecular and Cellular Biology vol 22 no 17 pp 6023ndash60332002

[38] M Cargnello and P P Roux ldquoActivation and function ofthe MAPKs and their substrates the MAPK-activated proteinkinasesrdquo Microbiology and Molecular Biology Reviews vol 75no 1 pp 50ndash83 2011

[39] A V Khokhlatchev B Canagarajah J Wilsbacher et al ldquoPhos-phorylation of theMAP kinase ERK2 promotes its homodimer-ization and nuclear translocationrdquo Cell vol 93 no 4 pp 605ndash615 1998

[40] R H Chen C Sarnecki and J Blenis ldquoNuclear localization andregulation of erk- and rsk-encoded protein kinasesrdquoMolecularand Cellular Biology vol 12 no 3 pp 915ndash927 1992

[41] S Traverse K Seedorf H Paterson C J Marshall P Cohenand A Ullrich ldquoEGF triggers neuronal differentiation of PC12cells that overexpress the EGF receptorrdquo Current Biology vol 4no 8 pp 694ndash701 1994

[42] T T Nguyen J C Scimeca C Filloux P Peraldi J L Car-pentier and E Van Obberghen ldquoCo-regulation of the mitogen-activated protein kinase extracellular signal-regulated kinase 1

and the 90-kDa ribosomal S6 kinase in PC12 cells Distincteffects of the neurotrophic factor nerve growth factor and themitogenic factor epidermal growth factorrdquo Journal of BiologicalChemistry vol 268 no 13 pp 9803ndash9810 1993

[43] I Dikic J Schlessinger and I Lax ldquoPC12 cells overexpressingthe insulin receptor undergo insulin-dependent neuronal dif-ferentiationrdquo Current Biology vol 4 no 8 pp 702ndash708 1994

[44] M H Shen Z R Li X R Xiang W M Niu and C B ZhangldquoEffect of electro-acupuncture on ERK protein expression ofMCAO ratsrdquo Journal of Clinical Acupuncture and Moxibustionvol 23 no 7 pp 62ndash63 2007

[45] M H Shen Z R Li X R Xiang W M Niu and C BZhang ldquoInvolvement of extracellular regulated protein kinasesignal transduction pathway in electroacupuncture-inducedimprovement of ischemic cerebral injury in rats with cerebralischemia reperfusionrdquo Acupuncture Research vol 32 no 6 pp368ndash372 2007

[46] Z B Yang J Yan and S X Yi ldquoUp-regulation of the ERK phos-phorylation in gastric mucosal cell by the serum of rats treatedwith electroacupuncture at stomach meridian acupointsrdquo Basicamp Clinical Medicine vol 29 no 2 pp 135ndash138 2009

[47] C L Wang S X Wang and W Gong ldquoChanges in phos-phatized ERK in the spinal dorsal horn in adjuvant arthritisrats during electroacupuncture of Jiaji points for analgesiardquoShanghai Journal of Acupuncture and Moxibustion vol 24 no12 pp 33ndash35 2005

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

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Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

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Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 3

Table 1 The sequence of the forward primer and reverse primer used for fluorescent quantitative PCR assays

Name Primer sense Sequence (51015840rarr31015840) Amplification product (bp)

GAPDH Forward 51015840-CCGAGGGCCCACTAAAGG-31015840 116 bpReverse 51015840-GCTGTTGAAGTCACAGGAGACAA-31015840

Ras Forward 51015840-TGGTGGGCAACAAGTGTGAC-31015840 181 bpReverse 51015840-GGGTTCAGTTTCCGCAGTTTATG-31015840

Raf-1 Forward 51015840-CAGGAGCACAAAGGTAAGAAAGC-31015840 201 bpReverse 51015840-GCCACAAGTCTGACATCGAAATC-31015840

MEK-1 Forward 51015840-AATATGTCAGGTTCAAGGAAAGGAC-31015840 169 bpReverse 51015840-AGGTAAGGACGAAGCCACAAG-31015840

ERK-12 Forward 51015840-TATATACATTCAGCTAATGTTCTGC-31015840 169 bpReverse 51015840-TCTACCTGCAGCTGGAACCTCTC-31015840

the rat colon Green staining was interpreted as collagenmucus and cartilage red staining denotes muscle and nervefibers and black indicates nuclei

26 EnVision Plus Immunohistochemical Detection of RasRaf MEK and ERK-12 in Colonic Tissue Fixed tissues wereconventionally deparaffinized in water and washed threetimes for 3min in 001M PBS pH 74 The tissue sampleswere heated and allowed to equilibrate to room temperaturefor 20min and then washed three times in PBS for 3min Toinhibit endogenous peroxidase the samples were treatedwith03 H

2O2at room temperature for 20min and then rinsed

three times with PBS for 3min The tissue samples wereincubated in 20normal goat serum at room temperature for30min and then with one drop of the appropriately dilutedprimary antibody (Santa Cruz Bio Inc Santa Cruz USA) at4∘C for 18 h The tissues were washed three times with PBSfor 3min mixed with one drop of Envision reagent (HRP-M) and incubated in a humidified chamber for 30min at37∘C After washing three times with PBS for 3min the assaywas developed in 004 DAB and 03 H

2O2for 8ndash12min

then washed in water stained with hematoxylin for 1minand bathed in hot water for 3 sec to allow the blue color todevelop Finally the tissue sections were dried and mountedwith a neutral resin All the samples were analyzed by aMoticMed 60 image analysis system (MoticGroupCo Ltd)Threefields were randomly selected the positive target value ofthe integral optical density was calculated under an opticalmicroscope (Olympus Co Ltd) at 200x magnification

27 qPCR Detection of Ras Raf MEK and ERK-12 mRNALevels in Colonic Tissues The total RNA was extracted fromcolon tissue samples The cDNA was synthesized by reversetranscription (RT) according to the qPCR kit (ShanghaiDaweike Bio Inc Shanghai China) instructions and ampli-fied by PCR according to the kit instructions (Table 1) Thefollowing cycling conditions were used RT at 37∘C for 1 h95∘C for 5min to inactivate the MMLV RT enzyme 50∘C for2min 95∘C for 5min and 40 cycles of 95∘C for 15 sec and60∘C for 45 sec

28 Statistical Analysis SPSS 180 statistical software (SPSSInc Chicago USA) was used for statistical analysis The

012345678

NC MC MM EA HSP

Gro

ss in

jury

scor

es

lowast

Figure 1 The gross injury scores of the colon tissue in eachrat group NC normal control group MC model control groupMM mild moxibustion EA electroacupuncture and HSM herb-separated moxibustion lowast119875 lt 001 versus NC 119875 lt 001 versus MC

measurement data are presented as the mean plusmn SD Fornormally distributed data one-way analysis of variance(ANOVA) was applied The least significant differencemethod (LSD) was used for the homogeneity of variance andGames-Howell was used to compare the differences betweengroups for the heterogeneity of variance A value of 119875 lt 005was considered statistically significant The nonparametricMann-Whitney U-test was used for abnormal distributions

3 Results

31 Gross Injury in the Colon Tissue of Rat When the colonictissues were observed by visual inspection the rats in thenormal group exhibited a uniformity of colon bowel andintestinal wall a smooth colonic mucosa surface clear vascu-lar texture no bleeder and no erosion or ulcers The modelgroup exhibited partial intestinal canal stenosis or intestinalwall thicken and harden fuzzy vascular texture edematouscolonic mucosa serious congestion erosion and differentsize shape and depth ulcer formation After treatmentsthere were lacked smooth colonicmucosa surface edematouscolonic mucosa and erosion in both MM and EA groupand lacked smooth colonic mucosa surface with no obviouserosion and ulcer in HPM group

As shown in Figure 1 the gross injury score of the colonictissue in the NC group was significantly higher than the MCgroup (119875 lt 001) After treatment the scores were lower in

4 Evidence-Based Complementary and Alternative Medicine

(a) (b)

(c) (d)

(e)

Figure 2 Expression of collagen fibers in the colon tissue of rat Masson trichrome staining method times100 (a) Normal (b) Crohnrsquos disease(c) mild moxibustion (d) electroacupuncture (e) herb-separated moxibustion

both the HPM group MM group and the EA group (119875 lt001) It suggests that these treatments are able to improve thetissue damage of CD

32 Expression of Collagen Fibers in the Colon Tissue of RatAs shown in Figure 2 Masson trichrome staining revealedthat in the MC group a large number of collagen fibers werepresent in the mucosa muscularis mucosa submucosa andmuscularis of the rat colon with a disordered arrangementIn the MM group and the HSM group there were smallnumbers of collagen fibers in the colonic lamina propria andsubmucosa at the original ulcer-healing location which wasless severe compared with that of the model group In the EAgroup there were a small number of collagen fibers in the

mucosa submucosa muscularis and serosa of the rat colonand the effect was less severe than in the MC group

33 Ras Expression in the Colonic Mucosa of Rat As shownin Figures 3 and 4 the phosphorylation of Ras protein inthe rat colon was significantly higher in the MC group thanin the NC group (119875 lt 001) Ras protein expression wassignificantly reduced (119875 lt 001) to normal levels after acu-puncture treatment

Figure 5 indicates that Ras mRNA expression in the MCgroup was higher than that in the NC group (119875 lt 001) andthat HSM effectively reduced the Ras mRNA levels in the ratcolon (119875 lt 005) whereas there was no significant differencebetween the EA group and the NC group

Evidence-Based Complementary and Alternative Medicine 5

0

100

200

300

400

500

600

700

NC MC MM EA HSPRa

s pho

spho

ryla

tion

prot

ein

(IO

D)

lowast

Figure 3The integral optical density (IOD) of Ras phosphorylation in each rat group NC normal control group MC model control groupMM mild moxibustion EA electroacupuncture and HSM herb-separated moxibustion lowast119875 lt 001 versus NC 119875 lt 001 versus MC

(a) (b)

(c) (d)

(e)

Figure 4 Expression of phosphorylated Ras protein in the colonic mucosa of rat The phosphorylation of Ras protein was expressedin epithelial cells and lymphocyte in the colonic mucosa of rats EnVision Plus method times200 (a) Normal (b) Crohnrsquos disease (c) mildmoxibustion (d) electroacupuncture (e) herb-separated moxibustion

6 Evidence-Based Complementary and Alternative Medicine

0

2

4

Ras m

RNA

leve

l (

)

6

8

10

12 17

25

21

NC MC MM EA HSP

lowast

lowast

Figure 5 The Ras mRNA levels in the rat colon NC normalcontrol group MC model control group MM mild moxibustionEA electroacupuncture and HSM herb-separated moxibustionlowast119875 lt 001 versus NC 119875 lt 005 versus MC

0100200300400500600700800900

NC MC MM EA HSP

Raf-1

pro

tein

(IO

D)

lowast

998771998771 998771

Figure 6 The IOD of Raf-1 protein expression in rat colonNC normal control group MC model control group MM mildmoxibustion EA electroacupuncture and HSM herb-separatedmoxibustion lowast119875 lt 001 versus NC 119875 lt 005 119875 lt 001 versusMC 998771998771119875 lt 005 998771119875 lt 001 versus EA

34 The Raf-1 Expression in the Colonic Mucosa of Rat Asshown in Figures 6 and 7 the Raf-1 protein expression inthe colonic tissue of the MC group was significantly higherthan in the NC group (119875 lt 001) and that the Raf-1 proteinexpression in the colon was reduced to varying degrees afteracupuncture treatment (119875 lt 005 or 119875 lt 005)

The results in Figure 8 show that the Raf-1 mRNAlevels in the MC group were significantly higher than in theNC group (119875 lt 001) and that MM and HSM treatmentssignificantly reduced rat colon Raf-1 mRNA levels (119875 lt 005)whereas EA treatment did not significantly affect rat colonRaf-1 mRNA levels

35 The MEK-1 Expression in the Colonic Mucosa of Rat Asshown in Figures 9 and 10 the MEK-1 protein expression inthe colonic tissue of the MC group was significantly higherthan in NC rats (119875 lt 001) and MEK-1 protein expressionwas significantly reduced in each group after acupuncturetreatment (119875 lt 001) although the levels were still higherthan normal levels (119875 lt 001)

The results in Figure 11 show that theMEK-1mRNA levelsin the model group were significantly higher than those inthe NC group (119875 lt 001) and that MM HSM and EAtreatment reduced MEK-1 mRNA levels although there was

no statistically significant difference between the treatmentand no treatment groups or among the treatment groups

36 The ERK-12 Expression in the Colonic Mucosa of Rat Asshown in Figures 12 and 13 the phosphorylation of ERK-12significantly increased in the MC group compared with theNC group (119875 lt 001) and that the levels of phosphorylatedERK-12 proteins were significantly reduced after the MMand HSM treatments (119875 lt 001)

The results in Figure 14 show that the ERK-12 mRNAexpression in the MC group was significantly higher thanthat in the NC group (119875 lt 001) and that ERK-12 mRNAexpression was significantly lower in the MM group afteracupuncture treatment compared with the MC group (119875 lt005)

4 Discussion

Extracellular signal-regulated kinase (ERK) a member ofthe mitogen-activated protein kinase (MAPK) family playsan important biological role in transmitting signals fromsurface receptors to the nucleus The ERK12 pathway is themost thoroughly researched pathway in the ERK family andplays a role in a wide range of cellular functions includ-ing regulating meiosis mitosis and anaphase processes indifferent cells A variety of stimulating factors such as growthfactors cytokines viruses G protein-coupled receptor lig-ands and oncogenes can activate the ERK12 pathway [18]The ERK signaling pathway is a Ras-Raf-MEK-ERK path-way Phosphorylation-activated ERK12 translocates fromthe cytoplasm to the nucleus and thereby mediates thetranscriptional activation of Elk-1 ATF NF-120581B AP-1 and c-fos which in turn regulate cell proliferation and differentia-tion the maintenance of cell shape the construction of thecytoskeleton apoptosis and the malignant transformation ofcells among other biological functions

One of the features of CD is the transformation of smoothmuscle cells into fibroblasts andor myofibroblasts [8] Thisabundance of fibroblasts and myofibroblasts increases theexpression of type I collagen mRNA and produces collagentissue within the muscle [19] There are a large number offibroblast cells and type I III IV and V collagen transcriptsin the small intestines of CD patients [20] which producemore ECM including collagen elastin and glycoproteinsthat are deposited intramurally to produce a thickening of theintestinal wall [10] Based on a large number of experimentalreports the ERK-12 signaling pathway appears to participatein myofibroblast proliferation differentiation and collagenproduction [10 11 21ndash25] extracellular matrix synthesis [2627] smooth muscle cell proliferation and apoptosis [28]and the migration of vascular smooth muscle cells [29]Inhibiting the expression and activity of ERK can inhibit theproliferation of vascular smooth muscle cells [30] to reducethe accumulation of ECM [31] Therefore it is inferred thatthe ERK-12 signaling pathway may induce the activationand proliferation of CD intestinal myofibroblasts and theirtransformation intomuscle fiber cells Additionally the ERK-12 signaling pathway may regulate the proliferation andapoptosis of smooth muscle cells

Evidence-Based Complementary and Alternative Medicine 7

(a) (b)

(c) (d)

(e)

Figure 7 Expression of Raf-1 protein in the colonic mucosa of rat The Raf-1 protein was expressed in epithelial cells in the colonic mucosaof rats EnVision Plus method times200 (a) Normal (b) Crohnrsquos disease (c) mild moxibustion (d) electroacupuncture and (e) herb-separatedmoxibustion

005

115

225

335

4

NC MC MM EA HSP

Raf-1

mRN

A le

vels

()

lowast

Figure 8 The Raf-1 mRNA levels in the rat colon NC normalcontrol group MC model control group MM mild moxibustionEA electroacupuncture and HSM herb-separated moxibustionlowast119875 lt 001 versus NC 119875 lt 005 versus MC

050

100150200250300350400450500

NC MC MM EA HSP

MEK

-1 p

rote

in (I

OD

)

lowast

Figure 9 The IOD of MEK-1 protein expression in the rat colonNC normal control group MC model control group MM mildmoxibustion EA electroacupuncture and HSM herb-separatedmoxibustion lowast119875 lt 001 versus NC 119875 lt 001 versus MC

8 Evidence-Based Complementary and Alternative Medicine

(a) (b)

(c) (d)

(e)

Figure 10 Expression of MEK-1 protein in the colonic mucosa of rat The MEK-1 protein was expressed in epithelial cells in the colonicmucosa of rats EnVision Plus method times200 (a) Normal (b) Crohnrsquos disease (c) mild moxibustion (d) electroacupuncture and (e) herb-separated moxibustion

As an upstream protein in the Raf-MEK-ERK pathwayRas is the product of the proto-oncogene c-ras A variety ofstimulating factors particularly growth factors (eg PDGFEGF TNF and PKC activators as well as Src family members)can activate Ras which further activates Raf-1 ActivatedRaf-1 then activates MEK and ERK downstream [32 33] toinitiate the MAPK pathway Raf-1 protein kinase is widelyexpressed in various types of cells [34] Ras and Raf-1 oftenexist in the form of complexes Ras the upstream activatorprotein can translocate Raf from the cytoplasm to theplasma membrane where the latter is activated Howeverthe mechanism by which Raf is activated remains unclearalthough it may include phosphorylation [35] All that isknown is that Raf activation is related to its phosphorylation

on SerThr residues [33] Recent studies have shown thatRaf-1 protein kinase may be a point of crosstalk with othersignaling pathways This protein may participate in a varietyof biological processes in signal transduction including itsown regulation by interactingwith other signalingmoleculesor via reciprocal regulation

The MAPK kinases include MEK1 (MKK1) and MEK2(MKK2)MEK1 andMEK2 are the central links in theMAPKpathway The activated upstream Raf-1 protein kinase cancombine with MEK1 and 2 and doubly phosphorylate Tyrand Thr its two regulatory sites to activate ERK [36] MEKsare not only ERK activators but may also relocalize theactivated ERKs to the nucleus or other active sites to furtherphosphorylate downstream substrates [37]

Evidence-Based Complementary and Alternative Medicine 9

0

2

1

3

5

4

6

19

NC MC MM EA HSP

MEK

-1 m

RNA

leve

ls (

)

lowast

Figure 11 The MEK-1 mRNA levels in the rat colon NC normalcontrol group MC model control group MM mild moxibustionEA electroacupuncture and HSM herb-separated moxibustionlowast119875 lt 001 versus NC

0200400600800

100012001400

NC MC MM EA HSPPhos

phor

ylat

ed E

RK-1

2 (I

OD

)

lowast

Figure 12 The IOD of phosphorylated ERK-12 levels in therat colon NC normal control group MC model control groupMM mild moxibustion EA electroacupuncture and HSM herb-separated moxibustion lowast119875 lt 001 versus NC 119875 lt 005 119875 lt 001versus MC

The upstream kinase MEK1 is a dual-specificity enzymeActivatedMEK1 can phosphorylate ERK1 and ERK2There isevidence that the Tyr residues of ERK1 and 2 are phosphory-lated before the Thr residues both intracellularly and extra-cellularly [38] ERK translocates into the nucleus by forminga homodimer of phosphorylated ERK and phosphorylatedor unphosphorylated ERK [39] ERK is evenly distributedin quiescent cells but when the cells are stimulated byserum persistently activated ERK can partially translocateinto the nucleus whereas transiently activated ERK cannotrelocalize Activated ERKs accumulate in the nucleus andthen phosphorylate the corresponding transcription factorswhich drive expression of specific gene products Becauseof the difference in nuclear localization transiently activated(or low-activity) ERK and persistently activated (or high-activity) ERK cause cells to express genes at a different qualityor quantity resulting in different biological effects [40ndash43]

The ERK1-ERK2-MAPK pathway incorporates a mech-anism for inactivation after activation which can preventexcessive activation-induced adverse consequences At thesame time the inactivated kinase is prepared for the nextactivation The mechanisms of inhibition of this pathwayinclude the following (1) the activation of ERK12-MAPK is

the result of the phosphorylation cascade and can be inac-tivated by dephosphorylation by the corresponding phos-phatases (2) Raf-1 is the direct substrate of PKA which canphosphorylate Ser43 of c-Raf an inhibitory phosphorylationsite in the N-terminus of Raf that interacts with Ras andthe phosphorylation of this site can inhibit the bindingbetween RasGTP and the N-terminus of Raf which blocksthe activation of ERK (3) the phosphatase PP2 can inactivateMEK and ERK via dephosphorylation (4) activated ERKphosphorylates Sos and Raf while ERK dephosphorylationinactivates them thereby generates negative feedback regula-tion of the pathwayTherefore the study of themechanism bywhich acupuncture regulates the expression of ERKupstreamreceptors the phosphorylation of ERK cascade members thetargets of downstream signal transduction and the potentialimpact of other signaling pathways on this pathway is neces-sary to confirm acupuncture as an antifibrotic treatment

Our experiments studied the regulation of Ras RafMEKand ERK protein and gene expression by acupuncture inCD intestinal fibrosis in the rat colon Our results showthat the mRNA levels of Ras Raf-1 MEK-1 and ERK-12 were significantly increased in CD rat intestinal tissueand that the expression of p-Ras Raf-1 MEK-1 and p-ERK-12 proteins in intestinal epithelial cells were also sig-nificantly increased These results suggest that the activityof the ERK pathway in CD intestinal fibrosis process wasaltered Medicine-separated moxibustion mild moxibustionand electroacupuncture treatments could effectively down-regulate the expression of p-Ras Raf-1 MEK-1 and p-ERK-12 but different inhibitory effects on Ras Raf-1 MEK-1and ERK-12 mRNA levels were shown in ERK pathwayactivity changes In this discussion we propose the following(1) medicine-separated moxibustion mild moxibustion andelectroacupuncture can inhibit CD intestinal fibrosis byregulating ERK pathway activity and (2) the acupuncture-induced inhibition of p-Ras Raf-1 MEK-1 and p-ERK-12 expression may be the key elements in the treatmentrsquosinhibition of CD intestinal fibrosis At present most reportsregarding the role of acupuncture in the regulation of theERK signaling pathway have focused on EA such as EAmodel rats After EA on the Dazhui (DU14) and Baihui(DU20) acupuncture points the number of ERK-positivestriatal cells increased significantly in the cerebral cortex andERK protein expression was increased in rats with damagedneurons after cerebral ischemia and reperfusion thereforeEA can increase ERK pathway activity [44 45] After the EAtreatment of gastric mucosa-injured rats the phosphoryla-tion levels of ERK in gastric mucosal cells from extractedserum were significantly increased and were significantlydifferent between the serum of the normal rats and that ofthe model rats These observations suggest that ERK maybe involved in signal transduction in the EA stimulation atacupoints of stomach meridian of foot yangming repairinggastric mucosal injury [46] and that EA had an analgesiceffect by downregulating p-ERK expression levels in thedorsal horn of the spinal cord of rats with adjuvant arthritis[47] The previous reports have demonstrated that EA canregulate the ERK signal transduction pathway The results ofour experiments show that medicine-separated moxibustion

10 Evidence-Based Complementary and Alternative Medicine

(a) (b)

(c) (d)

(e)

Figure 13 Expression of phosphorylated ERK-12 protein in the colonicmucosa of ratThe phosphorylation of ERK-12 proteinwas expressedin epithelial cells and lymphocyte in the colonic mucosa of rats EnVision Plus method times200 (a) Normal (b) Crohnrsquos disease (c) mildmoxibustion (d) electroacupuncture and (e) herb-separated moxibustion

005

115

225

335

4

NC MC MM EA HSP

ERK-

12

mRN

A le

vels

()

lowast

Figure 14 The ERK-12 mRNA levels in the rat colon NC normalcontrol group MC model control group MM mild moxibustionEA electroacupuncture and HSM herb-separated moxibustionlowast119875 lt 001 versus NC 119875 lt 005 versus MC

andmildmoxibustion can also regulate the ERK signal trans-duction pathway and inhibit Ras and Raf-1 better than EAThe functional mechanisms underlying these observationsneed to be explored further

Abbreviations

CD Crohnrsquos diseaseNC Normal control groupMC Model control groupEA ElectroacupunctureMM Mild moxibustionHSM Herb-separated moxibustionST25 Tianshu acupoint

Evidence-Based Complementary and Alternative Medicine 11

RN6 Qihai acupointERK Extracellular signal-regulated kinaseMAPK Mitogen-activated protein kinaseTNBS 246-Trinitrobenzene sulfonic acid

Acknowledgments

The authors thank Professor Shuhui Jiang for technical assis-tance and the support of the National Natural Science Foun-dation of China (no 81173331) and National Basic ResearchProgram of China (973 program no 2009CB522900)Xiaomei Wang and Yuan Lu have the same right

References

[1] G Manning D B Whyte R Martinez T Hunter and SSudarsanam ldquoThe protein kinase complement of the humangenomerdquo Science vol 298 no 5600 pp 1912ndash1934 2002

[2] C Mowat A Cole A Windsor et al ldquoGuidelines for themanagement of inflammatory bowel disease in adultsrdquoGut vol60 no 5 pp 571ndash607 2011

[3] Inflammatory Bowel Disease Co-operation Group ChineseSociety of Gastroenterology ldquoChinese consensus on diagnosisand treatment standard of inflammatory bowel diseaserdquoChineseJournal of Gastroenterology vol 12 no 8 pp 490ndash494 2007

[4] J J Zheng X S Zhu Z Huangfu Z X Gao Z R Guo andZ Wang ldquoCrohnrsquos disease in mainland China a systematicanalysis of 50 years of researchrdquo Chinese Journal of DigestiveDiseases vol 6 no 4 pp 175ndash181 2005

[5] M Dai and B M Li ldquoResearch progress in mechanisms ofintestinal fibrosis in Crohnrsquos diseaserdquo Shijie Huaren XiaohuaZazhi vol 17 no 11 pp 1117ndash1121 2009

[6] O Grip J Malm B Veress A Bjartell S Lindgren and AEgesten ldquoIncreased presence of cells containing transforminggrowth factor alpha (TGF-120572) in ulcerative colitis both duringactive inflammation and in remissionrdquo European Journal ofGastroenterology and Hepatology vol 12 no 7 pp 761ndash7662000

[7] I E Koutroubakis E Petinaki P Dimoulios et al ldquoSerumlaminin and collagen IV in inflammatory bowel diseaserdquoJournal of Clinical Pathology vol 56 no 11 pp 817ndash820 2003

[8] S Speca I Giusti F Rieder and G Latella ldquoCellular andmolecular mechanisms of intestinal fibrosisrdquo World Journal ofGastroenterology vol 18 no 28 pp 3635ndash3661 2012

[9] Y F Song J H Hong and L M Xu ldquoTGF-120573 1ERK pathwaysand its inhibitors and fibrosisrdquo Chinese Journal of ClinicalHepatology vol 21 no 2 pp 125ndash127 2005

[10] H C Huang M Yang J Z Li and H Y Wang ldquoConnectivetissue growth factor promotes the proliferation ofmyofibroblastthrough Erk-12 signaling pathwayrdquoNational Medical Journal ofChina vol 85 no 19 pp 1322ndash1326 2005

[11] L P Guo H C Huang and J Z Li ldquoHypoxia inducesmyofibroblast formation and stimulates production of collagenI in myofibroblasts through ERK12 pathwayrdquo Chinese Journalof Pathophysiology vol 24 no 12 pp 2428ndash2432 2008

[12] H R Liu X P Ma T P Zhao and H G Wu ldquoThe adjustmentof acupuncture on colon collagen synthesis and TGF-120573mRNAexpression in Crohnrsquos disease ratrdquo Journal of Anhui TraditionalChinese Medical College vol 24 no 4 pp 25ndash30 2005

[13] X P Ma C P An H G Wu et al ldquoHerbs-partitionedmoxibustion and electro-acupuncture effects on transforming

growth factor-beta 1 connective tissue growth factor collagentype I and fibronectin expression in the colon of Crohnrsquo sdisease ratsrdquo Journal of Clinical Rehabilitative Tissue EngineeringResearch vol 12 no 20 pp 3853ndash3858 2008

[14] G P Morris P L Beck M S Herridge W T Depew M RSzewczuk and J LWallace ldquoHapten-inducedmodel of chronicinflammation and ulceration in the rat colonrdquoGastroenterologyvol 96 no 3 pp 795ndash803 1989

[15] TheMinistry of Science andTechnology of the Peoplersquos Republicof China Guidance Suggestions for the Care and Use ofLaboratory Animals 2006

[16] J D Butzner R Parmar C J Bell andVDalal ldquoButyrate enematherapy stimulates mucosal repair in experimental colitis in theratrdquo Gut vol 38 no 4 pp 568ndash573 1996

[17] X M Wang Y Lu L Y Wu et al ldquoMoxibustion inhibitsinterleukin-12 and tumor necrosis factor alpha and modulatesintestinal flora in rat with ulcerative colitisrdquo World Journal ofGastroenterology vol 18 no 46 pp 6826ndash6835 2012

[18] J B Pucilowska K K McNaughton N K Mohapatra et alldquoIGF-I and procollagen 1205721(I) are coexpressed in a subset ofmesenchymal cells in active Crohnrsquos diseaserdquo American Journalof Physiology vol 279 no 6 pp G1307ndashG1322 2000

[19] M Gharaee-Kermani B Hu S H Phan and M R GyetkoldquoRecent advances in molecular targets and treatment of idio-pathic pulmonary fibrosis focus on TGF120573 signaling and themyofibroblastrdquo Current Medicinal Chemistry vol 16 no 11 pp1400ndash1417 2009

[20] E M Zimmermann L Li Y T Hou M Cannon G MChristman andKN Bitar ldquoIGF-I induces collagen and IGFBP-5 mRNA in rat intestinal smooth musclerdquo American Journal ofPhysiology vol 273 no 4 pp G875ndashG882 1997

[21] L J ZhangQ Li P Chen et al ldquoMediating effect of extracellularsignal-regulated kinase 12 in PDGF-induced proliferation andcollagen synthesis of cultured rat cardiac fibroblastsrdquo ChineseJournal of Anatomy vol 13 no 4 p 223 2007

[22] M Tang W Zhang H Lin H Jiang H Dai and Y ZhangldquoHigh glucose promotes the production of collagen types Iand III by cardiac fibroblasts through a pathway dependenton extracellular-signal-regulated kinase 12rdquo Molecular andCellular Biochemistry vol 301 no 1-2 pp 109ndash114 2007

[23] H Y Dai Z M Ge and Y H Li ldquoEffect of urotensin II on pro-liferative potential and phosphorylation of extracellular signal-regulated kinase 12 adventitial fibroblasts from spontaneouslyhypertensive ratrdquoActaAcademiaeMedicinae Sinicae vol 28 no6 pp 776ndash780 2006

[24] I Fuentes-Calvo A M Blazquez-Medela N Eleno E SantosJ M Lopez-Novoa and C Martınez-Salgado ldquoH-Ras isoformmodulates extracellular matrix synthesis proliferation andmigration in fibroblastsrdquo American Journal of Physiology vol302 no 4 pp C686ndashC697 2012

[25] Y W Ma G T Bai and M X Zhang ldquoStudies on signal trans-duction pathway induced by fibronectin in human embryoniclung fibroblastsrdquo Shanxi Medical Journal vol 38 no 7 pp 587ndash589 2009

[26] J H Jang and C P Chung ldquoFibronectin-mediated adhesionrescues cell cycle arrest induced by fibroblast growth factor-1 bydecreased expression of P21CIPWAF in human chondrocytesrdquoIn Vitro Cellular and Developmental Biology vol 41 no 5-6 pp126ndash129 2005

[27] B Zhu Y J Wang C F Zhu et al ldquoTriptolide inhibitsextracellularmatrix protein synthesis by suppressing the Smad2

12 Evidence-Based Complementary and Alternative Medicine

but not the MAPK pathway in TGF-1205731-stimulated NRK-49Fcellsrdquo Nephrology Dialysis Transplantation vol 25 no 10 pp3180ndash3191 2010

[28] J Bai X S Liu Y J Xu M Xie and W Ni ldquoEffects ofextracellular signal regulated kinase signaling pathway on cellcycle of airway smooth muscle cells in asthmatic ratsrdquo ChineseJournal of Respiratory and Critical Care Medicine vol 9 no 1pp 23ndash27 2010

[29] S Matsusaka and I Wakabayashi ldquoEnhancement of vascu-lar smooth muscle cell migration by urotensin IIrdquo Naunyn-Schmiedebergrsquos Archives of Pharmacology vol 373 no 5 pp 381ndash386 2006

[30] Y Chen Z Dai Y M Liu et al ldquoInhibitory effects ofCGRP on vascular smooth muscle cell proliferation role ofcaveolaecaveolin-1ERK12 signal pathwayrdquo Progress in Bio-chemistry and Biophysics vol 40 no 5 pp 445ndash453 2013

[31] W L Wu A Q Zhang Q Yin and W H Gan ldquoEffectsof ERK signal pathway on extracellular matrix synthesis ofrat mesangial cells treated with PDGF-BBrdquo ACTA UniversityMedicinalis Nanjing vol 27 no 10 pp 1126ndash1129 2007

[32] L S Steelman W H Chappell S L Abrams et al ldquoRoles ofthe RafMEKERK and PI3KPTENAktmTOR pathways incontrolling growth and sensitivity to therapy-implications forcancer and agingrdquo Aging vol 3 no 3 pp 192ndash222 2011

[33] W Kolch ldquoMeaningful relationships the regulation of theRasRafMEKERK pathway by protein interactionsrdquo Biochem-ical Journal vol 351 no 2 pp 289ndash305 2000

[34] D Matallanas M Birtwistle D Romano et al ldquoRaf familykinases old dogs have learned new tricksrdquo Genes and Cancervol 2 no 3 pp 232ndash260 2011

[35] C S Mason C J Springer R G Cooper G Superti-Furga C JMarshall and RMarais ldquoSerine and tyrosine phosphorylationscooperate in Raf-1 but not B-Raf activationrdquo The EMBOJournal vol 18 no 8 pp 2137ndash2148 1999

[36] R J Fiddes P W Janes S P Sivertsen R L Sutherland EA Musgrove and R J Daly ldquoInhibition of the MAP kinasecascade blocks heregulin-induced cell cycle progression in T-47D human breast cancer cellsrdquo Oncogene vol 16 no 21 pp2803ndash2813 1998

[37] S T Eblen J K SlackM JWeber andA D Catling ldquoRac-PAKsignaling stimulates extracellular signal-regulated kinase (ERK)activation by regulating formation of MEK1-ERK complexesrdquoMolecular and Cellular Biology vol 22 no 17 pp 6023ndash60332002

[38] M Cargnello and P P Roux ldquoActivation and function ofthe MAPKs and their substrates the MAPK-activated proteinkinasesrdquo Microbiology and Molecular Biology Reviews vol 75no 1 pp 50ndash83 2011

[39] A V Khokhlatchev B Canagarajah J Wilsbacher et al ldquoPhos-phorylation of theMAP kinase ERK2 promotes its homodimer-ization and nuclear translocationrdquo Cell vol 93 no 4 pp 605ndash615 1998

[40] R H Chen C Sarnecki and J Blenis ldquoNuclear localization andregulation of erk- and rsk-encoded protein kinasesrdquoMolecularand Cellular Biology vol 12 no 3 pp 915ndash927 1992

[41] S Traverse K Seedorf H Paterson C J Marshall P Cohenand A Ullrich ldquoEGF triggers neuronal differentiation of PC12cells that overexpress the EGF receptorrdquo Current Biology vol 4no 8 pp 694ndash701 1994

[42] T T Nguyen J C Scimeca C Filloux P Peraldi J L Car-pentier and E Van Obberghen ldquoCo-regulation of the mitogen-activated protein kinase extracellular signal-regulated kinase 1

and the 90-kDa ribosomal S6 kinase in PC12 cells Distincteffects of the neurotrophic factor nerve growth factor and themitogenic factor epidermal growth factorrdquo Journal of BiologicalChemistry vol 268 no 13 pp 9803ndash9810 1993

[43] I Dikic J Schlessinger and I Lax ldquoPC12 cells overexpressingthe insulin receptor undergo insulin-dependent neuronal dif-ferentiationrdquo Current Biology vol 4 no 8 pp 702ndash708 1994

[44] M H Shen Z R Li X R Xiang W M Niu and C B ZhangldquoEffect of electro-acupuncture on ERK protein expression ofMCAO ratsrdquo Journal of Clinical Acupuncture and Moxibustionvol 23 no 7 pp 62ndash63 2007

[45] M H Shen Z R Li X R Xiang W M Niu and C BZhang ldquoInvolvement of extracellular regulated protein kinasesignal transduction pathway in electroacupuncture-inducedimprovement of ischemic cerebral injury in rats with cerebralischemia reperfusionrdquo Acupuncture Research vol 32 no 6 pp368ndash372 2007

[46] Z B Yang J Yan and S X Yi ldquoUp-regulation of the ERK phos-phorylation in gastric mucosal cell by the serum of rats treatedwith electroacupuncture at stomach meridian acupointsrdquo Basicamp Clinical Medicine vol 29 no 2 pp 135ndash138 2009

[47] C L Wang S X Wang and W Gong ldquoChanges in phos-phatized ERK in the spinal dorsal horn in adjuvant arthritisrats during electroacupuncture of Jiaji points for analgesiardquoShanghai Journal of Acupuncture and Moxibustion vol 24 no12 pp 33ndash35 2005

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

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Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Research and TreatmentAIDS

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Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Evidence-Based Complementary and Alternative Medicine

(a) (b)

(c) (d)

(e)

Figure 2 Expression of collagen fibers in the colon tissue of rat Masson trichrome staining method times100 (a) Normal (b) Crohnrsquos disease(c) mild moxibustion (d) electroacupuncture (e) herb-separated moxibustion

both the HPM group MM group and the EA group (119875 lt001) It suggests that these treatments are able to improve thetissue damage of CD

32 Expression of Collagen Fibers in the Colon Tissue of RatAs shown in Figure 2 Masson trichrome staining revealedthat in the MC group a large number of collagen fibers werepresent in the mucosa muscularis mucosa submucosa andmuscularis of the rat colon with a disordered arrangementIn the MM group and the HSM group there were smallnumbers of collagen fibers in the colonic lamina propria andsubmucosa at the original ulcer-healing location which wasless severe compared with that of the model group In the EAgroup there were a small number of collagen fibers in the

mucosa submucosa muscularis and serosa of the rat colonand the effect was less severe than in the MC group

33 Ras Expression in the Colonic Mucosa of Rat As shownin Figures 3 and 4 the phosphorylation of Ras protein inthe rat colon was significantly higher in the MC group thanin the NC group (119875 lt 001) Ras protein expression wassignificantly reduced (119875 lt 001) to normal levels after acu-puncture treatment

Figure 5 indicates that Ras mRNA expression in the MCgroup was higher than that in the NC group (119875 lt 001) andthat HSM effectively reduced the Ras mRNA levels in the ratcolon (119875 lt 005) whereas there was no significant differencebetween the EA group and the NC group

Evidence-Based Complementary and Alternative Medicine 5

0

100

200

300

400

500

600

700

NC MC MM EA HSPRa

s pho

spho

ryla

tion

prot

ein

(IO

D)

lowast

Figure 3The integral optical density (IOD) of Ras phosphorylation in each rat group NC normal control group MC model control groupMM mild moxibustion EA electroacupuncture and HSM herb-separated moxibustion lowast119875 lt 001 versus NC 119875 lt 001 versus MC

(a) (b)

(c) (d)

(e)

Figure 4 Expression of phosphorylated Ras protein in the colonic mucosa of rat The phosphorylation of Ras protein was expressedin epithelial cells and lymphocyte in the colonic mucosa of rats EnVision Plus method times200 (a) Normal (b) Crohnrsquos disease (c) mildmoxibustion (d) electroacupuncture (e) herb-separated moxibustion

6 Evidence-Based Complementary and Alternative Medicine

0

2

4

Ras m

RNA

leve

l (

)

6

8

10

12 17

25

21

NC MC MM EA HSP

lowast

lowast

Figure 5 The Ras mRNA levels in the rat colon NC normalcontrol group MC model control group MM mild moxibustionEA electroacupuncture and HSM herb-separated moxibustionlowast119875 lt 001 versus NC 119875 lt 005 versus MC

0100200300400500600700800900

NC MC MM EA HSP

Raf-1

pro

tein

(IO

D)

lowast

998771998771 998771

Figure 6 The IOD of Raf-1 protein expression in rat colonNC normal control group MC model control group MM mildmoxibustion EA electroacupuncture and HSM herb-separatedmoxibustion lowast119875 lt 001 versus NC 119875 lt 005 119875 lt 001 versusMC 998771998771119875 lt 005 998771119875 lt 001 versus EA

34 The Raf-1 Expression in the Colonic Mucosa of Rat Asshown in Figures 6 and 7 the Raf-1 protein expression inthe colonic tissue of the MC group was significantly higherthan in the NC group (119875 lt 001) and that the Raf-1 proteinexpression in the colon was reduced to varying degrees afteracupuncture treatment (119875 lt 005 or 119875 lt 005)

The results in Figure 8 show that the Raf-1 mRNAlevels in the MC group were significantly higher than in theNC group (119875 lt 001) and that MM and HSM treatmentssignificantly reduced rat colon Raf-1 mRNA levels (119875 lt 005)whereas EA treatment did not significantly affect rat colonRaf-1 mRNA levels

35 The MEK-1 Expression in the Colonic Mucosa of Rat Asshown in Figures 9 and 10 the MEK-1 protein expression inthe colonic tissue of the MC group was significantly higherthan in NC rats (119875 lt 001) and MEK-1 protein expressionwas significantly reduced in each group after acupuncturetreatment (119875 lt 001) although the levels were still higherthan normal levels (119875 lt 001)

The results in Figure 11 show that theMEK-1mRNA levelsin the model group were significantly higher than those inthe NC group (119875 lt 001) and that MM HSM and EAtreatment reduced MEK-1 mRNA levels although there was

no statistically significant difference between the treatmentand no treatment groups or among the treatment groups

36 The ERK-12 Expression in the Colonic Mucosa of Rat Asshown in Figures 12 and 13 the phosphorylation of ERK-12significantly increased in the MC group compared with theNC group (119875 lt 001) and that the levels of phosphorylatedERK-12 proteins were significantly reduced after the MMand HSM treatments (119875 lt 001)

The results in Figure 14 show that the ERK-12 mRNAexpression in the MC group was significantly higher thanthat in the NC group (119875 lt 001) and that ERK-12 mRNAexpression was significantly lower in the MM group afteracupuncture treatment compared with the MC group (119875 lt005)

4 Discussion

Extracellular signal-regulated kinase (ERK) a member ofthe mitogen-activated protein kinase (MAPK) family playsan important biological role in transmitting signals fromsurface receptors to the nucleus The ERK12 pathway is themost thoroughly researched pathway in the ERK family andplays a role in a wide range of cellular functions includ-ing regulating meiosis mitosis and anaphase processes indifferent cells A variety of stimulating factors such as growthfactors cytokines viruses G protein-coupled receptor lig-ands and oncogenes can activate the ERK12 pathway [18]The ERK signaling pathway is a Ras-Raf-MEK-ERK path-way Phosphorylation-activated ERK12 translocates fromthe cytoplasm to the nucleus and thereby mediates thetranscriptional activation of Elk-1 ATF NF-120581B AP-1 and c-fos which in turn regulate cell proliferation and differentia-tion the maintenance of cell shape the construction of thecytoskeleton apoptosis and the malignant transformation ofcells among other biological functions

One of the features of CD is the transformation of smoothmuscle cells into fibroblasts andor myofibroblasts [8] Thisabundance of fibroblasts and myofibroblasts increases theexpression of type I collagen mRNA and produces collagentissue within the muscle [19] There are a large number offibroblast cells and type I III IV and V collagen transcriptsin the small intestines of CD patients [20] which producemore ECM including collagen elastin and glycoproteinsthat are deposited intramurally to produce a thickening of theintestinal wall [10] Based on a large number of experimentalreports the ERK-12 signaling pathway appears to participatein myofibroblast proliferation differentiation and collagenproduction [10 11 21ndash25] extracellular matrix synthesis [2627] smooth muscle cell proliferation and apoptosis [28]and the migration of vascular smooth muscle cells [29]Inhibiting the expression and activity of ERK can inhibit theproliferation of vascular smooth muscle cells [30] to reducethe accumulation of ECM [31] Therefore it is inferred thatthe ERK-12 signaling pathway may induce the activationand proliferation of CD intestinal myofibroblasts and theirtransformation intomuscle fiber cells Additionally the ERK-12 signaling pathway may regulate the proliferation andapoptosis of smooth muscle cells

Evidence-Based Complementary and Alternative Medicine 7

(a) (b)

(c) (d)

(e)

Figure 7 Expression of Raf-1 protein in the colonic mucosa of rat The Raf-1 protein was expressed in epithelial cells in the colonic mucosaof rats EnVision Plus method times200 (a) Normal (b) Crohnrsquos disease (c) mild moxibustion (d) electroacupuncture and (e) herb-separatedmoxibustion

005

115

225

335

4

NC MC MM EA HSP

Raf-1

mRN

A le

vels

()

lowast

Figure 8 The Raf-1 mRNA levels in the rat colon NC normalcontrol group MC model control group MM mild moxibustionEA electroacupuncture and HSM herb-separated moxibustionlowast119875 lt 001 versus NC 119875 lt 005 versus MC

050

100150200250300350400450500

NC MC MM EA HSP

MEK

-1 p

rote

in (I

OD

)

lowast

Figure 9 The IOD of MEK-1 protein expression in the rat colonNC normal control group MC model control group MM mildmoxibustion EA electroacupuncture and HSM herb-separatedmoxibustion lowast119875 lt 001 versus NC 119875 lt 001 versus MC

8 Evidence-Based Complementary and Alternative Medicine

(a) (b)

(c) (d)

(e)

Figure 10 Expression of MEK-1 protein in the colonic mucosa of rat The MEK-1 protein was expressed in epithelial cells in the colonicmucosa of rats EnVision Plus method times200 (a) Normal (b) Crohnrsquos disease (c) mild moxibustion (d) electroacupuncture and (e) herb-separated moxibustion

As an upstream protein in the Raf-MEK-ERK pathwayRas is the product of the proto-oncogene c-ras A variety ofstimulating factors particularly growth factors (eg PDGFEGF TNF and PKC activators as well as Src family members)can activate Ras which further activates Raf-1 ActivatedRaf-1 then activates MEK and ERK downstream [32 33] toinitiate the MAPK pathway Raf-1 protein kinase is widelyexpressed in various types of cells [34] Ras and Raf-1 oftenexist in the form of complexes Ras the upstream activatorprotein can translocate Raf from the cytoplasm to theplasma membrane where the latter is activated Howeverthe mechanism by which Raf is activated remains unclearalthough it may include phosphorylation [35] All that isknown is that Raf activation is related to its phosphorylation

on SerThr residues [33] Recent studies have shown thatRaf-1 protein kinase may be a point of crosstalk with othersignaling pathways This protein may participate in a varietyof biological processes in signal transduction including itsown regulation by interactingwith other signalingmoleculesor via reciprocal regulation

The MAPK kinases include MEK1 (MKK1) and MEK2(MKK2)MEK1 andMEK2 are the central links in theMAPKpathway The activated upstream Raf-1 protein kinase cancombine with MEK1 and 2 and doubly phosphorylate Tyrand Thr its two regulatory sites to activate ERK [36] MEKsare not only ERK activators but may also relocalize theactivated ERKs to the nucleus or other active sites to furtherphosphorylate downstream substrates [37]

Evidence-Based Complementary and Alternative Medicine 9

0

2

1

3

5

4

6

19

NC MC MM EA HSP

MEK

-1 m

RNA

leve

ls (

)

lowast

Figure 11 The MEK-1 mRNA levels in the rat colon NC normalcontrol group MC model control group MM mild moxibustionEA electroacupuncture and HSM herb-separated moxibustionlowast119875 lt 001 versus NC

0200400600800

100012001400

NC MC MM EA HSPPhos

phor

ylat

ed E

RK-1

2 (I

OD

)

lowast

Figure 12 The IOD of phosphorylated ERK-12 levels in therat colon NC normal control group MC model control groupMM mild moxibustion EA electroacupuncture and HSM herb-separated moxibustion lowast119875 lt 001 versus NC 119875 lt 005 119875 lt 001versus MC

The upstream kinase MEK1 is a dual-specificity enzymeActivatedMEK1 can phosphorylate ERK1 and ERK2There isevidence that the Tyr residues of ERK1 and 2 are phosphory-lated before the Thr residues both intracellularly and extra-cellularly [38] ERK translocates into the nucleus by forminga homodimer of phosphorylated ERK and phosphorylatedor unphosphorylated ERK [39] ERK is evenly distributedin quiescent cells but when the cells are stimulated byserum persistently activated ERK can partially translocateinto the nucleus whereas transiently activated ERK cannotrelocalize Activated ERKs accumulate in the nucleus andthen phosphorylate the corresponding transcription factorswhich drive expression of specific gene products Becauseof the difference in nuclear localization transiently activated(or low-activity) ERK and persistently activated (or high-activity) ERK cause cells to express genes at a different qualityor quantity resulting in different biological effects [40ndash43]

The ERK1-ERK2-MAPK pathway incorporates a mech-anism for inactivation after activation which can preventexcessive activation-induced adverse consequences At thesame time the inactivated kinase is prepared for the nextactivation The mechanisms of inhibition of this pathwayinclude the following (1) the activation of ERK12-MAPK is

the result of the phosphorylation cascade and can be inac-tivated by dephosphorylation by the corresponding phos-phatases (2) Raf-1 is the direct substrate of PKA which canphosphorylate Ser43 of c-Raf an inhibitory phosphorylationsite in the N-terminus of Raf that interacts with Ras andthe phosphorylation of this site can inhibit the bindingbetween RasGTP and the N-terminus of Raf which blocksthe activation of ERK (3) the phosphatase PP2 can inactivateMEK and ERK via dephosphorylation (4) activated ERKphosphorylates Sos and Raf while ERK dephosphorylationinactivates them thereby generates negative feedback regula-tion of the pathwayTherefore the study of themechanism bywhich acupuncture regulates the expression of ERKupstreamreceptors the phosphorylation of ERK cascade members thetargets of downstream signal transduction and the potentialimpact of other signaling pathways on this pathway is neces-sary to confirm acupuncture as an antifibrotic treatment

Our experiments studied the regulation of Ras RafMEKand ERK protein and gene expression by acupuncture inCD intestinal fibrosis in the rat colon Our results showthat the mRNA levels of Ras Raf-1 MEK-1 and ERK-12 were significantly increased in CD rat intestinal tissueand that the expression of p-Ras Raf-1 MEK-1 and p-ERK-12 proteins in intestinal epithelial cells were also sig-nificantly increased These results suggest that the activityof the ERK pathway in CD intestinal fibrosis process wasaltered Medicine-separated moxibustion mild moxibustionand electroacupuncture treatments could effectively down-regulate the expression of p-Ras Raf-1 MEK-1 and p-ERK-12 but different inhibitory effects on Ras Raf-1 MEK-1and ERK-12 mRNA levels were shown in ERK pathwayactivity changes In this discussion we propose the following(1) medicine-separated moxibustion mild moxibustion andelectroacupuncture can inhibit CD intestinal fibrosis byregulating ERK pathway activity and (2) the acupuncture-induced inhibition of p-Ras Raf-1 MEK-1 and p-ERK-12 expression may be the key elements in the treatmentrsquosinhibition of CD intestinal fibrosis At present most reportsregarding the role of acupuncture in the regulation of theERK signaling pathway have focused on EA such as EAmodel rats After EA on the Dazhui (DU14) and Baihui(DU20) acupuncture points the number of ERK-positivestriatal cells increased significantly in the cerebral cortex andERK protein expression was increased in rats with damagedneurons after cerebral ischemia and reperfusion thereforeEA can increase ERK pathway activity [44 45] After the EAtreatment of gastric mucosa-injured rats the phosphoryla-tion levels of ERK in gastric mucosal cells from extractedserum were significantly increased and were significantlydifferent between the serum of the normal rats and that ofthe model rats These observations suggest that ERK maybe involved in signal transduction in the EA stimulation atacupoints of stomach meridian of foot yangming repairinggastric mucosal injury [46] and that EA had an analgesiceffect by downregulating p-ERK expression levels in thedorsal horn of the spinal cord of rats with adjuvant arthritis[47] The previous reports have demonstrated that EA canregulate the ERK signal transduction pathway The results ofour experiments show that medicine-separated moxibustion

10 Evidence-Based Complementary and Alternative Medicine

(a) (b)

(c) (d)

(e)

Figure 13 Expression of phosphorylated ERK-12 protein in the colonicmucosa of ratThe phosphorylation of ERK-12 proteinwas expressedin epithelial cells and lymphocyte in the colonic mucosa of rats EnVision Plus method times200 (a) Normal (b) Crohnrsquos disease (c) mildmoxibustion (d) electroacupuncture and (e) herb-separated moxibustion

005

115

225

335

4

NC MC MM EA HSP

ERK-

12

mRN

A le

vels

()

lowast

Figure 14 The ERK-12 mRNA levels in the rat colon NC normalcontrol group MC model control group MM mild moxibustionEA electroacupuncture and HSM herb-separated moxibustionlowast119875 lt 001 versus NC 119875 lt 005 versus MC

andmildmoxibustion can also regulate the ERK signal trans-duction pathway and inhibit Ras and Raf-1 better than EAThe functional mechanisms underlying these observationsneed to be explored further

Abbreviations

CD Crohnrsquos diseaseNC Normal control groupMC Model control groupEA ElectroacupunctureMM Mild moxibustionHSM Herb-separated moxibustionST25 Tianshu acupoint

Evidence-Based Complementary and Alternative Medicine 11

RN6 Qihai acupointERK Extracellular signal-regulated kinaseMAPK Mitogen-activated protein kinaseTNBS 246-Trinitrobenzene sulfonic acid

Acknowledgments

The authors thank Professor Shuhui Jiang for technical assis-tance and the support of the National Natural Science Foun-dation of China (no 81173331) and National Basic ResearchProgram of China (973 program no 2009CB522900)Xiaomei Wang and Yuan Lu have the same right

References

[1] G Manning D B Whyte R Martinez T Hunter and SSudarsanam ldquoThe protein kinase complement of the humangenomerdquo Science vol 298 no 5600 pp 1912ndash1934 2002

[2] C Mowat A Cole A Windsor et al ldquoGuidelines for themanagement of inflammatory bowel disease in adultsrdquoGut vol60 no 5 pp 571ndash607 2011

[3] Inflammatory Bowel Disease Co-operation Group ChineseSociety of Gastroenterology ldquoChinese consensus on diagnosisand treatment standard of inflammatory bowel diseaserdquoChineseJournal of Gastroenterology vol 12 no 8 pp 490ndash494 2007

[4] J J Zheng X S Zhu Z Huangfu Z X Gao Z R Guo andZ Wang ldquoCrohnrsquos disease in mainland China a systematicanalysis of 50 years of researchrdquo Chinese Journal of DigestiveDiseases vol 6 no 4 pp 175ndash181 2005

[5] M Dai and B M Li ldquoResearch progress in mechanisms ofintestinal fibrosis in Crohnrsquos diseaserdquo Shijie Huaren XiaohuaZazhi vol 17 no 11 pp 1117ndash1121 2009

[6] O Grip J Malm B Veress A Bjartell S Lindgren and AEgesten ldquoIncreased presence of cells containing transforminggrowth factor alpha (TGF-120572) in ulcerative colitis both duringactive inflammation and in remissionrdquo European Journal ofGastroenterology and Hepatology vol 12 no 7 pp 761ndash7662000

[7] I E Koutroubakis E Petinaki P Dimoulios et al ldquoSerumlaminin and collagen IV in inflammatory bowel diseaserdquoJournal of Clinical Pathology vol 56 no 11 pp 817ndash820 2003

[8] S Speca I Giusti F Rieder and G Latella ldquoCellular andmolecular mechanisms of intestinal fibrosisrdquo World Journal ofGastroenterology vol 18 no 28 pp 3635ndash3661 2012

[9] Y F Song J H Hong and L M Xu ldquoTGF-120573 1ERK pathwaysand its inhibitors and fibrosisrdquo Chinese Journal of ClinicalHepatology vol 21 no 2 pp 125ndash127 2005

[10] H C Huang M Yang J Z Li and H Y Wang ldquoConnectivetissue growth factor promotes the proliferation ofmyofibroblastthrough Erk-12 signaling pathwayrdquoNational Medical Journal ofChina vol 85 no 19 pp 1322ndash1326 2005

[11] L P Guo H C Huang and J Z Li ldquoHypoxia inducesmyofibroblast formation and stimulates production of collagenI in myofibroblasts through ERK12 pathwayrdquo Chinese Journalof Pathophysiology vol 24 no 12 pp 2428ndash2432 2008

[12] H R Liu X P Ma T P Zhao and H G Wu ldquoThe adjustmentof acupuncture on colon collagen synthesis and TGF-120573mRNAexpression in Crohnrsquos disease ratrdquo Journal of Anhui TraditionalChinese Medical College vol 24 no 4 pp 25ndash30 2005

[13] X P Ma C P An H G Wu et al ldquoHerbs-partitionedmoxibustion and electro-acupuncture effects on transforming

growth factor-beta 1 connective tissue growth factor collagentype I and fibronectin expression in the colon of Crohnrsquo sdisease ratsrdquo Journal of Clinical Rehabilitative Tissue EngineeringResearch vol 12 no 20 pp 3853ndash3858 2008

[14] G P Morris P L Beck M S Herridge W T Depew M RSzewczuk and J LWallace ldquoHapten-inducedmodel of chronicinflammation and ulceration in the rat colonrdquoGastroenterologyvol 96 no 3 pp 795ndash803 1989

[15] TheMinistry of Science andTechnology of the Peoplersquos Republicof China Guidance Suggestions for the Care and Use ofLaboratory Animals 2006

[16] J D Butzner R Parmar C J Bell andVDalal ldquoButyrate enematherapy stimulates mucosal repair in experimental colitis in theratrdquo Gut vol 38 no 4 pp 568ndash573 1996

[17] X M Wang Y Lu L Y Wu et al ldquoMoxibustion inhibitsinterleukin-12 and tumor necrosis factor alpha and modulatesintestinal flora in rat with ulcerative colitisrdquo World Journal ofGastroenterology vol 18 no 46 pp 6826ndash6835 2012

[18] J B Pucilowska K K McNaughton N K Mohapatra et alldquoIGF-I and procollagen 1205721(I) are coexpressed in a subset ofmesenchymal cells in active Crohnrsquos diseaserdquo American Journalof Physiology vol 279 no 6 pp G1307ndashG1322 2000

[19] M Gharaee-Kermani B Hu S H Phan and M R GyetkoldquoRecent advances in molecular targets and treatment of idio-pathic pulmonary fibrosis focus on TGF120573 signaling and themyofibroblastrdquo Current Medicinal Chemistry vol 16 no 11 pp1400ndash1417 2009

[20] E M Zimmermann L Li Y T Hou M Cannon G MChristman andKN Bitar ldquoIGF-I induces collagen and IGFBP-5 mRNA in rat intestinal smooth musclerdquo American Journal ofPhysiology vol 273 no 4 pp G875ndashG882 1997

[21] L J ZhangQ Li P Chen et al ldquoMediating effect of extracellularsignal-regulated kinase 12 in PDGF-induced proliferation andcollagen synthesis of cultured rat cardiac fibroblastsrdquo ChineseJournal of Anatomy vol 13 no 4 p 223 2007

[22] M Tang W Zhang H Lin H Jiang H Dai and Y ZhangldquoHigh glucose promotes the production of collagen types Iand III by cardiac fibroblasts through a pathway dependenton extracellular-signal-regulated kinase 12rdquo Molecular andCellular Biochemistry vol 301 no 1-2 pp 109ndash114 2007

[23] H Y Dai Z M Ge and Y H Li ldquoEffect of urotensin II on pro-liferative potential and phosphorylation of extracellular signal-regulated kinase 12 adventitial fibroblasts from spontaneouslyhypertensive ratrdquoActaAcademiaeMedicinae Sinicae vol 28 no6 pp 776ndash780 2006

[24] I Fuentes-Calvo A M Blazquez-Medela N Eleno E SantosJ M Lopez-Novoa and C Martınez-Salgado ldquoH-Ras isoformmodulates extracellular matrix synthesis proliferation andmigration in fibroblastsrdquo American Journal of Physiology vol302 no 4 pp C686ndashC697 2012

[25] Y W Ma G T Bai and M X Zhang ldquoStudies on signal trans-duction pathway induced by fibronectin in human embryoniclung fibroblastsrdquo Shanxi Medical Journal vol 38 no 7 pp 587ndash589 2009

[26] J H Jang and C P Chung ldquoFibronectin-mediated adhesionrescues cell cycle arrest induced by fibroblast growth factor-1 bydecreased expression of P21CIPWAF in human chondrocytesrdquoIn Vitro Cellular and Developmental Biology vol 41 no 5-6 pp126ndash129 2005

[27] B Zhu Y J Wang C F Zhu et al ldquoTriptolide inhibitsextracellularmatrix protein synthesis by suppressing the Smad2

12 Evidence-Based Complementary and Alternative Medicine

but not the MAPK pathway in TGF-1205731-stimulated NRK-49Fcellsrdquo Nephrology Dialysis Transplantation vol 25 no 10 pp3180ndash3191 2010

[28] J Bai X S Liu Y J Xu M Xie and W Ni ldquoEffects ofextracellular signal regulated kinase signaling pathway on cellcycle of airway smooth muscle cells in asthmatic ratsrdquo ChineseJournal of Respiratory and Critical Care Medicine vol 9 no 1pp 23ndash27 2010

[29] S Matsusaka and I Wakabayashi ldquoEnhancement of vascu-lar smooth muscle cell migration by urotensin IIrdquo Naunyn-Schmiedebergrsquos Archives of Pharmacology vol 373 no 5 pp 381ndash386 2006

[30] Y Chen Z Dai Y M Liu et al ldquoInhibitory effects ofCGRP on vascular smooth muscle cell proliferation role ofcaveolaecaveolin-1ERK12 signal pathwayrdquo Progress in Bio-chemistry and Biophysics vol 40 no 5 pp 445ndash453 2013

[31] W L Wu A Q Zhang Q Yin and W H Gan ldquoEffectsof ERK signal pathway on extracellular matrix synthesis ofrat mesangial cells treated with PDGF-BBrdquo ACTA UniversityMedicinalis Nanjing vol 27 no 10 pp 1126ndash1129 2007

[32] L S Steelman W H Chappell S L Abrams et al ldquoRoles ofthe RafMEKERK and PI3KPTENAktmTOR pathways incontrolling growth and sensitivity to therapy-implications forcancer and agingrdquo Aging vol 3 no 3 pp 192ndash222 2011

[33] W Kolch ldquoMeaningful relationships the regulation of theRasRafMEKERK pathway by protein interactionsrdquo Biochem-ical Journal vol 351 no 2 pp 289ndash305 2000

[34] D Matallanas M Birtwistle D Romano et al ldquoRaf familykinases old dogs have learned new tricksrdquo Genes and Cancervol 2 no 3 pp 232ndash260 2011

[35] C S Mason C J Springer R G Cooper G Superti-Furga C JMarshall and RMarais ldquoSerine and tyrosine phosphorylationscooperate in Raf-1 but not B-Raf activationrdquo The EMBOJournal vol 18 no 8 pp 2137ndash2148 1999

[36] R J Fiddes P W Janes S P Sivertsen R L Sutherland EA Musgrove and R J Daly ldquoInhibition of the MAP kinasecascade blocks heregulin-induced cell cycle progression in T-47D human breast cancer cellsrdquo Oncogene vol 16 no 21 pp2803ndash2813 1998

[37] S T Eblen J K SlackM JWeber andA D Catling ldquoRac-PAKsignaling stimulates extracellular signal-regulated kinase (ERK)activation by regulating formation of MEK1-ERK complexesrdquoMolecular and Cellular Biology vol 22 no 17 pp 6023ndash60332002

[38] M Cargnello and P P Roux ldquoActivation and function ofthe MAPKs and their substrates the MAPK-activated proteinkinasesrdquo Microbiology and Molecular Biology Reviews vol 75no 1 pp 50ndash83 2011

[39] A V Khokhlatchev B Canagarajah J Wilsbacher et al ldquoPhos-phorylation of theMAP kinase ERK2 promotes its homodimer-ization and nuclear translocationrdquo Cell vol 93 no 4 pp 605ndash615 1998

[40] R H Chen C Sarnecki and J Blenis ldquoNuclear localization andregulation of erk- and rsk-encoded protein kinasesrdquoMolecularand Cellular Biology vol 12 no 3 pp 915ndash927 1992

[41] S Traverse K Seedorf H Paterson C J Marshall P Cohenand A Ullrich ldquoEGF triggers neuronal differentiation of PC12cells that overexpress the EGF receptorrdquo Current Biology vol 4no 8 pp 694ndash701 1994

[42] T T Nguyen J C Scimeca C Filloux P Peraldi J L Car-pentier and E Van Obberghen ldquoCo-regulation of the mitogen-activated protein kinase extracellular signal-regulated kinase 1

and the 90-kDa ribosomal S6 kinase in PC12 cells Distincteffects of the neurotrophic factor nerve growth factor and themitogenic factor epidermal growth factorrdquo Journal of BiologicalChemistry vol 268 no 13 pp 9803ndash9810 1993

[43] I Dikic J Schlessinger and I Lax ldquoPC12 cells overexpressingthe insulin receptor undergo insulin-dependent neuronal dif-ferentiationrdquo Current Biology vol 4 no 8 pp 702ndash708 1994

[44] M H Shen Z R Li X R Xiang W M Niu and C B ZhangldquoEffect of electro-acupuncture on ERK protein expression ofMCAO ratsrdquo Journal of Clinical Acupuncture and Moxibustionvol 23 no 7 pp 62ndash63 2007

[45] M H Shen Z R Li X R Xiang W M Niu and C BZhang ldquoInvolvement of extracellular regulated protein kinasesignal transduction pathway in electroacupuncture-inducedimprovement of ischemic cerebral injury in rats with cerebralischemia reperfusionrdquo Acupuncture Research vol 32 no 6 pp368ndash372 2007

[46] Z B Yang J Yan and S X Yi ldquoUp-regulation of the ERK phos-phorylation in gastric mucosal cell by the serum of rats treatedwith electroacupuncture at stomach meridian acupointsrdquo Basicamp Clinical Medicine vol 29 no 2 pp 135ndash138 2009

[47] C L Wang S X Wang and W Gong ldquoChanges in phos-phatized ERK in the spinal dorsal horn in adjuvant arthritisrats during electroacupuncture of Jiaji points for analgesiardquoShanghai Journal of Acupuncture and Moxibustion vol 24 no12 pp 33ndash35 2005

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 5

0

100

200

300

400

500

600

700

NC MC MM EA HSPRa

s pho

spho

ryla

tion

prot

ein

(IO

D)

lowast

Figure 3The integral optical density (IOD) of Ras phosphorylation in each rat group NC normal control group MC model control groupMM mild moxibustion EA electroacupuncture and HSM herb-separated moxibustion lowast119875 lt 001 versus NC 119875 lt 001 versus MC

(a) (b)

(c) (d)

(e)

Figure 4 Expression of phosphorylated Ras protein in the colonic mucosa of rat The phosphorylation of Ras protein was expressedin epithelial cells and lymphocyte in the colonic mucosa of rats EnVision Plus method times200 (a) Normal (b) Crohnrsquos disease (c) mildmoxibustion (d) electroacupuncture (e) herb-separated moxibustion

6 Evidence-Based Complementary and Alternative Medicine

0

2

4

Ras m

RNA

leve

l (

)

6

8

10

12 17

25

21

NC MC MM EA HSP

lowast

lowast

Figure 5 The Ras mRNA levels in the rat colon NC normalcontrol group MC model control group MM mild moxibustionEA electroacupuncture and HSM herb-separated moxibustionlowast119875 lt 001 versus NC 119875 lt 005 versus MC

0100200300400500600700800900

NC MC MM EA HSP

Raf-1

pro

tein

(IO

D)

lowast

998771998771 998771

Figure 6 The IOD of Raf-1 protein expression in rat colonNC normal control group MC model control group MM mildmoxibustion EA electroacupuncture and HSM herb-separatedmoxibustion lowast119875 lt 001 versus NC 119875 lt 005 119875 lt 001 versusMC 998771998771119875 lt 005 998771119875 lt 001 versus EA

34 The Raf-1 Expression in the Colonic Mucosa of Rat Asshown in Figures 6 and 7 the Raf-1 protein expression inthe colonic tissue of the MC group was significantly higherthan in the NC group (119875 lt 001) and that the Raf-1 proteinexpression in the colon was reduced to varying degrees afteracupuncture treatment (119875 lt 005 or 119875 lt 005)

The results in Figure 8 show that the Raf-1 mRNAlevels in the MC group were significantly higher than in theNC group (119875 lt 001) and that MM and HSM treatmentssignificantly reduced rat colon Raf-1 mRNA levels (119875 lt 005)whereas EA treatment did not significantly affect rat colonRaf-1 mRNA levels

35 The MEK-1 Expression in the Colonic Mucosa of Rat Asshown in Figures 9 and 10 the MEK-1 protein expression inthe colonic tissue of the MC group was significantly higherthan in NC rats (119875 lt 001) and MEK-1 protein expressionwas significantly reduced in each group after acupuncturetreatment (119875 lt 001) although the levels were still higherthan normal levels (119875 lt 001)

The results in Figure 11 show that theMEK-1mRNA levelsin the model group were significantly higher than those inthe NC group (119875 lt 001) and that MM HSM and EAtreatment reduced MEK-1 mRNA levels although there was

no statistically significant difference between the treatmentand no treatment groups or among the treatment groups

36 The ERK-12 Expression in the Colonic Mucosa of Rat Asshown in Figures 12 and 13 the phosphorylation of ERK-12significantly increased in the MC group compared with theNC group (119875 lt 001) and that the levels of phosphorylatedERK-12 proteins were significantly reduced after the MMand HSM treatments (119875 lt 001)

The results in Figure 14 show that the ERK-12 mRNAexpression in the MC group was significantly higher thanthat in the NC group (119875 lt 001) and that ERK-12 mRNAexpression was significantly lower in the MM group afteracupuncture treatment compared with the MC group (119875 lt005)

4 Discussion

Extracellular signal-regulated kinase (ERK) a member ofthe mitogen-activated protein kinase (MAPK) family playsan important biological role in transmitting signals fromsurface receptors to the nucleus The ERK12 pathway is themost thoroughly researched pathway in the ERK family andplays a role in a wide range of cellular functions includ-ing regulating meiosis mitosis and anaphase processes indifferent cells A variety of stimulating factors such as growthfactors cytokines viruses G protein-coupled receptor lig-ands and oncogenes can activate the ERK12 pathway [18]The ERK signaling pathway is a Ras-Raf-MEK-ERK path-way Phosphorylation-activated ERK12 translocates fromthe cytoplasm to the nucleus and thereby mediates thetranscriptional activation of Elk-1 ATF NF-120581B AP-1 and c-fos which in turn regulate cell proliferation and differentia-tion the maintenance of cell shape the construction of thecytoskeleton apoptosis and the malignant transformation ofcells among other biological functions

One of the features of CD is the transformation of smoothmuscle cells into fibroblasts andor myofibroblasts [8] Thisabundance of fibroblasts and myofibroblasts increases theexpression of type I collagen mRNA and produces collagentissue within the muscle [19] There are a large number offibroblast cells and type I III IV and V collagen transcriptsin the small intestines of CD patients [20] which producemore ECM including collagen elastin and glycoproteinsthat are deposited intramurally to produce a thickening of theintestinal wall [10] Based on a large number of experimentalreports the ERK-12 signaling pathway appears to participatein myofibroblast proliferation differentiation and collagenproduction [10 11 21ndash25] extracellular matrix synthesis [2627] smooth muscle cell proliferation and apoptosis [28]and the migration of vascular smooth muscle cells [29]Inhibiting the expression and activity of ERK can inhibit theproliferation of vascular smooth muscle cells [30] to reducethe accumulation of ECM [31] Therefore it is inferred thatthe ERK-12 signaling pathway may induce the activationand proliferation of CD intestinal myofibroblasts and theirtransformation intomuscle fiber cells Additionally the ERK-12 signaling pathway may regulate the proliferation andapoptosis of smooth muscle cells

Evidence-Based Complementary and Alternative Medicine 7

(a) (b)

(c) (d)

(e)

Figure 7 Expression of Raf-1 protein in the colonic mucosa of rat The Raf-1 protein was expressed in epithelial cells in the colonic mucosaof rats EnVision Plus method times200 (a) Normal (b) Crohnrsquos disease (c) mild moxibustion (d) electroacupuncture and (e) herb-separatedmoxibustion

005

115

225

335

4

NC MC MM EA HSP

Raf-1

mRN

A le

vels

()

lowast

Figure 8 The Raf-1 mRNA levels in the rat colon NC normalcontrol group MC model control group MM mild moxibustionEA electroacupuncture and HSM herb-separated moxibustionlowast119875 lt 001 versus NC 119875 lt 005 versus MC

050

100150200250300350400450500

NC MC MM EA HSP

MEK

-1 p

rote

in (I

OD

)

lowast

Figure 9 The IOD of MEK-1 protein expression in the rat colonNC normal control group MC model control group MM mildmoxibustion EA electroacupuncture and HSM herb-separatedmoxibustion lowast119875 lt 001 versus NC 119875 lt 001 versus MC

8 Evidence-Based Complementary and Alternative Medicine

(a) (b)

(c) (d)

(e)

Figure 10 Expression of MEK-1 protein in the colonic mucosa of rat The MEK-1 protein was expressed in epithelial cells in the colonicmucosa of rats EnVision Plus method times200 (a) Normal (b) Crohnrsquos disease (c) mild moxibustion (d) electroacupuncture and (e) herb-separated moxibustion

As an upstream protein in the Raf-MEK-ERK pathwayRas is the product of the proto-oncogene c-ras A variety ofstimulating factors particularly growth factors (eg PDGFEGF TNF and PKC activators as well as Src family members)can activate Ras which further activates Raf-1 ActivatedRaf-1 then activates MEK and ERK downstream [32 33] toinitiate the MAPK pathway Raf-1 protein kinase is widelyexpressed in various types of cells [34] Ras and Raf-1 oftenexist in the form of complexes Ras the upstream activatorprotein can translocate Raf from the cytoplasm to theplasma membrane where the latter is activated Howeverthe mechanism by which Raf is activated remains unclearalthough it may include phosphorylation [35] All that isknown is that Raf activation is related to its phosphorylation

on SerThr residues [33] Recent studies have shown thatRaf-1 protein kinase may be a point of crosstalk with othersignaling pathways This protein may participate in a varietyof biological processes in signal transduction including itsown regulation by interactingwith other signalingmoleculesor via reciprocal regulation

The MAPK kinases include MEK1 (MKK1) and MEK2(MKK2)MEK1 andMEK2 are the central links in theMAPKpathway The activated upstream Raf-1 protein kinase cancombine with MEK1 and 2 and doubly phosphorylate Tyrand Thr its two regulatory sites to activate ERK [36] MEKsare not only ERK activators but may also relocalize theactivated ERKs to the nucleus or other active sites to furtherphosphorylate downstream substrates [37]

Evidence-Based Complementary and Alternative Medicine 9

0

2

1

3

5

4

6

19

NC MC MM EA HSP

MEK

-1 m

RNA

leve

ls (

)

lowast

Figure 11 The MEK-1 mRNA levels in the rat colon NC normalcontrol group MC model control group MM mild moxibustionEA electroacupuncture and HSM herb-separated moxibustionlowast119875 lt 001 versus NC

0200400600800

100012001400

NC MC MM EA HSPPhos

phor

ylat

ed E

RK-1

2 (I

OD

)

lowast

Figure 12 The IOD of phosphorylated ERK-12 levels in therat colon NC normal control group MC model control groupMM mild moxibustion EA electroacupuncture and HSM herb-separated moxibustion lowast119875 lt 001 versus NC 119875 lt 005 119875 lt 001versus MC

The upstream kinase MEK1 is a dual-specificity enzymeActivatedMEK1 can phosphorylate ERK1 and ERK2There isevidence that the Tyr residues of ERK1 and 2 are phosphory-lated before the Thr residues both intracellularly and extra-cellularly [38] ERK translocates into the nucleus by forminga homodimer of phosphorylated ERK and phosphorylatedor unphosphorylated ERK [39] ERK is evenly distributedin quiescent cells but when the cells are stimulated byserum persistently activated ERK can partially translocateinto the nucleus whereas transiently activated ERK cannotrelocalize Activated ERKs accumulate in the nucleus andthen phosphorylate the corresponding transcription factorswhich drive expression of specific gene products Becauseof the difference in nuclear localization transiently activated(or low-activity) ERK and persistently activated (or high-activity) ERK cause cells to express genes at a different qualityor quantity resulting in different biological effects [40ndash43]

The ERK1-ERK2-MAPK pathway incorporates a mech-anism for inactivation after activation which can preventexcessive activation-induced adverse consequences At thesame time the inactivated kinase is prepared for the nextactivation The mechanisms of inhibition of this pathwayinclude the following (1) the activation of ERK12-MAPK is

the result of the phosphorylation cascade and can be inac-tivated by dephosphorylation by the corresponding phos-phatases (2) Raf-1 is the direct substrate of PKA which canphosphorylate Ser43 of c-Raf an inhibitory phosphorylationsite in the N-terminus of Raf that interacts with Ras andthe phosphorylation of this site can inhibit the bindingbetween RasGTP and the N-terminus of Raf which blocksthe activation of ERK (3) the phosphatase PP2 can inactivateMEK and ERK via dephosphorylation (4) activated ERKphosphorylates Sos and Raf while ERK dephosphorylationinactivates them thereby generates negative feedback regula-tion of the pathwayTherefore the study of themechanism bywhich acupuncture regulates the expression of ERKupstreamreceptors the phosphorylation of ERK cascade members thetargets of downstream signal transduction and the potentialimpact of other signaling pathways on this pathway is neces-sary to confirm acupuncture as an antifibrotic treatment

Our experiments studied the regulation of Ras RafMEKand ERK protein and gene expression by acupuncture inCD intestinal fibrosis in the rat colon Our results showthat the mRNA levels of Ras Raf-1 MEK-1 and ERK-12 were significantly increased in CD rat intestinal tissueand that the expression of p-Ras Raf-1 MEK-1 and p-ERK-12 proteins in intestinal epithelial cells were also sig-nificantly increased These results suggest that the activityof the ERK pathway in CD intestinal fibrosis process wasaltered Medicine-separated moxibustion mild moxibustionand electroacupuncture treatments could effectively down-regulate the expression of p-Ras Raf-1 MEK-1 and p-ERK-12 but different inhibitory effects on Ras Raf-1 MEK-1and ERK-12 mRNA levels were shown in ERK pathwayactivity changes In this discussion we propose the following(1) medicine-separated moxibustion mild moxibustion andelectroacupuncture can inhibit CD intestinal fibrosis byregulating ERK pathway activity and (2) the acupuncture-induced inhibition of p-Ras Raf-1 MEK-1 and p-ERK-12 expression may be the key elements in the treatmentrsquosinhibition of CD intestinal fibrosis At present most reportsregarding the role of acupuncture in the regulation of theERK signaling pathway have focused on EA such as EAmodel rats After EA on the Dazhui (DU14) and Baihui(DU20) acupuncture points the number of ERK-positivestriatal cells increased significantly in the cerebral cortex andERK protein expression was increased in rats with damagedneurons after cerebral ischemia and reperfusion thereforeEA can increase ERK pathway activity [44 45] After the EAtreatment of gastric mucosa-injured rats the phosphoryla-tion levels of ERK in gastric mucosal cells from extractedserum were significantly increased and were significantlydifferent between the serum of the normal rats and that ofthe model rats These observations suggest that ERK maybe involved in signal transduction in the EA stimulation atacupoints of stomach meridian of foot yangming repairinggastric mucosal injury [46] and that EA had an analgesiceffect by downregulating p-ERK expression levels in thedorsal horn of the spinal cord of rats with adjuvant arthritis[47] The previous reports have demonstrated that EA canregulate the ERK signal transduction pathway The results ofour experiments show that medicine-separated moxibustion

10 Evidence-Based Complementary and Alternative Medicine

(a) (b)

(c) (d)

(e)

Figure 13 Expression of phosphorylated ERK-12 protein in the colonicmucosa of ratThe phosphorylation of ERK-12 proteinwas expressedin epithelial cells and lymphocyte in the colonic mucosa of rats EnVision Plus method times200 (a) Normal (b) Crohnrsquos disease (c) mildmoxibustion (d) electroacupuncture and (e) herb-separated moxibustion

005

115

225

335

4

NC MC MM EA HSP

ERK-

12

mRN

A le

vels

()

lowast

Figure 14 The ERK-12 mRNA levels in the rat colon NC normalcontrol group MC model control group MM mild moxibustionEA electroacupuncture and HSM herb-separated moxibustionlowast119875 lt 001 versus NC 119875 lt 005 versus MC

andmildmoxibustion can also regulate the ERK signal trans-duction pathway and inhibit Ras and Raf-1 better than EAThe functional mechanisms underlying these observationsneed to be explored further

Abbreviations

CD Crohnrsquos diseaseNC Normal control groupMC Model control groupEA ElectroacupunctureMM Mild moxibustionHSM Herb-separated moxibustionST25 Tianshu acupoint

Evidence-Based Complementary and Alternative Medicine 11

RN6 Qihai acupointERK Extracellular signal-regulated kinaseMAPK Mitogen-activated protein kinaseTNBS 246-Trinitrobenzene sulfonic acid

Acknowledgments

The authors thank Professor Shuhui Jiang for technical assis-tance and the support of the National Natural Science Foun-dation of China (no 81173331) and National Basic ResearchProgram of China (973 program no 2009CB522900)Xiaomei Wang and Yuan Lu have the same right

References

[1] G Manning D B Whyte R Martinez T Hunter and SSudarsanam ldquoThe protein kinase complement of the humangenomerdquo Science vol 298 no 5600 pp 1912ndash1934 2002

[2] C Mowat A Cole A Windsor et al ldquoGuidelines for themanagement of inflammatory bowel disease in adultsrdquoGut vol60 no 5 pp 571ndash607 2011

[3] Inflammatory Bowel Disease Co-operation Group ChineseSociety of Gastroenterology ldquoChinese consensus on diagnosisand treatment standard of inflammatory bowel diseaserdquoChineseJournal of Gastroenterology vol 12 no 8 pp 490ndash494 2007

[4] J J Zheng X S Zhu Z Huangfu Z X Gao Z R Guo andZ Wang ldquoCrohnrsquos disease in mainland China a systematicanalysis of 50 years of researchrdquo Chinese Journal of DigestiveDiseases vol 6 no 4 pp 175ndash181 2005

[5] M Dai and B M Li ldquoResearch progress in mechanisms ofintestinal fibrosis in Crohnrsquos diseaserdquo Shijie Huaren XiaohuaZazhi vol 17 no 11 pp 1117ndash1121 2009

[6] O Grip J Malm B Veress A Bjartell S Lindgren and AEgesten ldquoIncreased presence of cells containing transforminggrowth factor alpha (TGF-120572) in ulcerative colitis both duringactive inflammation and in remissionrdquo European Journal ofGastroenterology and Hepatology vol 12 no 7 pp 761ndash7662000

[7] I E Koutroubakis E Petinaki P Dimoulios et al ldquoSerumlaminin and collagen IV in inflammatory bowel diseaserdquoJournal of Clinical Pathology vol 56 no 11 pp 817ndash820 2003

[8] S Speca I Giusti F Rieder and G Latella ldquoCellular andmolecular mechanisms of intestinal fibrosisrdquo World Journal ofGastroenterology vol 18 no 28 pp 3635ndash3661 2012

[9] Y F Song J H Hong and L M Xu ldquoTGF-120573 1ERK pathwaysand its inhibitors and fibrosisrdquo Chinese Journal of ClinicalHepatology vol 21 no 2 pp 125ndash127 2005

[10] H C Huang M Yang J Z Li and H Y Wang ldquoConnectivetissue growth factor promotes the proliferation ofmyofibroblastthrough Erk-12 signaling pathwayrdquoNational Medical Journal ofChina vol 85 no 19 pp 1322ndash1326 2005

[11] L P Guo H C Huang and J Z Li ldquoHypoxia inducesmyofibroblast formation and stimulates production of collagenI in myofibroblasts through ERK12 pathwayrdquo Chinese Journalof Pathophysiology vol 24 no 12 pp 2428ndash2432 2008

[12] H R Liu X P Ma T P Zhao and H G Wu ldquoThe adjustmentof acupuncture on colon collagen synthesis and TGF-120573mRNAexpression in Crohnrsquos disease ratrdquo Journal of Anhui TraditionalChinese Medical College vol 24 no 4 pp 25ndash30 2005

[13] X P Ma C P An H G Wu et al ldquoHerbs-partitionedmoxibustion and electro-acupuncture effects on transforming

growth factor-beta 1 connective tissue growth factor collagentype I and fibronectin expression in the colon of Crohnrsquo sdisease ratsrdquo Journal of Clinical Rehabilitative Tissue EngineeringResearch vol 12 no 20 pp 3853ndash3858 2008

[14] G P Morris P L Beck M S Herridge W T Depew M RSzewczuk and J LWallace ldquoHapten-inducedmodel of chronicinflammation and ulceration in the rat colonrdquoGastroenterologyvol 96 no 3 pp 795ndash803 1989

[15] TheMinistry of Science andTechnology of the Peoplersquos Republicof China Guidance Suggestions for the Care and Use ofLaboratory Animals 2006

[16] J D Butzner R Parmar C J Bell andVDalal ldquoButyrate enematherapy stimulates mucosal repair in experimental colitis in theratrdquo Gut vol 38 no 4 pp 568ndash573 1996

[17] X M Wang Y Lu L Y Wu et al ldquoMoxibustion inhibitsinterleukin-12 and tumor necrosis factor alpha and modulatesintestinal flora in rat with ulcerative colitisrdquo World Journal ofGastroenterology vol 18 no 46 pp 6826ndash6835 2012

[18] J B Pucilowska K K McNaughton N K Mohapatra et alldquoIGF-I and procollagen 1205721(I) are coexpressed in a subset ofmesenchymal cells in active Crohnrsquos diseaserdquo American Journalof Physiology vol 279 no 6 pp G1307ndashG1322 2000

[19] M Gharaee-Kermani B Hu S H Phan and M R GyetkoldquoRecent advances in molecular targets and treatment of idio-pathic pulmonary fibrosis focus on TGF120573 signaling and themyofibroblastrdquo Current Medicinal Chemistry vol 16 no 11 pp1400ndash1417 2009

[20] E M Zimmermann L Li Y T Hou M Cannon G MChristman andKN Bitar ldquoIGF-I induces collagen and IGFBP-5 mRNA in rat intestinal smooth musclerdquo American Journal ofPhysiology vol 273 no 4 pp G875ndashG882 1997

[21] L J ZhangQ Li P Chen et al ldquoMediating effect of extracellularsignal-regulated kinase 12 in PDGF-induced proliferation andcollagen synthesis of cultured rat cardiac fibroblastsrdquo ChineseJournal of Anatomy vol 13 no 4 p 223 2007

[22] M Tang W Zhang H Lin H Jiang H Dai and Y ZhangldquoHigh glucose promotes the production of collagen types Iand III by cardiac fibroblasts through a pathway dependenton extracellular-signal-regulated kinase 12rdquo Molecular andCellular Biochemistry vol 301 no 1-2 pp 109ndash114 2007

[23] H Y Dai Z M Ge and Y H Li ldquoEffect of urotensin II on pro-liferative potential and phosphorylation of extracellular signal-regulated kinase 12 adventitial fibroblasts from spontaneouslyhypertensive ratrdquoActaAcademiaeMedicinae Sinicae vol 28 no6 pp 776ndash780 2006

[24] I Fuentes-Calvo A M Blazquez-Medela N Eleno E SantosJ M Lopez-Novoa and C Martınez-Salgado ldquoH-Ras isoformmodulates extracellular matrix synthesis proliferation andmigration in fibroblastsrdquo American Journal of Physiology vol302 no 4 pp C686ndashC697 2012

[25] Y W Ma G T Bai and M X Zhang ldquoStudies on signal trans-duction pathway induced by fibronectin in human embryoniclung fibroblastsrdquo Shanxi Medical Journal vol 38 no 7 pp 587ndash589 2009

[26] J H Jang and C P Chung ldquoFibronectin-mediated adhesionrescues cell cycle arrest induced by fibroblast growth factor-1 bydecreased expression of P21CIPWAF in human chondrocytesrdquoIn Vitro Cellular and Developmental Biology vol 41 no 5-6 pp126ndash129 2005

[27] B Zhu Y J Wang C F Zhu et al ldquoTriptolide inhibitsextracellularmatrix protein synthesis by suppressing the Smad2

12 Evidence-Based Complementary and Alternative Medicine

but not the MAPK pathway in TGF-1205731-stimulated NRK-49Fcellsrdquo Nephrology Dialysis Transplantation vol 25 no 10 pp3180ndash3191 2010

[28] J Bai X S Liu Y J Xu M Xie and W Ni ldquoEffects ofextracellular signal regulated kinase signaling pathway on cellcycle of airway smooth muscle cells in asthmatic ratsrdquo ChineseJournal of Respiratory and Critical Care Medicine vol 9 no 1pp 23ndash27 2010

[29] S Matsusaka and I Wakabayashi ldquoEnhancement of vascu-lar smooth muscle cell migration by urotensin IIrdquo Naunyn-Schmiedebergrsquos Archives of Pharmacology vol 373 no 5 pp 381ndash386 2006

[30] Y Chen Z Dai Y M Liu et al ldquoInhibitory effects ofCGRP on vascular smooth muscle cell proliferation role ofcaveolaecaveolin-1ERK12 signal pathwayrdquo Progress in Bio-chemistry and Biophysics vol 40 no 5 pp 445ndash453 2013

[31] W L Wu A Q Zhang Q Yin and W H Gan ldquoEffectsof ERK signal pathway on extracellular matrix synthesis ofrat mesangial cells treated with PDGF-BBrdquo ACTA UniversityMedicinalis Nanjing vol 27 no 10 pp 1126ndash1129 2007

[32] L S Steelman W H Chappell S L Abrams et al ldquoRoles ofthe RafMEKERK and PI3KPTENAktmTOR pathways incontrolling growth and sensitivity to therapy-implications forcancer and agingrdquo Aging vol 3 no 3 pp 192ndash222 2011

[33] W Kolch ldquoMeaningful relationships the regulation of theRasRafMEKERK pathway by protein interactionsrdquo Biochem-ical Journal vol 351 no 2 pp 289ndash305 2000

[34] D Matallanas M Birtwistle D Romano et al ldquoRaf familykinases old dogs have learned new tricksrdquo Genes and Cancervol 2 no 3 pp 232ndash260 2011

[35] C S Mason C J Springer R G Cooper G Superti-Furga C JMarshall and RMarais ldquoSerine and tyrosine phosphorylationscooperate in Raf-1 but not B-Raf activationrdquo The EMBOJournal vol 18 no 8 pp 2137ndash2148 1999

[36] R J Fiddes P W Janes S P Sivertsen R L Sutherland EA Musgrove and R J Daly ldquoInhibition of the MAP kinasecascade blocks heregulin-induced cell cycle progression in T-47D human breast cancer cellsrdquo Oncogene vol 16 no 21 pp2803ndash2813 1998

[37] S T Eblen J K SlackM JWeber andA D Catling ldquoRac-PAKsignaling stimulates extracellular signal-regulated kinase (ERK)activation by regulating formation of MEK1-ERK complexesrdquoMolecular and Cellular Biology vol 22 no 17 pp 6023ndash60332002

[38] M Cargnello and P P Roux ldquoActivation and function ofthe MAPKs and their substrates the MAPK-activated proteinkinasesrdquo Microbiology and Molecular Biology Reviews vol 75no 1 pp 50ndash83 2011

[39] A V Khokhlatchev B Canagarajah J Wilsbacher et al ldquoPhos-phorylation of theMAP kinase ERK2 promotes its homodimer-ization and nuclear translocationrdquo Cell vol 93 no 4 pp 605ndash615 1998

[40] R H Chen C Sarnecki and J Blenis ldquoNuclear localization andregulation of erk- and rsk-encoded protein kinasesrdquoMolecularand Cellular Biology vol 12 no 3 pp 915ndash927 1992

[41] S Traverse K Seedorf H Paterson C J Marshall P Cohenand A Ullrich ldquoEGF triggers neuronal differentiation of PC12cells that overexpress the EGF receptorrdquo Current Biology vol 4no 8 pp 694ndash701 1994

[42] T T Nguyen J C Scimeca C Filloux P Peraldi J L Car-pentier and E Van Obberghen ldquoCo-regulation of the mitogen-activated protein kinase extracellular signal-regulated kinase 1

and the 90-kDa ribosomal S6 kinase in PC12 cells Distincteffects of the neurotrophic factor nerve growth factor and themitogenic factor epidermal growth factorrdquo Journal of BiologicalChemistry vol 268 no 13 pp 9803ndash9810 1993

[43] I Dikic J Schlessinger and I Lax ldquoPC12 cells overexpressingthe insulin receptor undergo insulin-dependent neuronal dif-ferentiationrdquo Current Biology vol 4 no 8 pp 702ndash708 1994

[44] M H Shen Z R Li X R Xiang W M Niu and C B ZhangldquoEffect of electro-acupuncture on ERK protein expression ofMCAO ratsrdquo Journal of Clinical Acupuncture and Moxibustionvol 23 no 7 pp 62ndash63 2007

[45] M H Shen Z R Li X R Xiang W M Niu and C BZhang ldquoInvolvement of extracellular regulated protein kinasesignal transduction pathway in electroacupuncture-inducedimprovement of ischemic cerebral injury in rats with cerebralischemia reperfusionrdquo Acupuncture Research vol 32 no 6 pp368ndash372 2007

[46] Z B Yang J Yan and S X Yi ldquoUp-regulation of the ERK phos-phorylation in gastric mucosal cell by the serum of rats treatedwith electroacupuncture at stomach meridian acupointsrdquo Basicamp Clinical Medicine vol 29 no 2 pp 135ndash138 2009

[47] C L Wang S X Wang and W Gong ldquoChanges in phos-phatized ERK in the spinal dorsal horn in adjuvant arthritisrats during electroacupuncture of Jiaji points for analgesiardquoShanghai Journal of Acupuncture and Moxibustion vol 24 no12 pp 33ndash35 2005

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Evidence-Based Complementary and Alternative Medicine

0

2

4

Ras m

RNA

leve

l (

)

6

8

10

12 17

25

21

NC MC MM EA HSP

lowast

lowast

Figure 5 The Ras mRNA levels in the rat colon NC normalcontrol group MC model control group MM mild moxibustionEA electroacupuncture and HSM herb-separated moxibustionlowast119875 lt 001 versus NC 119875 lt 005 versus MC

0100200300400500600700800900

NC MC MM EA HSP

Raf-1

pro

tein

(IO

D)

lowast

998771998771 998771

Figure 6 The IOD of Raf-1 protein expression in rat colonNC normal control group MC model control group MM mildmoxibustion EA electroacupuncture and HSM herb-separatedmoxibustion lowast119875 lt 001 versus NC 119875 lt 005 119875 lt 001 versusMC 998771998771119875 lt 005 998771119875 lt 001 versus EA

34 The Raf-1 Expression in the Colonic Mucosa of Rat Asshown in Figures 6 and 7 the Raf-1 protein expression inthe colonic tissue of the MC group was significantly higherthan in the NC group (119875 lt 001) and that the Raf-1 proteinexpression in the colon was reduced to varying degrees afteracupuncture treatment (119875 lt 005 or 119875 lt 005)

The results in Figure 8 show that the Raf-1 mRNAlevels in the MC group were significantly higher than in theNC group (119875 lt 001) and that MM and HSM treatmentssignificantly reduced rat colon Raf-1 mRNA levels (119875 lt 005)whereas EA treatment did not significantly affect rat colonRaf-1 mRNA levels

35 The MEK-1 Expression in the Colonic Mucosa of Rat Asshown in Figures 9 and 10 the MEK-1 protein expression inthe colonic tissue of the MC group was significantly higherthan in NC rats (119875 lt 001) and MEK-1 protein expressionwas significantly reduced in each group after acupuncturetreatment (119875 lt 001) although the levels were still higherthan normal levels (119875 lt 001)

The results in Figure 11 show that theMEK-1mRNA levelsin the model group were significantly higher than those inthe NC group (119875 lt 001) and that MM HSM and EAtreatment reduced MEK-1 mRNA levels although there was

no statistically significant difference between the treatmentand no treatment groups or among the treatment groups

36 The ERK-12 Expression in the Colonic Mucosa of Rat Asshown in Figures 12 and 13 the phosphorylation of ERK-12significantly increased in the MC group compared with theNC group (119875 lt 001) and that the levels of phosphorylatedERK-12 proteins were significantly reduced after the MMand HSM treatments (119875 lt 001)

The results in Figure 14 show that the ERK-12 mRNAexpression in the MC group was significantly higher thanthat in the NC group (119875 lt 001) and that ERK-12 mRNAexpression was significantly lower in the MM group afteracupuncture treatment compared with the MC group (119875 lt005)

4 Discussion

Extracellular signal-regulated kinase (ERK) a member ofthe mitogen-activated protein kinase (MAPK) family playsan important biological role in transmitting signals fromsurface receptors to the nucleus The ERK12 pathway is themost thoroughly researched pathway in the ERK family andplays a role in a wide range of cellular functions includ-ing regulating meiosis mitosis and anaphase processes indifferent cells A variety of stimulating factors such as growthfactors cytokines viruses G protein-coupled receptor lig-ands and oncogenes can activate the ERK12 pathway [18]The ERK signaling pathway is a Ras-Raf-MEK-ERK path-way Phosphorylation-activated ERK12 translocates fromthe cytoplasm to the nucleus and thereby mediates thetranscriptional activation of Elk-1 ATF NF-120581B AP-1 and c-fos which in turn regulate cell proliferation and differentia-tion the maintenance of cell shape the construction of thecytoskeleton apoptosis and the malignant transformation ofcells among other biological functions

One of the features of CD is the transformation of smoothmuscle cells into fibroblasts andor myofibroblasts [8] Thisabundance of fibroblasts and myofibroblasts increases theexpression of type I collagen mRNA and produces collagentissue within the muscle [19] There are a large number offibroblast cells and type I III IV and V collagen transcriptsin the small intestines of CD patients [20] which producemore ECM including collagen elastin and glycoproteinsthat are deposited intramurally to produce a thickening of theintestinal wall [10] Based on a large number of experimentalreports the ERK-12 signaling pathway appears to participatein myofibroblast proliferation differentiation and collagenproduction [10 11 21ndash25] extracellular matrix synthesis [2627] smooth muscle cell proliferation and apoptosis [28]and the migration of vascular smooth muscle cells [29]Inhibiting the expression and activity of ERK can inhibit theproliferation of vascular smooth muscle cells [30] to reducethe accumulation of ECM [31] Therefore it is inferred thatthe ERK-12 signaling pathway may induce the activationand proliferation of CD intestinal myofibroblasts and theirtransformation intomuscle fiber cells Additionally the ERK-12 signaling pathway may regulate the proliferation andapoptosis of smooth muscle cells

Evidence-Based Complementary and Alternative Medicine 7

(a) (b)

(c) (d)

(e)

Figure 7 Expression of Raf-1 protein in the colonic mucosa of rat The Raf-1 protein was expressed in epithelial cells in the colonic mucosaof rats EnVision Plus method times200 (a) Normal (b) Crohnrsquos disease (c) mild moxibustion (d) electroacupuncture and (e) herb-separatedmoxibustion

005

115

225

335

4

NC MC MM EA HSP

Raf-1

mRN

A le

vels

()

lowast

Figure 8 The Raf-1 mRNA levels in the rat colon NC normalcontrol group MC model control group MM mild moxibustionEA electroacupuncture and HSM herb-separated moxibustionlowast119875 lt 001 versus NC 119875 lt 005 versus MC

050

100150200250300350400450500

NC MC MM EA HSP

MEK

-1 p

rote

in (I

OD

)

lowast

Figure 9 The IOD of MEK-1 protein expression in the rat colonNC normal control group MC model control group MM mildmoxibustion EA electroacupuncture and HSM herb-separatedmoxibustion lowast119875 lt 001 versus NC 119875 lt 001 versus MC

8 Evidence-Based Complementary and Alternative Medicine

(a) (b)

(c) (d)

(e)

Figure 10 Expression of MEK-1 protein in the colonic mucosa of rat The MEK-1 protein was expressed in epithelial cells in the colonicmucosa of rats EnVision Plus method times200 (a) Normal (b) Crohnrsquos disease (c) mild moxibustion (d) electroacupuncture and (e) herb-separated moxibustion

As an upstream protein in the Raf-MEK-ERK pathwayRas is the product of the proto-oncogene c-ras A variety ofstimulating factors particularly growth factors (eg PDGFEGF TNF and PKC activators as well as Src family members)can activate Ras which further activates Raf-1 ActivatedRaf-1 then activates MEK and ERK downstream [32 33] toinitiate the MAPK pathway Raf-1 protein kinase is widelyexpressed in various types of cells [34] Ras and Raf-1 oftenexist in the form of complexes Ras the upstream activatorprotein can translocate Raf from the cytoplasm to theplasma membrane where the latter is activated Howeverthe mechanism by which Raf is activated remains unclearalthough it may include phosphorylation [35] All that isknown is that Raf activation is related to its phosphorylation

on SerThr residues [33] Recent studies have shown thatRaf-1 protein kinase may be a point of crosstalk with othersignaling pathways This protein may participate in a varietyof biological processes in signal transduction including itsown regulation by interactingwith other signalingmoleculesor via reciprocal regulation

The MAPK kinases include MEK1 (MKK1) and MEK2(MKK2)MEK1 andMEK2 are the central links in theMAPKpathway The activated upstream Raf-1 protein kinase cancombine with MEK1 and 2 and doubly phosphorylate Tyrand Thr its two regulatory sites to activate ERK [36] MEKsare not only ERK activators but may also relocalize theactivated ERKs to the nucleus or other active sites to furtherphosphorylate downstream substrates [37]

Evidence-Based Complementary and Alternative Medicine 9

0

2

1

3

5

4

6

19

NC MC MM EA HSP

MEK

-1 m

RNA

leve

ls (

)

lowast

Figure 11 The MEK-1 mRNA levels in the rat colon NC normalcontrol group MC model control group MM mild moxibustionEA electroacupuncture and HSM herb-separated moxibustionlowast119875 lt 001 versus NC

0200400600800

100012001400

NC MC MM EA HSPPhos

phor

ylat

ed E

RK-1

2 (I

OD

)

lowast

Figure 12 The IOD of phosphorylated ERK-12 levels in therat colon NC normal control group MC model control groupMM mild moxibustion EA electroacupuncture and HSM herb-separated moxibustion lowast119875 lt 001 versus NC 119875 lt 005 119875 lt 001versus MC

The upstream kinase MEK1 is a dual-specificity enzymeActivatedMEK1 can phosphorylate ERK1 and ERK2There isevidence that the Tyr residues of ERK1 and 2 are phosphory-lated before the Thr residues both intracellularly and extra-cellularly [38] ERK translocates into the nucleus by forminga homodimer of phosphorylated ERK and phosphorylatedor unphosphorylated ERK [39] ERK is evenly distributedin quiescent cells but when the cells are stimulated byserum persistently activated ERK can partially translocateinto the nucleus whereas transiently activated ERK cannotrelocalize Activated ERKs accumulate in the nucleus andthen phosphorylate the corresponding transcription factorswhich drive expression of specific gene products Becauseof the difference in nuclear localization transiently activated(or low-activity) ERK and persistently activated (or high-activity) ERK cause cells to express genes at a different qualityor quantity resulting in different biological effects [40ndash43]

The ERK1-ERK2-MAPK pathway incorporates a mech-anism for inactivation after activation which can preventexcessive activation-induced adverse consequences At thesame time the inactivated kinase is prepared for the nextactivation The mechanisms of inhibition of this pathwayinclude the following (1) the activation of ERK12-MAPK is

the result of the phosphorylation cascade and can be inac-tivated by dephosphorylation by the corresponding phos-phatases (2) Raf-1 is the direct substrate of PKA which canphosphorylate Ser43 of c-Raf an inhibitory phosphorylationsite in the N-terminus of Raf that interacts with Ras andthe phosphorylation of this site can inhibit the bindingbetween RasGTP and the N-terminus of Raf which blocksthe activation of ERK (3) the phosphatase PP2 can inactivateMEK and ERK via dephosphorylation (4) activated ERKphosphorylates Sos and Raf while ERK dephosphorylationinactivates them thereby generates negative feedback regula-tion of the pathwayTherefore the study of themechanism bywhich acupuncture regulates the expression of ERKupstreamreceptors the phosphorylation of ERK cascade members thetargets of downstream signal transduction and the potentialimpact of other signaling pathways on this pathway is neces-sary to confirm acupuncture as an antifibrotic treatment

Our experiments studied the regulation of Ras RafMEKand ERK protein and gene expression by acupuncture inCD intestinal fibrosis in the rat colon Our results showthat the mRNA levels of Ras Raf-1 MEK-1 and ERK-12 were significantly increased in CD rat intestinal tissueand that the expression of p-Ras Raf-1 MEK-1 and p-ERK-12 proteins in intestinal epithelial cells were also sig-nificantly increased These results suggest that the activityof the ERK pathway in CD intestinal fibrosis process wasaltered Medicine-separated moxibustion mild moxibustionand electroacupuncture treatments could effectively down-regulate the expression of p-Ras Raf-1 MEK-1 and p-ERK-12 but different inhibitory effects on Ras Raf-1 MEK-1and ERK-12 mRNA levels were shown in ERK pathwayactivity changes In this discussion we propose the following(1) medicine-separated moxibustion mild moxibustion andelectroacupuncture can inhibit CD intestinal fibrosis byregulating ERK pathway activity and (2) the acupuncture-induced inhibition of p-Ras Raf-1 MEK-1 and p-ERK-12 expression may be the key elements in the treatmentrsquosinhibition of CD intestinal fibrosis At present most reportsregarding the role of acupuncture in the regulation of theERK signaling pathway have focused on EA such as EAmodel rats After EA on the Dazhui (DU14) and Baihui(DU20) acupuncture points the number of ERK-positivestriatal cells increased significantly in the cerebral cortex andERK protein expression was increased in rats with damagedneurons after cerebral ischemia and reperfusion thereforeEA can increase ERK pathway activity [44 45] After the EAtreatment of gastric mucosa-injured rats the phosphoryla-tion levels of ERK in gastric mucosal cells from extractedserum were significantly increased and were significantlydifferent between the serum of the normal rats and that ofthe model rats These observations suggest that ERK maybe involved in signal transduction in the EA stimulation atacupoints of stomach meridian of foot yangming repairinggastric mucosal injury [46] and that EA had an analgesiceffect by downregulating p-ERK expression levels in thedorsal horn of the spinal cord of rats with adjuvant arthritis[47] The previous reports have demonstrated that EA canregulate the ERK signal transduction pathway The results ofour experiments show that medicine-separated moxibustion

10 Evidence-Based Complementary and Alternative Medicine

(a) (b)

(c) (d)

(e)

Figure 13 Expression of phosphorylated ERK-12 protein in the colonicmucosa of ratThe phosphorylation of ERK-12 proteinwas expressedin epithelial cells and lymphocyte in the colonic mucosa of rats EnVision Plus method times200 (a) Normal (b) Crohnrsquos disease (c) mildmoxibustion (d) electroacupuncture and (e) herb-separated moxibustion

005

115

225

335

4

NC MC MM EA HSP

ERK-

12

mRN

A le

vels

()

lowast

Figure 14 The ERK-12 mRNA levels in the rat colon NC normalcontrol group MC model control group MM mild moxibustionEA electroacupuncture and HSM herb-separated moxibustionlowast119875 lt 001 versus NC 119875 lt 005 versus MC

andmildmoxibustion can also regulate the ERK signal trans-duction pathway and inhibit Ras and Raf-1 better than EAThe functional mechanisms underlying these observationsneed to be explored further

Abbreviations

CD Crohnrsquos diseaseNC Normal control groupMC Model control groupEA ElectroacupunctureMM Mild moxibustionHSM Herb-separated moxibustionST25 Tianshu acupoint

Evidence-Based Complementary and Alternative Medicine 11

RN6 Qihai acupointERK Extracellular signal-regulated kinaseMAPK Mitogen-activated protein kinaseTNBS 246-Trinitrobenzene sulfonic acid

Acknowledgments

The authors thank Professor Shuhui Jiang for technical assis-tance and the support of the National Natural Science Foun-dation of China (no 81173331) and National Basic ResearchProgram of China (973 program no 2009CB522900)Xiaomei Wang and Yuan Lu have the same right

References

[1] G Manning D B Whyte R Martinez T Hunter and SSudarsanam ldquoThe protein kinase complement of the humangenomerdquo Science vol 298 no 5600 pp 1912ndash1934 2002

[2] C Mowat A Cole A Windsor et al ldquoGuidelines for themanagement of inflammatory bowel disease in adultsrdquoGut vol60 no 5 pp 571ndash607 2011

[3] Inflammatory Bowel Disease Co-operation Group ChineseSociety of Gastroenterology ldquoChinese consensus on diagnosisand treatment standard of inflammatory bowel diseaserdquoChineseJournal of Gastroenterology vol 12 no 8 pp 490ndash494 2007

[4] J J Zheng X S Zhu Z Huangfu Z X Gao Z R Guo andZ Wang ldquoCrohnrsquos disease in mainland China a systematicanalysis of 50 years of researchrdquo Chinese Journal of DigestiveDiseases vol 6 no 4 pp 175ndash181 2005

[5] M Dai and B M Li ldquoResearch progress in mechanisms ofintestinal fibrosis in Crohnrsquos diseaserdquo Shijie Huaren XiaohuaZazhi vol 17 no 11 pp 1117ndash1121 2009

[6] O Grip J Malm B Veress A Bjartell S Lindgren and AEgesten ldquoIncreased presence of cells containing transforminggrowth factor alpha (TGF-120572) in ulcerative colitis both duringactive inflammation and in remissionrdquo European Journal ofGastroenterology and Hepatology vol 12 no 7 pp 761ndash7662000

[7] I E Koutroubakis E Petinaki P Dimoulios et al ldquoSerumlaminin and collagen IV in inflammatory bowel diseaserdquoJournal of Clinical Pathology vol 56 no 11 pp 817ndash820 2003

[8] S Speca I Giusti F Rieder and G Latella ldquoCellular andmolecular mechanisms of intestinal fibrosisrdquo World Journal ofGastroenterology vol 18 no 28 pp 3635ndash3661 2012

[9] Y F Song J H Hong and L M Xu ldquoTGF-120573 1ERK pathwaysand its inhibitors and fibrosisrdquo Chinese Journal of ClinicalHepatology vol 21 no 2 pp 125ndash127 2005

[10] H C Huang M Yang J Z Li and H Y Wang ldquoConnectivetissue growth factor promotes the proliferation ofmyofibroblastthrough Erk-12 signaling pathwayrdquoNational Medical Journal ofChina vol 85 no 19 pp 1322ndash1326 2005

[11] L P Guo H C Huang and J Z Li ldquoHypoxia inducesmyofibroblast formation and stimulates production of collagenI in myofibroblasts through ERK12 pathwayrdquo Chinese Journalof Pathophysiology vol 24 no 12 pp 2428ndash2432 2008

[12] H R Liu X P Ma T P Zhao and H G Wu ldquoThe adjustmentof acupuncture on colon collagen synthesis and TGF-120573mRNAexpression in Crohnrsquos disease ratrdquo Journal of Anhui TraditionalChinese Medical College vol 24 no 4 pp 25ndash30 2005

[13] X P Ma C P An H G Wu et al ldquoHerbs-partitionedmoxibustion and electro-acupuncture effects on transforming

growth factor-beta 1 connective tissue growth factor collagentype I and fibronectin expression in the colon of Crohnrsquo sdisease ratsrdquo Journal of Clinical Rehabilitative Tissue EngineeringResearch vol 12 no 20 pp 3853ndash3858 2008

[14] G P Morris P L Beck M S Herridge W T Depew M RSzewczuk and J LWallace ldquoHapten-inducedmodel of chronicinflammation and ulceration in the rat colonrdquoGastroenterologyvol 96 no 3 pp 795ndash803 1989

[15] TheMinistry of Science andTechnology of the Peoplersquos Republicof China Guidance Suggestions for the Care and Use ofLaboratory Animals 2006

[16] J D Butzner R Parmar C J Bell andVDalal ldquoButyrate enematherapy stimulates mucosal repair in experimental colitis in theratrdquo Gut vol 38 no 4 pp 568ndash573 1996

[17] X M Wang Y Lu L Y Wu et al ldquoMoxibustion inhibitsinterleukin-12 and tumor necrosis factor alpha and modulatesintestinal flora in rat with ulcerative colitisrdquo World Journal ofGastroenterology vol 18 no 46 pp 6826ndash6835 2012

[18] J B Pucilowska K K McNaughton N K Mohapatra et alldquoIGF-I and procollagen 1205721(I) are coexpressed in a subset ofmesenchymal cells in active Crohnrsquos diseaserdquo American Journalof Physiology vol 279 no 6 pp G1307ndashG1322 2000

[19] M Gharaee-Kermani B Hu S H Phan and M R GyetkoldquoRecent advances in molecular targets and treatment of idio-pathic pulmonary fibrosis focus on TGF120573 signaling and themyofibroblastrdquo Current Medicinal Chemistry vol 16 no 11 pp1400ndash1417 2009

[20] E M Zimmermann L Li Y T Hou M Cannon G MChristman andKN Bitar ldquoIGF-I induces collagen and IGFBP-5 mRNA in rat intestinal smooth musclerdquo American Journal ofPhysiology vol 273 no 4 pp G875ndashG882 1997

[21] L J ZhangQ Li P Chen et al ldquoMediating effect of extracellularsignal-regulated kinase 12 in PDGF-induced proliferation andcollagen synthesis of cultured rat cardiac fibroblastsrdquo ChineseJournal of Anatomy vol 13 no 4 p 223 2007

[22] M Tang W Zhang H Lin H Jiang H Dai and Y ZhangldquoHigh glucose promotes the production of collagen types Iand III by cardiac fibroblasts through a pathway dependenton extracellular-signal-regulated kinase 12rdquo Molecular andCellular Biochemistry vol 301 no 1-2 pp 109ndash114 2007

[23] H Y Dai Z M Ge and Y H Li ldquoEffect of urotensin II on pro-liferative potential and phosphorylation of extracellular signal-regulated kinase 12 adventitial fibroblasts from spontaneouslyhypertensive ratrdquoActaAcademiaeMedicinae Sinicae vol 28 no6 pp 776ndash780 2006

[24] I Fuentes-Calvo A M Blazquez-Medela N Eleno E SantosJ M Lopez-Novoa and C Martınez-Salgado ldquoH-Ras isoformmodulates extracellular matrix synthesis proliferation andmigration in fibroblastsrdquo American Journal of Physiology vol302 no 4 pp C686ndashC697 2012

[25] Y W Ma G T Bai and M X Zhang ldquoStudies on signal trans-duction pathway induced by fibronectin in human embryoniclung fibroblastsrdquo Shanxi Medical Journal vol 38 no 7 pp 587ndash589 2009

[26] J H Jang and C P Chung ldquoFibronectin-mediated adhesionrescues cell cycle arrest induced by fibroblast growth factor-1 bydecreased expression of P21CIPWAF in human chondrocytesrdquoIn Vitro Cellular and Developmental Biology vol 41 no 5-6 pp126ndash129 2005

[27] B Zhu Y J Wang C F Zhu et al ldquoTriptolide inhibitsextracellularmatrix protein synthesis by suppressing the Smad2

12 Evidence-Based Complementary and Alternative Medicine

but not the MAPK pathway in TGF-1205731-stimulated NRK-49Fcellsrdquo Nephrology Dialysis Transplantation vol 25 no 10 pp3180ndash3191 2010

[28] J Bai X S Liu Y J Xu M Xie and W Ni ldquoEffects ofextracellular signal regulated kinase signaling pathway on cellcycle of airway smooth muscle cells in asthmatic ratsrdquo ChineseJournal of Respiratory and Critical Care Medicine vol 9 no 1pp 23ndash27 2010

[29] S Matsusaka and I Wakabayashi ldquoEnhancement of vascu-lar smooth muscle cell migration by urotensin IIrdquo Naunyn-Schmiedebergrsquos Archives of Pharmacology vol 373 no 5 pp 381ndash386 2006

[30] Y Chen Z Dai Y M Liu et al ldquoInhibitory effects ofCGRP on vascular smooth muscle cell proliferation role ofcaveolaecaveolin-1ERK12 signal pathwayrdquo Progress in Bio-chemistry and Biophysics vol 40 no 5 pp 445ndash453 2013

[31] W L Wu A Q Zhang Q Yin and W H Gan ldquoEffectsof ERK signal pathway on extracellular matrix synthesis ofrat mesangial cells treated with PDGF-BBrdquo ACTA UniversityMedicinalis Nanjing vol 27 no 10 pp 1126ndash1129 2007

[32] L S Steelman W H Chappell S L Abrams et al ldquoRoles ofthe RafMEKERK and PI3KPTENAktmTOR pathways incontrolling growth and sensitivity to therapy-implications forcancer and agingrdquo Aging vol 3 no 3 pp 192ndash222 2011

[33] W Kolch ldquoMeaningful relationships the regulation of theRasRafMEKERK pathway by protein interactionsrdquo Biochem-ical Journal vol 351 no 2 pp 289ndash305 2000

[34] D Matallanas M Birtwistle D Romano et al ldquoRaf familykinases old dogs have learned new tricksrdquo Genes and Cancervol 2 no 3 pp 232ndash260 2011

[35] C S Mason C J Springer R G Cooper G Superti-Furga C JMarshall and RMarais ldquoSerine and tyrosine phosphorylationscooperate in Raf-1 but not B-Raf activationrdquo The EMBOJournal vol 18 no 8 pp 2137ndash2148 1999

[36] R J Fiddes P W Janes S P Sivertsen R L Sutherland EA Musgrove and R J Daly ldquoInhibition of the MAP kinasecascade blocks heregulin-induced cell cycle progression in T-47D human breast cancer cellsrdquo Oncogene vol 16 no 21 pp2803ndash2813 1998

[37] S T Eblen J K SlackM JWeber andA D Catling ldquoRac-PAKsignaling stimulates extracellular signal-regulated kinase (ERK)activation by regulating formation of MEK1-ERK complexesrdquoMolecular and Cellular Biology vol 22 no 17 pp 6023ndash60332002

[38] M Cargnello and P P Roux ldquoActivation and function ofthe MAPKs and their substrates the MAPK-activated proteinkinasesrdquo Microbiology and Molecular Biology Reviews vol 75no 1 pp 50ndash83 2011

[39] A V Khokhlatchev B Canagarajah J Wilsbacher et al ldquoPhos-phorylation of theMAP kinase ERK2 promotes its homodimer-ization and nuclear translocationrdquo Cell vol 93 no 4 pp 605ndash615 1998

[40] R H Chen C Sarnecki and J Blenis ldquoNuclear localization andregulation of erk- and rsk-encoded protein kinasesrdquoMolecularand Cellular Biology vol 12 no 3 pp 915ndash927 1992

[41] S Traverse K Seedorf H Paterson C J Marshall P Cohenand A Ullrich ldquoEGF triggers neuronal differentiation of PC12cells that overexpress the EGF receptorrdquo Current Biology vol 4no 8 pp 694ndash701 1994

[42] T T Nguyen J C Scimeca C Filloux P Peraldi J L Car-pentier and E Van Obberghen ldquoCo-regulation of the mitogen-activated protein kinase extracellular signal-regulated kinase 1

and the 90-kDa ribosomal S6 kinase in PC12 cells Distincteffects of the neurotrophic factor nerve growth factor and themitogenic factor epidermal growth factorrdquo Journal of BiologicalChemistry vol 268 no 13 pp 9803ndash9810 1993

[43] I Dikic J Schlessinger and I Lax ldquoPC12 cells overexpressingthe insulin receptor undergo insulin-dependent neuronal dif-ferentiationrdquo Current Biology vol 4 no 8 pp 702ndash708 1994

[44] M H Shen Z R Li X R Xiang W M Niu and C B ZhangldquoEffect of electro-acupuncture on ERK protein expression ofMCAO ratsrdquo Journal of Clinical Acupuncture and Moxibustionvol 23 no 7 pp 62ndash63 2007

[45] M H Shen Z R Li X R Xiang W M Niu and C BZhang ldquoInvolvement of extracellular regulated protein kinasesignal transduction pathway in electroacupuncture-inducedimprovement of ischemic cerebral injury in rats with cerebralischemia reperfusionrdquo Acupuncture Research vol 32 no 6 pp368ndash372 2007

[46] Z B Yang J Yan and S X Yi ldquoUp-regulation of the ERK phos-phorylation in gastric mucosal cell by the serum of rats treatedwith electroacupuncture at stomach meridian acupointsrdquo Basicamp Clinical Medicine vol 29 no 2 pp 135ndash138 2009

[47] C L Wang S X Wang and W Gong ldquoChanges in phos-phatized ERK in the spinal dorsal horn in adjuvant arthritisrats during electroacupuncture of Jiaji points for analgesiardquoShanghai Journal of Acupuncture and Moxibustion vol 24 no12 pp 33ndash35 2005

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 7

(a) (b)

(c) (d)

(e)

Figure 7 Expression of Raf-1 protein in the colonic mucosa of rat The Raf-1 protein was expressed in epithelial cells in the colonic mucosaof rats EnVision Plus method times200 (a) Normal (b) Crohnrsquos disease (c) mild moxibustion (d) electroacupuncture and (e) herb-separatedmoxibustion

005

115

225

335

4

NC MC MM EA HSP

Raf-1

mRN

A le

vels

()

lowast

Figure 8 The Raf-1 mRNA levels in the rat colon NC normalcontrol group MC model control group MM mild moxibustionEA electroacupuncture and HSM herb-separated moxibustionlowast119875 lt 001 versus NC 119875 lt 005 versus MC

050

100150200250300350400450500

NC MC MM EA HSP

MEK

-1 p

rote

in (I

OD

)

lowast

Figure 9 The IOD of MEK-1 protein expression in the rat colonNC normal control group MC model control group MM mildmoxibustion EA electroacupuncture and HSM herb-separatedmoxibustion lowast119875 lt 001 versus NC 119875 lt 001 versus MC

8 Evidence-Based Complementary and Alternative Medicine

(a) (b)

(c) (d)

(e)

Figure 10 Expression of MEK-1 protein in the colonic mucosa of rat The MEK-1 protein was expressed in epithelial cells in the colonicmucosa of rats EnVision Plus method times200 (a) Normal (b) Crohnrsquos disease (c) mild moxibustion (d) electroacupuncture and (e) herb-separated moxibustion

As an upstream protein in the Raf-MEK-ERK pathwayRas is the product of the proto-oncogene c-ras A variety ofstimulating factors particularly growth factors (eg PDGFEGF TNF and PKC activators as well as Src family members)can activate Ras which further activates Raf-1 ActivatedRaf-1 then activates MEK and ERK downstream [32 33] toinitiate the MAPK pathway Raf-1 protein kinase is widelyexpressed in various types of cells [34] Ras and Raf-1 oftenexist in the form of complexes Ras the upstream activatorprotein can translocate Raf from the cytoplasm to theplasma membrane where the latter is activated Howeverthe mechanism by which Raf is activated remains unclearalthough it may include phosphorylation [35] All that isknown is that Raf activation is related to its phosphorylation

on SerThr residues [33] Recent studies have shown thatRaf-1 protein kinase may be a point of crosstalk with othersignaling pathways This protein may participate in a varietyof biological processes in signal transduction including itsown regulation by interactingwith other signalingmoleculesor via reciprocal regulation

The MAPK kinases include MEK1 (MKK1) and MEK2(MKK2)MEK1 andMEK2 are the central links in theMAPKpathway The activated upstream Raf-1 protein kinase cancombine with MEK1 and 2 and doubly phosphorylate Tyrand Thr its two regulatory sites to activate ERK [36] MEKsare not only ERK activators but may also relocalize theactivated ERKs to the nucleus or other active sites to furtherphosphorylate downstream substrates [37]

Evidence-Based Complementary and Alternative Medicine 9

0

2

1

3

5

4

6

19

NC MC MM EA HSP

MEK

-1 m

RNA

leve

ls (

)

lowast

Figure 11 The MEK-1 mRNA levels in the rat colon NC normalcontrol group MC model control group MM mild moxibustionEA electroacupuncture and HSM herb-separated moxibustionlowast119875 lt 001 versus NC

0200400600800

100012001400

NC MC MM EA HSPPhos

phor

ylat

ed E

RK-1

2 (I

OD

)

lowast

Figure 12 The IOD of phosphorylated ERK-12 levels in therat colon NC normal control group MC model control groupMM mild moxibustion EA electroacupuncture and HSM herb-separated moxibustion lowast119875 lt 001 versus NC 119875 lt 005 119875 lt 001versus MC

The upstream kinase MEK1 is a dual-specificity enzymeActivatedMEK1 can phosphorylate ERK1 and ERK2There isevidence that the Tyr residues of ERK1 and 2 are phosphory-lated before the Thr residues both intracellularly and extra-cellularly [38] ERK translocates into the nucleus by forminga homodimer of phosphorylated ERK and phosphorylatedor unphosphorylated ERK [39] ERK is evenly distributedin quiescent cells but when the cells are stimulated byserum persistently activated ERK can partially translocateinto the nucleus whereas transiently activated ERK cannotrelocalize Activated ERKs accumulate in the nucleus andthen phosphorylate the corresponding transcription factorswhich drive expression of specific gene products Becauseof the difference in nuclear localization transiently activated(or low-activity) ERK and persistently activated (or high-activity) ERK cause cells to express genes at a different qualityor quantity resulting in different biological effects [40ndash43]

The ERK1-ERK2-MAPK pathway incorporates a mech-anism for inactivation after activation which can preventexcessive activation-induced adverse consequences At thesame time the inactivated kinase is prepared for the nextactivation The mechanisms of inhibition of this pathwayinclude the following (1) the activation of ERK12-MAPK is

the result of the phosphorylation cascade and can be inac-tivated by dephosphorylation by the corresponding phos-phatases (2) Raf-1 is the direct substrate of PKA which canphosphorylate Ser43 of c-Raf an inhibitory phosphorylationsite in the N-terminus of Raf that interacts with Ras andthe phosphorylation of this site can inhibit the bindingbetween RasGTP and the N-terminus of Raf which blocksthe activation of ERK (3) the phosphatase PP2 can inactivateMEK and ERK via dephosphorylation (4) activated ERKphosphorylates Sos and Raf while ERK dephosphorylationinactivates them thereby generates negative feedback regula-tion of the pathwayTherefore the study of themechanism bywhich acupuncture regulates the expression of ERKupstreamreceptors the phosphorylation of ERK cascade members thetargets of downstream signal transduction and the potentialimpact of other signaling pathways on this pathway is neces-sary to confirm acupuncture as an antifibrotic treatment

Our experiments studied the regulation of Ras RafMEKand ERK protein and gene expression by acupuncture inCD intestinal fibrosis in the rat colon Our results showthat the mRNA levels of Ras Raf-1 MEK-1 and ERK-12 were significantly increased in CD rat intestinal tissueand that the expression of p-Ras Raf-1 MEK-1 and p-ERK-12 proteins in intestinal epithelial cells were also sig-nificantly increased These results suggest that the activityof the ERK pathway in CD intestinal fibrosis process wasaltered Medicine-separated moxibustion mild moxibustionand electroacupuncture treatments could effectively down-regulate the expression of p-Ras Raf-1 MEK-1 and p-ERK-12 but different inhibitory effects on Ras Raf-1 MEK-1and ERK-12 mRNA levels were shown in ERK pathwayactivity changes In this discussion we propose the following(1) medicine-separated moxibustion mild moxibustion andelectroacupuncture can inhibit CD intestinal fibrosis byregulating ERK pathway activity and (2) the acupuncture-induced inhibition of p-Ras Raf-1 MEK-1 and p-ERK-12 expression may be the key elements in the treatmentrsquosinhibition of CD intestinal fibrosis At present most reportsregarding the role of acupuncture in the regulation of theERK signaling pathway have focused on EA such as EAmodel rats After EA on the Dazhui (DU14) and Baihui(DU20) acupuncture points the number of ERK-positivestriatal cells increased significantly in the cerebral cortex andERK protein expression was increased in rats with damagedneurons after cerebral ischemia and reperfusion thereforeEA can increase ERK pathway activity [44 45] After the EAtreatment of gastric mucosa-injured rats the phosphoryla-tion levels of ERK in gastric mucosal cells from extractedserum were significantly increased and were significantlydifferent between the serum of the normal rats and that ofthe model rats These observations suggest that ERK maybe involved in signal transduction in the EA stimulation atacupoints of stomach meridian of foot yangming repairinggastric mucosal injury [46] and that EA had an analgesiceffect by downregulating p-ERK expression levels in thedorsal horn of the spinal cord of rats with adjuvant arthritis[47] The previous reports have demonstrated that EA canregulate the ERK signal transduction pathway The results ofour experiments show that medicine-separated moxibustion

10 Evidence-Based Complementary and Alternative Medicine

(a) (b)

(c) (d)

(e)

Figure 13 Expression of phosphorylated ERK-12 protein in the colonicmucosa of ratThe phosphorylation of ERK-12 proteinwas expressedin epithelial cells and lymphocyte in the colonic mucosa of rats EnVision Plus method times200 (a) Normal (b) Crohnrsquos disease (c) mildmoxibustion (d) electroacupuncture and (e) herb-separated moxibustion

005

115

225

335

4

NC MC MM EA HSP

ERK-

12

mRN

A le

vels

()

lowast

Figure 14 The ERK-12 mRNA levels in the rat colon NC normalcontrol group MC model control group MM mild moxibustionEA electroacupuncture and HSM herb-separated moxibustionlowast119875 lt 001 versus NC 119875 lt 005 versus MC

andmildmoxibustion can also regulate the ERK signal trans-duction pathway and inhibit Ras and Raf-1 better than EAThe functional mechanisms underlying these observationsneed to be explored further

Abbreviations

CD Crohnrsquos diseaseNC Normal control groupMC Model control groupEA ElectroacupunctureMM Mild moxibustionHSM Herb-separated moxibustionST25 Tianshu acupoint

Evidence-Based Complementary and Alternative Medicine 11

RN6 Qihai acupointERK Extracellular signal-regulated kinaseMAPK Mitogen-activated protein kinaseTNBS 246-Trinitrobenzene sulfonic acid

Acknowledgments

The authors thank Professor Shuhui Jiang for technical assis-tance and the support of the National Natural Science Foun-dation of China (no 81173331) and National Basic ResearchProgram of China (973 program no 2009CB522900)Xiaomei Wang and Yuan Lu have the same right

References

[1] G Manning D B Whyte R Martinez T Hunter and SSudarsanam ldquoThe protein kinase complement of the humangenomerdquo Science vol 298 no 5600 pp 1912ndash1934 2002

[2] C Mowat A Cole A Windsor et al ldquoGuidelines for themanagement of inflammatory bowel disease in adultsrdquoGut vol60 no 5 pp 571ndash607 2011

[3] Inflammatory Bowel Disease Co-operation Group ChineseSociety of Gastroenterology ldquoChinese consensus on diagnosisand treatment standard of inflammatory bowel diseaserdquoChineseJournal of Gastroenterology vol 12 no 8 pp 490ndash494 2007

[4] J J Zheng X S Zhu Z Huangfu Z X Gao Z R Guo andZ Wang ldquoCrohnrsquos disease in mainland China a systematicanalysis of 50 years of researchrdquo Chinese Journal of DigestiveDiseases vol 6 no 4 pp 175ndash181 2005

[5] M Dai and B M Li ldquoResearch progress in mechanisms ofintestinal fibrosis in Crohnrsquos diseaserdquo Shijie Huaren XiaohuaZazhi vol 17 no 11 pp 1117ndash1121 2009

[6] O Grip J Malm B Veress A Bjartell S Lindgren and AEgesten ldquoIncreased presence of cells containing transforminggrowth factor alpha (TGF-120572) in ulcerative colitis both duringactive inflammation and in remissionrdquo European Journal ofGastroenterology and Hepatology vol 12 no 7 pp 761ndash7662000

[7] I E Koutroubakis E Petinaki P Dimoulios et al ldquoSerumlaminin and collagen IV in inflammatory bowel diseaserdquoJournal of Clinical Pathology vol 56 no 11 pp 817ndash820 2003

[8] S Speca I Giusti F Rieder and G Latella ldquoCellular andmolecular mechanisms of intestinal fibrosisrdquo World Journal ofGastroenterology vol 18 no 28 pp 3635ndash3661 2012

[9] Y F Song J H Hong and L M Xu ldquoTGF-120573 1ERK pathwaysand its inhibitors and fibrosisrdquo Chinese Journal of ClinicalHepatology vol 21 no 2 pp 125ndash127 2005

[10] H C Huang M Yang J Z Li and H Y Wang ldquoConnectivetissue growth factor promotes the proliferation ofmyofibroblastthrough Erk-12 signaling pathwayrdquoNational Medical Journal ofChina vol 85 no 19 pp 1322ndash1326 2005

[11] L P Guo H C Huang and J Z Li ldquoHypoxia inducesmyofibroblast formation and stimulates production of collagenI in myofibroblasts through ERK12 pathwayrdquo Chinese Journalof Pathophysiology vol 24 no 12 pp 2428ndash2432 2008

[12] H R Liu X P Ma T P Zhao and H G Wu ldquoThe adjustmentof acupuncture on colon collagen synthesis and TGF-120573mRNAexpression in Crohnrsquos disease ratrdquo Journal of Anhui TraditionalChinese Medical College vol 24 no 4 pp 25ndash30 2005

[13] X P Ma C P An H G Wu et al ldquoHerbs-partitionedmoxibustion and electro-acupuncture effects on transforming

growth factor-beta 1 connective tissue growth factor collagentype I and fibronectin expression in the colon of Crohnrsquo sdisease ratsrdquo Journal of Clinical Rehabilitative Tissue EngineeringResearch vol 12 no 20 pp 3853ndash3858 2008

[14] G P Morris P L Beck M S Herridge W T Depew M RSzewczuk and J LWallace ldquoHapten-inducedmodel of chronicinflammation and ulceration in the rat colonrdquoGastroenterologyvol 96 no 3 pp 795ndash803 1989

[15] TheMinistry of Science andTechnology of the Peoplersquos Republicof China Guidance Suggestions for the Care and Use ofLaboratory Animals 2006

[16] J D Butzner R Parmar C J Bell andVDalal ldquoButyrate enematherapy stimulates mucosal repair in experimental colitis in theratrdquo Gut vol 38 no 4 pp 568ndash573 1996

[17] X M Wang Y Lu L Y Wu et al ldquoMoxibustion inhibitsinterleukin-12 and tumor necrosis factor alpha and modulatesintestinal flora in rat with ulcerative colitisrdquo World Journal ofGastroenterology vol 18 no 46 pp 6826ndash6835 2012

[18] J B Pucilowska K K McNaughton N K Mohapatra et alldquoIGF-I and procollagen 1205721(I) are coexpressed in a subset ofmesenchymal cells in active Crohnrsquos diseaserdquo American Journalof Physiology vol 279 no 6 pp G1307ndashG1322 2000

[19] M Gharaee-Kermani B Hu S H Phan and M R GyetkoldquoRecent advances in molecular targets and treatment of idio-pathic pulmonary fibrosis focus on TGF120573 signaling and themyofibroblastrdquo Current Medicinal Chemistry vol 16 no 11 pp1400ndash1417 2009

[20] E M Zimmermann L Li Y T Hou M Cannon G MChristman andKN Bitar ldquoIGF-I induces collagen and IGFBP-5 mRNA in rat intestinal smooth musclerdquo American Journal ofPhysiology vol 273 no 4 pp G875ndashG882 1997

[21] L J ZhangQ Li P Chen et al ldquoMediating effect of extracellularsignal-regulated kinase 12 in PDGF-induced proliferation andcollagen synthesis of cultured rat cardiac fibroblastsrdquo ChineseJournal of Anatomy vol 13 no 4 p 223 2007

[22] M Tang W Zhang H Lin H Jiang H Dai and Y ZhangldquoHigh glucose promotes the production of collagen types Iand III by cardiac fibroblasts through a pathway dependenton extracellular-signal-regulated kinase 12rdquo Molecular andCellular Biochemistry vol 301 no 1-2 pp 109ndash114 2007

[23] H Y Dai Z M Ge and Y H Li ldquoEffect of urotensin II on pro-liferative potential and phosphorylation of extracellular signal-regulated kinase 12 adventitial fibroblasts from spontaneouslyhypertensive ratrdquoActaAcademiaeMedicinae Sinicae vol 28 no6 pp 776ndash780 2006

[24] I Fuentes-Calvo A M Blazquez-Medela N Eleno E SantosJ M Lopez-Novoa and C Martınez-Salgado ldquoH-Ras isoformmodulates extracellular matrix synthesis proliferation andmigration in fibroblastsrdquo American Journal of Physiology vol302 no 4 pp C686ndashC697 2012

[25] Y W Ma G T Bai and M X Zhang ldquoStudies on signal trans-duction pathway induced by fibronectin in human embryoniclung fibroblastsrdquo Shanxi Medical Journal vol 38 no 7 pp 587ndash589 2009

[26] J H Jang and C P Chung ldquoFibronectin-mediated adhesionrescues cell cycle arrest induced by fibroblast growth factor-1 bydecreased expression of P21CIPWAF in human chondrocytesrdquoIn Vitro Cellular and Developmental Biology vol 41 no 5-6 pp126ndash129 2005

[27] B Zhu Y J Wang C F Zhu et al ldquoTriptolide inhibitsextracellularmatrix protein synthesis by suppressing the Smad2

12 Evidence-Based Complementary and Alternative Medicine

but not the MAPK pathway in TGF-1205731-stimulated NRK-49Fcellsrdquo Nephrology Dialysis Transplantation vol 25 no 10 pp3180ndash3191 2010

[28] J Bai X S Liu Y J Xu M Xie and W Ni ldquoEffects ofextracellular signal regulated kinase signaling pathway on cellcycle of airway smooth muscle cells in asthmatic ratsrdquo ChineseJournal of Respiratory and Critical Care Medicine vol 9 no 1pp 23ndash27 2010

[29] S Matsusaka and I Wakabayashi ldquoEnhancement of vascu-lar smooth muscle cell migration by urotensin IIrdquo Naunyn-Schmiedebergrsquos Archives of Pharmacology vol 373 no 5 pp 381ndash386 2006

[30] Y Chen Z Dai Y M Liu et al ldquoInhibitory effects ofCGRP on vascular smooth muscle cell proliferation role ofcaveolaecaveolin-1ERK12 signal pathwayrdquo Progress in Bio-chemistry and Biophysics vol 40 no 5 pp 445ndash453 2013

[31] W L Wu A Q Zhang Q Yin and W H Gan ldquoEffectsof ERK signal pathway on extracellular matrix synthesis ofrat mesangial cells treated with PDGF-BBrdquo ACTA UniversityMedicinalis Nanjing vol 27 no 10 pp 1126ndash1129 2007

[32] L S Steelman W H Chappell S L Abrams et al ldquoRoles ofthe RafMEKERK and PI3KPTENAktmTOR pathways incontrolling growth and sensitivity to therapy-implications forcancer and agingrdquo Aging vol 3 no 3 pp 192ndash222 2011

[33] W Kolch ldquoMeaningful relationships the regulation of theRasRafMEKERK pathway by protein interactionsrdquo Biochem-ical Journal vol 351 no 2 pp 289ndash305 2000

[34] D Matallanas M Birtwistle D Romano et al ldquoRaf familykinases old dogs have learned new tricksrdquo Genes and Cancervol 2 no 3 pp 232ndash260 2011

[35] C S Mason C J Springer R G Cooper G Superti-Furga C JMarshall and RMarais ldquoSerine and tyrosine phosphorylationscooperate in Raf-1 but not B-Raf activationrdquo The EMBOJournal vol 18 no 8 pp 2137ndash2148 1999

[36] R J Fiddes P W Janes S P Sivertsen R L Sutherland EA Musgrove and R J Daly ldquoInhibition of the MAP kinasecascade blocks heregulin-induced cell cycle progression in T-47D human breast cancer cellsrdquo Oncogene vol 16 no 21 pp2803ndash2813 1998

[37] S T Eblen J K SlackM JWeber andA D Catling ldquoRac-PAKsignaling stimulates extracellular signal-regulated kinase (ERK)activation by regulating formation of MEK1-ERK complexesrdquoMolecular and Cellular Biology vol 22 no 17 pp 6023ndash60332002

[38] M Cargnello and P P Roux ldquoActivation and function ofthe MAPKs and their substrates the MAPK-activated proteinkinasesrdquo Microbiology and Molecular Biology Reviews vol 75no 1 pp 50ndash83 2011

[39] A V Khokhlatchev B Canagarajah J Wilsbacher et al ldquoPhos-phorylation of theMAP kinase ERK2 promotes its homodimer-ization and nuclear translocationrdquo Cell vol 93 no 4 pp 605ndash615 1998

[40] R H Chen C Sarnecki and J Blenis ldquoNuclear localization andregulation of erk- and rsk-encoded protein kinasesrdquoMolecularand Cellular Biology vol 12 no 3 pp 915ndash927 1992

[41] S Traverse K Seedorf H Paterson C J Marshall P Cohenand A Ullrich ldquoEGF triggers neuronal differentiation of PC12cells that overexpress the EGF receptorrdquo Current Biology vol 4no 8 pp 694ndash701 1994

[42] T T Nguyen J C Scimeca C Filloux P Peraldi J L Car-pentier and E Van Obberghen ldquoCo-regulation of the mitogen-activated protein kinase extracellular signal-regulated kinase 1

and the 90-kDa ribosomal S6 kinase in PC12 cells Distincteffects of the neurotrophic factor nerve growth factor and themitogenic factor epidermal growth factorrdquo Journal of BiologicalChemistry vol 268 no 13 pp 9803ndash9810 1993

[43] I Dikic J Schlessinger and I Lax ldquoPC12 cells overexpressingthe insulin receptor undergo insulin-dependent neuronal dif-ferentiationrdquo Current Biology vol 4 no 8 pp 702ndash708 1994

[44] M H Shen Z R Li X R Xiang W M Niu and C B ZhangldquoEffect of electro-acupuncture on ERK protein expression ofMCAO ratsrdquo Journal of Clinical Acupuncture and Moxibustionvol 23 no 7 pp 62ndash63 2007

[45] M H Shen Z R Li X R Xiang W M Niu and C BZhang ldquoInvolvement of extracellular regulated protein kinasesignal transduction pathway in electroacupuncture-inducedimprovement of ischemic cerebral injury in rats with cerebralischemia reperfusionrdquo Acupuncture Research vol 32 no 6 pp368ndash372 2007

[46] Z B Yang J Yan and S X Yi ldquoUp-regulation of the ERK phos-phorylation in gastric mucosal cell by the serum of rats treatedwith electroacupuncture at stomach meridian acupointsrdquo Basicamp Clinical Medicine vol 29 no 2 pp 135ndash138 2009

[47] C L Wang S X Wang and W Gong ldquoChanges in phos-phatized ERK in the spinal dorsal horn in adjuvant arthritisrats during electroacupuncture of Jiaji points for analgesiardquoShanghai Journal of Acupuncture and Moxibustion vol 24 no12 pp 33ndash35 2005

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 Evidence-Based Complementary and Alternative Medicine

(a) (b)

(c) (d)

(e)

Figure 10 Expression of MEK-1 protein in the colonic mucosa of rat The MEK-1 protein was expressed in epithelial cells in the colonicmucosa of rats EnVision Plus method times200 (a) Normal (b) Crohnrsquos disease (c) mild moxibustion (d) electroacupuncture and (e) herb-separated moxibustion

As an upstream protein in the Raf-MEK-ERK pathwayRas is the product of the proto-oncogene c-ras A variety ofstimulating factors particularly growth factors (eg PDGFEGF TNF and PKC activators as well as Src family members)can activate Ras which further activates Raf-1 ActivatedRaf-1 then activates MEK and ERK downstream [32 33] toinitiate the MAPK pathway Raf-1 protein kinase is widelyexpressed in various types of cells [34] Ras and Raf-1 oftenexist in the form of complexes Ras the upstream activatorprotein can translocate Raf from the cytoplasm to theplasma membrane where the latter is activated Howeverthe mechanism by which Raf is activated remains unclearalthough it may include phosphorylation [35] All that isknown is that Raf activation is related to its phosphorylation

on SerThr residues [33] Recent studies have shown thatRaf-1 protein kinase may be a point of crosstalk with othersignaling pathways This protein may participate in a varietyof biological processes in signal transduction including itsown regulation by interactingwith other signalingmoleculesor via reciprocal regulation

The MAPK kinases include MEK1 (MKK1) and MEK2(MKK2)MEK1 andMEK2 are the central links in theMAPKpathway The activated upstream Raf-1 protein kinase cancombine with MEK1 and 2 and doubly phosphorylate Tyrand Thr its two regulatory sites to activate ERK [36] MEKsare not only ERK activators but may also relocalize theactivated ERKs to the nucleus or other active sites to furtherphosphorylate downstream substrates [37]

Evidence-Based Complementary and Alternative Medicine 9

0

2

1

3

5

4

6

19

NC MC MM EA HSP

MEK

-1 m

RNA

leve

ls (

)

lowast

Figure 11 The MEK-1 mRNA levels in the rat colon NC normalcontrol group MC model control group MM mild moxibustionEA electroacupuncture and HSM herb-separated moxibustionlowast119875 lt 001 versus NC

0200400600800

100012001400

NC MC MM EA HSPPhos

phor

ylat

ed E

RK-1

2 (I

OD

)

lowast

Figure 12 The IOD of phosphorylated ERK-12 levels in therat colon NC normal control group MC model control groupMM mild moxibustion EA electroacupuncture and HSM herb-separated moxibustion lowast119875 lt 001 versus NC 119875 lt 005 119875 lt 001versus MC

The upstream kinase MEK1 is a dual-specificity enzymeActivatedMEK1 can phosphorylate ERK1 and ERK2There isevidence that the Tyr residues of ERK1 and 2 are phosphory-lated before the Thr residues both intracellularly and extra-cellularly [38] ERK translocates into the nucleus by forminga homodimer of phosphorylated ERK and phosphorylatedor unphosphorylated ERK [39] ERK is evenly distributedin quiescent cells but when the cells are stimulated byserum persistently activated ERK can partially translocateinto the nucleus whereas transiently activated ERK cannotrelocalize Activated ERKs accumulate in the nucleus andthen phosphorylate the corresponding transcription factorswhich drive expression of specific gene products Becauseof the difference in nuclear localization transiently activated(or low-activity) ERK and persistently activated (or high-activity) ERK cause cells to express genes at a different qualityor quantity resulting in different biological effects [40ndash43]

The ERK1-ERK2-MAPK pathway incorporates a mech-anism for inactivation after activation which can preventexcessive activation-induced adverse consequences At thesame time the inactivated kinase is prepared for the nextactivation The mechanisms of inhibition of this pathwayinclude the following (1) the activation of ERK12-MAPK is

the result of the phosphorylation cascade and can be inac-tivated by dephosphorylation by the corresponding phos-phatases (2) Raf-1 is the direct substrate of PKA which canphosphorylate Ser43 of c-Raf an inhibitory phosphorylationsite in the N-terminus of Raf that interacts with Ras andthe phosphorylation of this site can inhibit the bindingbetween RasGTP and the N-terminus of Raf which blocksthe activation of ERK (3) the phosphatase PP2 can inactivateMEK and ERK via dephosphorylation (4) activated ERKphosphorylates Sos and Raf while ERK dephosphorylationinactivates them thereby generates negative feedback regula-tion of the pathwayTherefore the study of themechanism bywhich acupuncture regulates the expression of ERKupstreamreceptors the phosphorylation of ERK cascade members thetargets of downstream signal transduction and the potentialimpact of other signaling pathways on this pathway is neces-sary to confirm acupuncture as an antifibrotic treatment

Our experiments studied the regulation of Ras RafMEKand ERK protein and gene expression by acupuncture inCD intestinal fibrosis in the rat colon Our results showthat the mRNA levels of Ras Raf-1 MEK-1 and ERK-12 were significantly increased in CD rat intestinal tissueand that the expression of p-Ras Raf-1 MEK-1 and p-ERK-12 proteins in intestinal epithelial cells were also sig-nificantly increased These results suggest that the activityof the ERK pathway in CD intestinal fibrosis process wasaltered Medicine-separated moxibustion mild moxibustionand electroacupuncture treatments could effectively down-regulate the expression of p-Ras Raf-1 MEK-1 and p-ERK-12 but different inhibitory effects on Ras Raf-1 MEK-1and ERK-12 mRNA levels were shown in ERK pathwayactivity changes In this discussion we propose the following(1) medicine-separated moxibustion mild moxibustion andelectroacupuncture can inhibit CD intestinal fibrosis byregulating ERK pathway activity and (2) the acupuncture-induced inhibition of p-Ras Raf-1 MEK-1 and p-ERK-12 expression may be the key elements in the treatmentrsquosinhibition of CD intestinal fibrosis At present most reportsregarding the role of acupuncture in the regulation of theERK signaling pathway have focused on EA such as EAmodel rats After EA on the Dazhui (DU14) and Baihui(DU20) acupuncture points the number of ERK-positivestriatal cells increased significantly in the cerebral cortex andERK protein expression was increased in rats with damagedneurons after cerebral ischemia and reperfusion thereforeEA can increase ERK pathway activity [44 45] After the EAtreatment of gastric mucosa-injured rats the phosphoryla-tion levels of ERK in gastric mucosal cells from extractedserum were significantly increased and were significantlydifferent between the serum of the normal rats and that ofthe model rats These observations suggest that ERK maybe involved in signal transduction in the EA stimulation atacupoints of stomach meridian of foot yangming repairinggastric mucosal injury [46] and that EA had an analgesiceffect by downregulating p-ERK expression levels in thedorsal horn of the spinal cord of rats with adjuvant arthritis[47] The previous reports have demonstrated that EA canregulate the ERK signal transduction pathway The results ofour experiments show that medicine-separated moxibustion

10 Evidence-Based Complementary and Alternative Medicine

(a) (b)

(c) (d)

(e)

Figure 13 Expression of phosphorylated ERK-12 protein in the colonicmucosa of ratThe phosphorylation of ERK-12 proteinwas expressedin epithelial cells and lymphocyte in the colonic mucosa of rats EnVision Plus method times200 (a) Normal (b) Crohnrsquos disease (c) mildmoxibustion (d) electroacupuncture and (e) herb-separated moxibustion

005

115

225

335

4

NC MC MM EA HSP

ERK-

12

mRN

A le

vels

()

lowast

Figure 14 The ERK-12 mRNA levels in the rat colon NC normalcontrol group MC model control group MM mild moxibustionEA electroacupuncture and HSM herb-separated moxibustionlowast119875 lt 001 versus NC 119875 lt 005 versus MC

andmildmoxibustion can also regulate the ERK signal trans-duction pathway and inhibit Ras and Raf-1 better than EAThe functional mechanisms underlying these observationsneed to be explored further

Abbreviations

CD Crohnrsquos diseaseNC Normal control groupMC Model control groupEA ElectroacupunctureMM Mild moxibustionHSM Herb-separated moxibustionST25 Tianshu acupoint

Evidence-Based Complementary and Alternative Medicine 11

RN6 Qihai acupointERK Extracellular signal-regulated kinaseMAPK Mitogen-activated protein kinaseTNBS 246-Trinitrobenzene sulfonic acid

Acknowledgments

The authors thank Professor Shuhui Jiang for technical assis-tance and the support of the National Natural Science Foun-dation of China (no 81173331) and National Basic ResearchProgram of China (973 program no 2009CB522900)Xiaomei Wang and Yuan Lu have the same right

References

[1] G Manning D B Whyte R Martinez T Hunter and SSudarsanam ldquoThe protein kinase complement of the humangenomerdquo Science vol 298 no 5600 pp 1912ndash1934 2002

[2] C Mowat A Cole A Windsor et al ldquoGuidelines for themanagement of inflammatory bowel disease in adultsrdquoGut vol60 no 5 pp 571ndash607 2011

[3] Inflammatory Bowel Disease Co-operation Group ChineseSociety of Gastroenterology ldquoChinese consensus on diagnosisand treatment standard of inflammatory bowel diseaserdquoChineseJournal of Gastroenterology vol 12 no 8 pp 490ndash494 2007

[4] J J Zheng X S Zhu Z Huangfu Z X Gao Z R Guo andZ Wang ldquoCrohnrsquos disease in mainland China a systematicanalysis of 50 years of researchrdquo Chinese Journal of DigestiveDiseases vol 6 no 4 pp 175ndash181 2005

[5] M Dai and B M Li ldquoResearch progress in mechanisms ofintestinal fibrosis in Crohnrsquos diseaserdquo Shijie Huaren XiaohuaZazhi vol 17 no 11 pp 1117ndash1121 2009

[6] O Grip J Malm B Veress A Bjartell S Lindgren and AEgesten ldquoIncreased presence of cells containing transforminggrowth factor alpha (TGF-120572) in ulcerative colitis both duringactive inflammation and in remissionrdquo European Journal ofGastroenterology and Hepatology vol 12 no 7 pp 761ndash7662000

[7] I E Koutroubakis E Petinaki P Dimoulios et al ldquoSerumlaminin and collagen IV in inflammatory bowel diseaserdquoJournal of Clinical Pathology vol 56 no 11 pp 817ndash820 2003

[8] S Speca I Giusti F Rieder and G Latella ldquoCellular andmolecular mechanisms of intestinal fibrosisrdquo World Journal ofGastroenterology vol 18 no 28 pp 3635ndash3661 2012

[9] Y F Song J H Hong and L M Xu ldquoTGF-120573 1ERK pathwaysand its inhibitors and fibrosisrdquo Chinese Journal of ClinicalHepatology vol 21 no 2 pp 125ndash127 2005

[10] H C Huang M Yang J Z Li and H Y Wang ldquoConnectivetissue growth factor promotes the proliferation ofmyofibroblastthrough Erk-12 signaling pathwayrdquoNational Medical Journal ofChina vol 85 no 19 pp 1322ndash1326 2005

[11] L P Guo H C Huang and J Z Li ldquoHypoxia inducesmyofibroblast formation and stimulates production of collagenI in myofibroblasts through ERK12 pathwayrdquo Chinese Journalof Pathophysiology vol 24 no 12 pp 2428ndash2432 2008

[12] H R Liu X P Ma T P Zhao and H G Wu ldquoThe adjustmentof acupuncture on colon collagen synthesis and TGF-120573mRNAexpression in Crohnrsquos disease ratrdquo Journal of Anhui TraditionalChinese Medical College vol 24 no 4 pp 25ndash30 2005

[13] X P Ma C P An H G Wu et al ldquoHerbs-partitionedmoxibustion and electro-acupuncture effects on transforming

growth factor-beta 1 connective tissue growth factor collagentype I and fibronectin expression in the colon of Crohnrsquo sdisease ratsrdquo Journal of Clinical Rehabilitative Tissue EngineeringResearch vol 12 no 20 pp 3853ndash3858 2008

[14] G P Morris P L Beck M S Herridge W T Depew M RSzewczuk and J LWallace ldquoHapten-inducedmodel of chronicinflammation and ulceration in the rat colonrdquoGastroenterologyvol 96 no 3 pp 795ndash803 1989

[15] TheMinistry of Science andTechnology of the Peoplersquos Republicof China Guidance Suggestions for the Care and Use ofLaboratory Animals 2006

[16] J D Butzner R Parmar C J Bell andVDalal ldquoButyrate enematherapy stimulates mucosal repair in experimental colitis in theratrdquo Gut vol 38 no 4 pp 568ndash573 1996

[17] X M Wang Y Lu L Y Wu et al ldquoMoxibustion inhibitsinterleukin-12 and tumor necrosis factor alpha and modulatesintestinal flora in rat with ulcerative colitisrdquo World Journal ofGastroenterology vol 18 no 46 pp 6826ndash6835 2012

[18] J B Pucilowska K K McNaughton N K Mohapatra et alldquoIGF-I and procollagen 1205721(I) are coexpressed in a subset ofmesenchymal cells in active Crohnrsquos diseaserdquo American Journalof Physiology vol 279 no 6 pp G1307ndashG1322 2000

[19] M Gharaee-Kermani B Hu S H Phan and M R GyetkoldquoRecent advances in molecular targets and treatment of idio-pathic pulmonary fibrosis focus on TGF120573 signaling and themyofibroblastrdquo Current Medicinal Chemistry vol 16 no 11 pp1400ndash1417 2009

[20] E M Zimmermann L Li Y T Hou M Cannon G MChristman andKN Bitar ldquoIGF-I induces collagen and IGFBP-5 mRNA in rat intestinal smooth musclerdquo American Journal ofPhysiology vol 273 no 4 pp G875ndashG882 1997

[21] L J ZhangQ Li P Chen et al ldquoMediating effect of extracellularsignal-regulated kinase 12 in PDGF-induced proliferation andcollagen synthesis of cultured rat cardiac fibroblastsrdquo ChineseJournal of Anatomy vol 13 no 4 p 223 2007

[22] M Tang W Zhang H Lin H Jiang H Dai and Y ZhangldquoHigh glucose promotes the production of collagen types Iand III by cardiac fibroblasts through a pathway dependenton extracellular-signal-regulated kinase 12rdquo Molecular andCellular Biochemistry vol 301 no 1-2 pp 109ndash114 2007

[23] H Y Dai Z M Ge and Y H Li ldquoEffect of urotensin II on pro-liferative potential and phosphorylation of extracellular signal-regulated kinase 12 adventitial fibroblasts from spontaneouslyhypertensive ratrdquoActaAcademiaeMedicinae Sinicae vol 28 no6 pp 776ndash780 2006

[24] I Fuentes-Calvo A M Blazquez-Medela N Eleno E SantosJ M Lopez-Novoa and C Martınez-Salgado ldquoH-Ras isoformmodulates extracellular matrix synthesis proliferation andmigration in fibroblastsrdquo American Journal of Physiology vol302 no 4 pp C686ndashC697 2012

[25] Y W Ma G T Bai and M X Zhang ldquoStudies on signal trans-duction pathway induced by fibronectin in human embryoniclung fibroblastsrdquo Shanxi Medical Journal vol 38 no 7 pp 587ndash589 2009

[26] J H Jang and C P Chung ldquoFibronectin-mediated adhesionrescues cell cycle arrest induced by fibroblast growth factor-1 bydecreased expression of P21CIPWAF in human chondrocytesrdquoIn Vitro Cellular and Developmental Biology vol 41 no 5-6 pp126ndash129 2005

[27] B Zhu Y J Wang C F Zhu et al ldquoTriptolide inhibitsextracellularmatrix protein synthesis by suppressing the Smad2

12 Evidence-Based Complementary and Alternative Medicine

but not the MAPK pathway in TGF-1205731-stimulated NRK-49Fcellsrdquo Nephrology Dialysis Transplantation vol 25 no 10 pp3180ndash3191 2010

[28] J Bai X S Liu Y J Xu M Xie and W Ni ldquoEffects ofextracellular signal regulated kinase signaling pathway on cellcycle of airway smooth muscle cells in asthmatic ratsrdquo ChineseJournal of Respiratory and Critical Care Medicine vol 9 no 1pp 23ndash27 2010

[29] S Matsusaka and I Wakabayashi ldquoEnhancement of vascu-lar smooth muscle cell migration by urotensin IIrdquo Naunyn-Schmiedebergrsquos Archives of Pharmacology vol 373 no 5 pp 381ndash386 2006

[30] Y Chen Z Dai Y M Liu et al ldquoInhibitory effects ofCGRP on vascular smooth muscle cell proliferation role ofcaveolaecaveolin-1ERK12 signal pathwayrdquo Progress in Bio-chemistry and Biophysics vol 40 no 5 pp 445ndash453 2013

[31] W L Wu A Q Zhang Q Yin and W H Gan ldquoEffectsof ERK signal pathway on extracellular matrix synthesis ofrat mesangial cells treated with PDGF-BBrdquo ACTA UniversityMedicinalis Nanjing vol 27 no 10 pp 1126ndash1129 2007

[32] L S Steelman W H Chappell S L Abrams et al ldquoRoles ofthe RafMEKERK and PI3KPTENAktmTOR pathways incontrolling growth and sensitivity to therapy-implications forcancer and agingrdquo Aging vol 3 no 3 pp 192ndash222 2011

[33] W Kolch ldquoMeaningful relationships the regulation of theRasRafMEKERK pathway by protein interactionsrdquo Biochem-ical Journal vol 351 no 2 pp 289ndash305 2000

[34] D Matallanas M Birtwistle D Romano et al ldquoRaf familykinases old dogs have learned new tricksrdquo Genes and Cancervol 2 no 3 pp 232ndash260 2011

[35] C S Mason C J Springer R G Cooper G Superti-Furga C JMarshall and RMarais ldquoSerine and tyrosine phosphorylationscooperate in Raf-1 but not B-Raf activationrdquo The EMBOJournal vol 18 no 8 pp 2137ndash2148 1999

[36] R J Fiddes P W Janes S P Sivertsen R L Sutherland EA Musgrove and R J Daly ldquoInhibition of the MAP kinasecascade blocks heregulin-induced cell cycle progression in T-47D human breast cancer cellsrdquo Oncogene vol 16 no 21 pp2803ndash2813 1998

[37] S T Eblen J K SlackM JWeber andA D Catling ldquoRac-PAKsignaling stimulates extracellular signal-regulated kinase (ERK)activation by regulating formation of MEK1-ERK complexesrdquoMolecular and Cellular Biology vol 22 no 17 pp 6023ndash60332002

[38] M Cargnello and P P Roux ldquoActivation and function ofthe MAPKs and their substrates the MAPK-activated proteinkinasesrdquo Microbiology and Molecular Biology Reviews vol 75no 1 pp 50ndash83 2011

[39] A V Khokhlatchev B Canagarajah J Wilsbacher et al ldquoPhos-phorylation of theMAP kinase ERK2 promotes its homodimer-ization and nuclear translocationrdquo Cell vol 93 no 4 pp 605ndash615 1998

[40] R H Chen C Sarnecki and J Blenis ldquoNuclear localization andregulation of erk- and rsk-encoded protein kinasesrdquoMolecularand Cellular Biology vol 12 no 3 pp 915ndash927 1992

[41] S Traverse K Seedorf H Paterson C J Marshall P Cohenand A Ullrich ldquoEGF triggers neuronal differentiation of PC12cells that overexpress the EGF receptorrdquo Current Biology vol 4no 8 pp 694ndash701 1994

[42] T T Nguyen J C Scimeca C Filloux P Peraldi J L Car-pentier and E Van Obberghen ldquoCo-regulation of the mitogen-activated protein kinase extracellular signal-regulated kinase 1

and the 90-kDa ribosomal S6 kinase in PC12 cells Distincteffects of the neurotrophic factor nerve growth factor and themitogenic factor epidermal growth factorrdquo Journal of BiologicalChemistry vol 268 no 13 pp 9803ndash9810 1993

[43] I Dikic J Schlessinger and I Lax ldquoPC12 cells overexpressingthe insulin receptor undergo insulin-dependent neuronal dif-ferentiationrdquo Current Biology vol 4 no 8 pp 702ndash708 1994

[44] M H Shen Z R Li X R Xiang W M Niu and C B ZhangldquoEffect of electro-acupuncture on ERK protein expression ofMCAO ratsrdquo Journal of Clinical Acupuncture and Moxibustionvol 23 no 7 pp 62ndash63 2007

[45] M H Shen Z R Li X R Xiang W M Niu and C BZhang ldquoInvolvement of extracellular regulated protein kinasesignal transduction pathway in electroacupuncture-inducedimprovement of ischemic cerebral injury in rats with cerebralischemia reperfusionrdquo Acupuncture Research vol 32 no 6 pp368ndash372 2007

[46] Z B Yang J Yan and S X Yi ldquoUp-regulation of the ERK phos-phorylation in gastric mucosal cell by the serum of rats treatedwith electroacupuncture at stomach meridian acupointsrdquo Basicamp Clinical Medicine vol 29 no 2 pp 135ndash138 2009

[47] C L Wang S X Wang and W Gong ldquoChanges in phos-phatized ERK in the spinal dorsal horn in adjuvant arthritisrats during electroacupuncture of Jiaji points for analgesiardquoShanghai Journal of Acupuncture and Moxibustion vol 24 no12 pp 33ndash35 2005

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 9

0

2

1

3

5

4

6

19

NC MC MM EA HSP

MEK

-1 m

RNA

leve

ls (

)

lowast

Figure 11 The MEK-1 mRNA levels in the rat colon NC normalcontrol group MC model control group MM mild moxibustionEA electroacupuncture and HSM herb-separated moxibustionlowast119875 lt 001 versus NC

0200400600800

100012001400

NC MC MM EA HSPPhos

phor

ylat

ed E

RK-1

2 (I

OD

)

lowast

Figure 12 The IOD of phosphorylated ERK-12 levels in therat colon NC normal control group MC model control groupMM mild moxibustion EA electroacupuncture and HSM herb-separated moxibustion lowast119875 lt 001 versus NC 119875 lt 005 119875 lt 001versus MC

The upstream kinase MEK1 is a dual-specificity enzymeActivatedMEK1 can phosphorylate ERK1 and ERK2There isevidence that the Tyr residues of ERK1 and 2 are phosphory-lated before the Thr residues both intracellularly and extra-cellularly [38] ERK translocates into the nucleus by forminga homodimer of phosphorylated ERK and phosphorylatedor unphosphorylated ERK [39] ERK is evenly distributedin quiescent cells but when the cells are stimulated byserum persistently activated ERK can partially translocateinto the nucleus whereas transiently activated ERK cannotrelocalize Activated ERKs accumulate in the nucleus andthen phosphorylate the corresponding transcription factorswhich drive expression of specific gene products Becauseof the difference in nuclear localization transiently activated(or low-activity) ERK and persistently activated (or high-activity) ERK cause cells to express genes at a different qualityor quantity resulting in different biological effects [40ndash43]

The ERK1-ERK2-MAPK pathway incorporates a mech-anism for inactivation after activation which can preventexcessive activation-induced adverse consequences At thesame time the inactivated kinase is prepared for the nextactivation The mechanisms of inhibition of this pathwayinclude the following (1) the activation of ERK12-MAPK is

the result of the phosphorylation cascade and can be inac-tivated by dephosphorylation by the corresponding phos-phatases (2) Raf-1 is the direct substrate of PKA which canphosphorylate Ser43 of c-Raf an inhibitory phosphorylationsite in the N-terminus of Raf that interacts with Ras andthe phosphorylation of this site can inhibit the bindingbetween RasGTP and the N-terminus of Raf which blocksthe activation of ERK (3) the phosphatase PP2 can inactivateMEK and ERK via dephosphorylation (4) activated ERKphosphorylates Sos and Raf while ERK dephosphorylationinactivates them thereby generates negative feedback regula-tion of the pathwayTherefore the study of themechanism bywhich acupuncture regulates the expression of ERKupstreamreceptors the phosphorylation of ERK cascade members thetargets of downstream signal transduction and the potentialimpact of other signaling pathways on this pathway is neces-sary to confirm acupuncture as an antifibrotic treatment

Our experiments studied the regulation of Ras RafMEKand ERK protein and gene expression by acupuncture inCD intestinal fibrosis in the rat colon Our results showthat the mRNA levels of Ras Raf-1 MEK-1 and ERK-12 were significantly increased in CD rat intestinal tissueand that the expression of p-Ras Raf-1 MEK-1 and p-ERK-12 proteins in intestinal epithelial cells were also sig-nificantly increased These results suggest that the activityof the ERK pathway in CD intestinal fibrosis process wasaltered Medicine-separated moxibustion mild moxibustionand electroacupuncture treatments could effectively down-regulate the expression of p-Ras Raf-1 MEK-1 and p-ERK-12 but different inhibitory effects on Ras Raf-1 MEK-1and ERK-12 mRNA levels were shown in ERK pathwayactivity changes In this discussion we propose the following(1) medicine-separated moxibustion mild moxibustion andelectroacupuncture can inhibit CD intestinal fibrosis byregulating ERK pathway activity and (2) the acupuncture-induced inhibition of p-Ras Raf-1 MEK-1 and p-ERK-12 expression may be the key elements in the treatmentrsquosinhibition of CD intestinal fibrosis At present most reportsregarding the role of acupuncture in the regulation of theERK signaling pathway have focused on EA such as EAmodel rats After EA on the Dazhui (DU14) and Baihui(DU20) acupuncture points the number of ERK-positivestriatal cells increased significantly in the cerebral cortex andERK protein expression was increased in rats with damagedneurons after cerebral ischemia and reperfusion thereforeEA can increase ERK pathway activity [44 45] After the EAtreatment of gastric mucosa-injured rats the phosphoryla-tion levels of ERK in gastric mucosal cells from extractedserum were significantly increased and were significantlydifferent between the serum of the normal rats and that ofthe model rats These observations suggest that ERK maybe involved in signal transduction in the EA stimulation atacupoints of stomach meridian of foot yangming repairinggastric mucosal injury [46] and that EA had an analgesiceffect by downregulating p-ERK expression levels in thedorsal horn of the spinal cord of rats with adjuvant arthritis[47] The previous reports have demonstrated that EA canregulate the ERK signal transduction pathway The results ofour experiments show that medicine-separated moxibustion

10 Evidence-Based Complementary and Alternative Medicine

(a) (b)

(c) (d)

(e)

Figure 13 Expression of phosphorylated ERK-12 protein in the colonicmucosa of ratThe phosphorylation of ERK-12 proteinwas expressedin epithelial cells and lymphocyte in the colonic mucosa of rats EnVision Plus method times200 (a) Normal (b) Crohnrsquos disease (c) mildmoxibustion (d) electroacupuncture and (e) herb-separated moxibustion

005

115

225

335

4

NC MC MM EA HSP

ERK-

12

mRN

A le

vels

()

lowast

Figure 14 The ERK-12 mRNA levels in the rat colon NC normalcontrol group MC model control group MM mild moxibustionEA electroacupuncture and HSM herb-separated moxibustionlowast119875 lt 001 versus NC 119875 lt 005 versus MC

andmildmoxibustion can also regulate the ERK signal trans-duction pathway and inhibit Ras and Raf-1 better than EAThe functional mechanisms underlying these observationsneed to be explored further

Abbreviations

CD Crohnrsquos diseaseNC Normal control groupMC Model control groupEA ElectroacupunctureMM Mild moxibustionHSM Herb-separated moxibustionST25 Tianshu acupoint

Evidence-Based Complementary and Alternative Medicine 11

RN6 Qihai acupointERK Extracellular signal-regulated kinaseMAPK Mitogen-activated protein kinaseTNBS 246-Trinitrobenzene sulfonic acid

Acknowledgments

The authors thank Professor Shuhui Jiang for technical assis-tance and the support of the National Natural Science Foun-dation of China (no 81173331) and National Basic ResearchProgram of China (973 program no 2009CB522900)Xiaomei Wang and Yuan Lu have the same right

References

[1] G Manning D B Whyte R Martinez T Hunter and SSudarsanam ldquoThe protein kinase complement of the humangenomerdquo Science vol 298 no 5600 pp 1912ndash1934 2002

[2] C Mowat A Cole A Windsor et al ldquoGuidelines for themanagement of inflammatory bowel disease in adultsrdquoGut vol60 no 5 pp 571ndash607 2011

[3] Inflammatory Bowel Disease Co-operation Group ChineseSociety of Gastroenterology ldquoChinese consensus on diagnosisand treatment standard of inflammatory bowel diseaserdquoChineseJournal of Gastroenterology vol 12 no 8 pp 490ndash494 2007

[4] J J Zheng X S Zhu Z Huangfu Z X Gao Z R Guo andZ Wang ldquoCrohnrsquos disease in mainland China a systematicanalysis of 50 years of researchrdquo Chinese Journal of DigestiveDiseases vol 6 no 4 pp 175ndash181 2005

[5] M Dai and B M Li ldquoResearch progress in mechanisms ofintestinal fibrosis in Crohnrsquos diseaserdquo Shijie Huaren XiaohuaZazhi vol 17 no 11 pp 1117ndash1121 2009

[6] O Grip J Malm B Veress A Bjartell S Lindgren and AEgesten ldquoIncreased presence of cells containing transforminggrowth factor alpha (TGF-120572) in ulcerative colitis both duringactive inflammation and in remissionrdquo European Journal ofGastroenterology and Hepatology vol 12 no 7 pp 761ndash7662000

[7] I E Koutroubakis E Petinaki P Dimoulios et al ldquoSerumlaminin and collagen IV in inflammatory bowel diseaserdquoJournal of Clinical Pathology vol 56 no 11 pp 817ndash820 2003

[8] S Speca I Giusti F Rieder and G Latella ldquoCellular andmolecular mechanisms of intestinal fibrosisrdquo World Journal ofGastroenterology vol 18 no 28 pp 3635ndash3661 2012

[9] Y F Song J H Hong and L M Xu ldquoTGF-120573 1ERK pathwaysand its inhibitors and fibrosisrdquo Chinese Journal of ClinicalHepatology vol 21 no 2 pp 125ndash127 2005

[10] H C Huang M Yang J Z Li and H Y Wang ldquoConnectivetissue growth factor promotes the proliferation ofmyofibroblastthrough Erk-12 signaling pathwayrdquoNational Medical Journal ofChina vol 85 no 19 pp 1322ndash1326 2005

[11] L P Guo H C Huang and J Z Li ldquoHypoxia inducesmyofibroblast formation and stimulates production of collagenI in myofibroblasts through ERK12 pathwayrdquo Chinese Journalof Pathophysiology vol 24 no 12 pp 2428ndash2432 2008

[12] H R Liu X P Ma T P Zhao and H G Wu ldquoThe adjustmentof acupuncture on colon collagen synthesis and TGF-120573mRNAexpression in Crohnrsquos disease ratrdquo Journal of Anhui TraditionalChinese Medical College vol 24 no 4 pp 25ndash30 2005

[13] X P Ma C P An H G Wu et al ldquoHerbs-partitionedmoxibustion and electro-acupuncture effects on transforming

growth factor-beta 1 connective tissue growth factor collagentype I and fibronectin expression in the colon of Crohnrsquo sdisease ratsrdquo Journal of Clinical Rehabilitative Tissue EngineeringResearch vol 12 no 20 pp 3853ndash3858 2008

[14] G P Morris P L Beck M S Herridge W T Depew M RSzewczuk and J LWallace ldquoHapten-inducedmodel of chronicinflammation and ulceration in the rat colonrdquoGastroenterologyvol 96 no 3 pp 795ndash803 1989

[15] TheMinistry of Science andTechnology of the Peoplersquos Republicof China Guidance Suggestions for the Care and Use ofLaboratory Animals 2006

[16] J D Butzner R Parmar C J Bell andVDalal ldquoButyrate enematherapy stimulates mucosal repair in experimental colitis in theratrdquo Gut vol 38 no 4 pp 568ndash573 1996

[17] X M Wang Y Lu L Y Wu et al ldquoMoxibustion inhibitsinterleukin-12 and tumor necrosis factor alpha and modulatesintestinal flora in rat with ulcerative colitisrdquo World Journal ofGastroenterology vol 18 no 46 pp 6826ndash6835 2012

[18] J B Pucilowska K K McNaughton N K Mohapatra et alldquoIGF-I and procollagen 1205721(I) are coexpressed in a subset ofmesenchymal cells in active Crohnrsquos diseaserdquo American Journalof Physiology vol 279 no 6 pp G1307ndashG1322 2000

[19] M Gharaee-Kermani B Hu S H Phan and M R GyetkoldquoRecent advances in molecular targets and treatment of idio-pathic pulmonary fibrosis focus on TGF120573 signaling and themyofibroblastrdquo Current Medicinal Chemistry vol 16 no 11 pp1400ndash1417 2009

[20] E M Zimmermann L Li Y T Hou M Cannon G MChristman andKN Bitar ldquoIGF-I induces collagen and IGFBP-5 mRNA in rat intestinal smooth musclerdquo American Journal ofPhysiology vol 273 no 4 pp G875ndashG882 1997

[21] L J ZhangQ Li P Chen et al ldquoMediating effect of extracellularsignal-regulated kinase 12 in PDGF-induced proliferation andcollagen synthesis of cultured rat cardiac fibroblastsrdquo ChineseJournal of Anatomy vol 13 no 4 p 223 2007

[22] M Tang W Zhang H Lin H Jiang H Dai and Y ZhangldquoHigh glucose promotes the production of collagen types Iand III by cardiac fibroblasts through a pathway dependenton extracellular-signal-regulated kinase 12rdquo Molecular andCellular Biochemistry vol 301 no 1-2 pp 109ndash114 2007

[23] H Y Dai Z M Ge and Y H Li ldquoEffect of urotensin II on pro-liferative potential and phosphorylation of extracellular signal-regulated kinase 12 adventitial fibroblasts from spontaneouslyhypertensive ratrdquoActaAcademiaeMedicinae Sinicae vol 28 no6 pp 776ndash780 2006

[24] I Fuentes-Calvo A M Blazquez-Medela N Eleno E SantosJ M Lopez-Novoa and C Martınez-Salgado ldquoH-Ras isoformmodulates extracellular matrix synthesis proliferation andmigration in fibroblastsrdquo American Journal of Physiology vol302 no 4 pp C686ndashC697 2012

[25] Y W Ma G T Bai and M X Zhang ldquoStudies on signal trans-duction pathway induced by fibronectin in human embryoniclung fibroblastsrdquo Shanxi Medical Journal vol 38 no 7 pp 587ndash589 2009

[26] J H Jang and C P Chung ldquoFibronectin-mediated adhesionrescues cell cycle arrest induced by fibroblast growth factor-1 bydecreased expression of P21CIPWAF in human chondrocytesrdquoIn Vitro Cellular and Developmental Biology vol 41 no 5-6 pp126ndash129 2005

[27] B Zhu Y J Wang C F Zhu et al ldquoTriptolide inhibitsextracellularmatrix protein synthesis by suppressing the Smad2

12 Evidence-Based Complementary and Alternative Medicine

but not the MAPK pathway in TGF-1205731-stimulated NRK-49Fcellsrdquo Nephrology Dialysis Transplantation vol 25 no 10 pp3180ndash3191 2010

[28] J Bai X S Liu Y J Xu M Xie and W Ni ldquoEffects ofextracellular signal regulated kinase signaling pathway on cellcycle of airway smooth muscle cells in asthmatic ratsrdquo ChineseJournal of Respiratory and Critical Care Medicine vol 9 no 1pp 23ndash27 2010

[29] S Matsusaka and I Wakabayashi ldquoEnhancement of vascu-lar smooth muscle cell migration by urotensin IIrdquo Naunyn-Schmiedebergrsquos Archives of Pharmacology vol 373 no 5 pp 381ndash386 2006

[30] Y Chen Z Dai Y M Liu et al ldquoInhibitory effects ofCGRP on vascular smooth muscle cell proliferation role ofcaveolaecaveolin-1ERK12 signal pathwayrdquo Progress in Bio-chemistry and Biophysics vol 40 no 5 pp 445ndash453 2013

[31] W L Wu A Q Zhang Q Yin and W H Gan ldquoEffectsof ERK signal pathway on extracellular matrix synthesis ofrat mesangial cells treated with PDGF-BBrdquo ACTA UniversityMedicinalis Nanjing vol 27 no 10 pp 1126ndash1129 2007

[32] L S Steelman W H Chappell S L Abrams et al ldquoRoles ofthe RafMEKERK and PI3KPTENAktmTOR pathways incontrolling growth and sensitivity to therapy-implications forcancer and agingrdquo Aging vol 3 no 3 pp 192ndash222 2011

[33] W Kolch ldquoMeaningful relationships the regulation of theRasRafMEKERK pathway by protein interactionsrdquo Biochem-ical Journal vol 351 no 2 pp 289ndash305 2000

[34] D Matallanas M Birtwistle D Romano et al ldquoRaf familykinases old dogs have learned new tricksrdquo Genes and Cancervol 2 no 3 pp 232ndash260 2011

[35] C S Mason C J Springer R G Cooper G Superti-Furga C JMarshall and RMarais ldquoSerine and tyrosine phosphorylationscooperate in Raf-1 but not B-Raf activationrdquo The EMBOJournal vol 18 no 8 pp 2137ndash2148 1999

[36] R J Fiddes P W Janes S P Sivertsen R L Sutherland EA Musgrove and R J Daly ldquoInhibition of the MAP kinasecascade blocks heregulin-induced cell cycle progression in T-47D human breast cancer cellsrdquo Oncogene vol 16 no 21 pp2803ndash2813 1998

[37] S T Eblen J K SlackM JWeber andA D Catling ldquoRac-PAKsignaling stimulates extracellular signal-regulated kinase (ERK)activation by regulating formation of MEK1-ERK complexesrdquoMolecular and Cellular Biology vol 22 no 17 pp 6023ndash60332002

[38] M Cargnello and P P Roux ldquoActivation and function ofthe MAPKs and their substrates the MAPK-activated proteinkinasesrdquo Microbiology and Molecular Biology Reviews vol 75no 1 pp 50ndash83 2011

[39] A V Khokhlatchev B Canagarajah J Wilsbacher et al ldquoPhos-phorylation of theMAP kinase ERK2 promotes its homodimer-ization and nuclear translocationrdquo Cell vol 93 no 4 pp 605ndash615 1998

[40] R H Chen C Sarnecki and J Blenis ldquoNuclear localization andregulation of erk- and rsk-encoded protein kinasesrdquoMolecularand Cellular Biology vol 12 no 3 pp 915ndash927 1992

[41] S Traverse K Seedorf H Paterson C J Marshall P Cohenand A Ullrich ldquoEGF triggers neuronal differentiation of PC12cells that overexpress the EGF receptorrdquo Current Biology vol 4no 8 pp 694ndash701 1994

[42] T T Nguyen J C Scimeca C Filloux P Peraldi J L Car-pentier and E Van Obberghen ldquoCo-regulation of the mitogen-activated protein kinase extracellular signal-regulated kinase 1

and the 90-kDa ribosomal S6 kinase in PC12 cells Distincteffects of the neurotrophic factor nerve growth factor and themitogenic factor epidermal growth factorrdquo Journal of BiologicalChemistry vol 268 no 13 pp 9803ndash9810 1993

[43] I Dikic J Schlessinger and I Lax ldquoPC12 cells overexpressingthe insulin receptor undergo insulin-dependent neuronal dif-ferentiationrdquo Current Biology vol 4 no 8 pp 702ndash708 1994

[44] M H Shen Z R Li X R Xiang W M Niu and C B ZhangldquoEffect of electro-acupuncture on ERK protein expression ofMCAO ratsrdquo Journal of Clinical Acupuncture and Moxibustionvol 23 no 7 pp 62ndash63 2007

[45] M H Shen Z R Li X R Xiang W M Niu and C BZhang ldquoInvolvement of extracellular regulated protein kinasesignal transduction pathway in electroacupuncture-inducedimprovement of ischemic cerebral injury in rats with cerebralischemia reperfusionrdquo Acupuncture Research vol 32 no 6 pp368ndash372 2007

[46] Z B Yang J Yan and S X Yi ldquoUp-regulation of the ERK phos-phorylation in gastric mucosal cell by the serum of rats treatedwith electroacupuncture at stomach meridian acupointsrdquo Basicamp Clinical Medicine vol 29 no 2 pp 135ndash138 2009

[47] C L Wang S X Wang and W Gong ldquoChanges in phos-phatized ERK in the spinal dorsal horn in adjuvant arthritisrats during electroacupuncture of Jiaji points for analgesiardquoShanghai Journal of Acupuncture and Moxibustion vol 24 no12 pp 33ndash35 2005

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

10 Evidence-Based Complementary and Alternative Medicine

(a) (b)

(c) (d)

(e)

Figure 13 Expression of phosphorylated ERK-12 protein in the colonicmucosa of ratThe phosphorylation of ERK-12 proteinwas expressedin epithelial cells and lymphocyte in the colonic mucosa of rats EnVision Plus method times200 (a) Normal (b) Crohnrsquos disease (c) mildmoxibustion (d) electroacupuncture and (e) herb-separated moxibustion

005

115

225

335

4

NC MC MM EA HSP

ERK-

12

mRN

A le

vels

()

lowast

Figure 14 The ERK-12 mRNA levels in the rat colon NC normalcontrol group MC model control group MM mild moxibustionEA electroacupuncture and HSM herb-separated moxibustionlowast119875 lt 001 versus NC 119875 lt 005 versus MC

andmildmoxibustion can also regulate the ERK signal trans-duction pathway and inhibit Ras and Raf-1 better than EAThe functional mechanisms underlying these observationsneed to be explored further

Abbreviations

CD Crohnrsquos diseaseNC Normal control groupMC Model control groupEA ElectroacupunctureMM Mild moxibustionHSM Herb-separated moxibustionST25 Tianshu acupoint

Evidence-Based Complementary and Alternative Medicine 11

RN6 Qihai acupointERK Extracellular signal-regulated kinaseMAPK Mitogen-activated protein kinaseTNBS 246-Trinitrobenzene sulfonic acid

Acknowledgments

The authors thank Professor Shuhui Jiang for technical assis-tance and the support of the National Natural Science Foun-dation of China (no 81173331) and National Basic ResearchProgram of China (973 program no 2009CB522900)Xiaomei Wang and Yuan Lu have the same right

References

[1] G Manning D B Whyte R Martinez T Hunter and SSudarsanam ldquoThe protein kinase complement of the humangenomerdquo Science vol 298 no 5600 pp 1912ndash1934 2002

[2] C Mowat A Cole A Windsor et al ldquoGuidelines for themanagement of inflammatory bowel disease in adultsrdquoGut vol60 no 5 pp 571ndash607 2011

[3] Inflammatory Bowel Disease Co-operation Group ChineseSociety of Gastroenterology ldquoChinese consensus on diagnosisand treatment standard of inflammatory bowel diseaserdquoChineseJournal of Gastroenterology vol 12 no 8 pp 490ndash494 2007

[4] J J Zheng X S Zhu Z Huangfu Z X Gao Z R Guo andZ Wang ldquoCrohnrsquos disease in mainland China a systematicanalysis of 50 years of researchrdquo Chinese Journal of DigestiveDiseases vol 6 no 4 pp 175ndash181 2005

[5] M Dai and B M Li ldquoResearch progress in mechanisms ofintestinal fibrosis in Crohnrsquos diseaserdquo Shijie Huaren XiaohuaZazhi vol 17 no 11 pp 1117ndash1121 2009

[6] O Grip J Malm B Veress A Bjartell S Lindgren and AEgesten ldquoIncreased presence of cells containing transforminggrowth factor alpha (TGF-120572) in ulcerative colitis both duringactive inflammation and in remissionrdquo European Journal ofGastroenterology and Hepatology vol 12 no 7 pp 761ndash7662000

[7] I E Koutroubakis E Petinaki P Dimoulios et al ldquoSerumlaminin and collagen IV in inflammatory bowel diseaserdquoJournal of Clinical Pathology vol 56 no 11 pp 817ndash820 2003

[8] S Speca I Giusti F Rieder and G Latella ldquoCellular andmolecular mechanisms of intestinal fibrosisrdquo World Journal ofGastroenterology vol 18 no 28 pp 3635ndash3661 2012

[9] Y F Song J H Hong and L M Xu ldquoTGF-120573 1ERK pathwaysand its inhibitors and fibrosisrdquo Chinese Journal of ClinicalHepatology vol 21 no 2 pp 125ndash127 2005

[10] H C Huang M Yang J Z Li and H Y Wang ldquoConnectivetissue growth factor promotes the proliferation ofmyofibroblastthrough Erk-12 signaling pathwayrdquoNational Medical Journal ofChina vol 85 no 19 pp 1322ndash1326 2005

[11] L P Guo H C Huang and J Z Li ldquoHypoxia inducesmyofibroblast formation and stimulates production of collagenI in myofibroblasts through ERK12 pathwayrdquo Chinese Journalof Pathophysiology vol 24 no 12 pp 2428ndash2432 2008

[12] H R Liu X P Ma T P Zhao and H G Wu ldquoThe adjustmentof acupuncture on colon collagen synthesis and TGF-120573mRNAexpression in Crohnrsquos disease ratrdquo Journal of Anhui TraditionalChinese Medical College vol 24 no 4 pp 25ndash30 2005

[13] X P Ma C P An H G Wu et al ldquoHerbs-partitionedmoxibustion and electro-acupuncture effects on transforming

growth factor-beta 1 connective tissue growth factor collagentype I and fibronectin expression in the colon of Crohnrsquo sdisease ratsrdquo Journal of Clinical Rehabilitative Tissue EngineeringResearch vol 12 no 20 pp 3853ndash3858 2008

[14] G P Morris P L Beck M S Herridge W T Depew M RSzewczuk and J LWallace ldquoHapten-inducedmodel of chronicinflammation and ulceration in the rat colonrdquoGastroenterologyvol 96 no 3 pp 795ndash803 1989

[15] TheMinistry of Science andTechnology of the Peoplersquos Republicof China Guidance Suggestions for the Care and Use ofLaboratory Animals 2006

[16] J D Butzner R Parmar C J Bell andVDalal ldquoButyrate enematherapy stimulates mucosal repair in experimental colitis in theratrdquo Gut vol 38 no 4 pp 568ndash573 1996

[17] X M Wang Y Lu L Y Wu et al ldquoMoxibustion inhibitsinterleukin-12 and tumor necrosis factor alpha and modulatesintestinal flora in rat with ulcerative colitisrdquo World Journal ofGastroenterology vol 18 no 46 pp 6826ndash6835 2012

[18] J B Pucilowska K K McNaughton N K Mohapatra et alldquoIGF-I and procollagen 1205721(I) are coexpressed in a subset ofmesenchymal cells in active Crohnrsquos diseaserdquo American Journalof Physiology vol 279 no 6 pp G1307ndashG1322 2000

[19] M Gharaee-Kermani B Hu S H Phan and M R GyetkoldquoRecent advances in molecular targets and treatment of idio-pathic pulmonary fibrosis focus on TGF120573 signaling and themyofibroblastrdquo Current Medicinal Chemistry vol 16 no 11 pp1400ndash1417 2009

[20] E M Zimmermann L Li Y T Hou M Cannon G MChristman andKN Bitar ldquoIGF-I induces collagen and IGFBP-5 mRNA in rat intestinal smooth musclerdquo American Journal ofPhysiology vol 273 no 4 pp G875ndashG882 1997

[21] L J ZhangQ Li P Chen et al ldquoMediating effect of extracellularsignal-regulated kinase 12 in PDGF-induced proliferation andcollagen synthesis of cultured rat cardiac fibroblastsrdquo ChineseJournal of Anatomy vol 13 no 4 p 223 2007

[22] M Tang W Zhang H Lin H Jiang H Dai and Y ZhangldquoHigh glucose promotes the production of collagen types Iand III by cardiac fibroblasts through a pathway dependenton extracellular-signal-regulated kinase 12rdquo Molecular andCellular Biochemistry vol 301 no 1-2 pp 109ndash114 2007

[23] H Y Dai Z M Ge and Y H Li ldquoEffect of urotensin II on pro-liferative potential and phosphorylation of extracellular signal-regulated kinase 12 adventitial fibroblasts from spontaneouslyhypertensive ratrdquoActaAcademiaeMedicinae Sinicae vol 28 no6 pp 776ndash780 2006

[24] I Fuentes-Calvo A M Blazquez-Medela N Eleno E SantosJ M Lopez-Novoa and C Martınez-Salgado ldquoH-Ras isoformmodulates extracellular matrix synthesis proliferation andmigration in fibroblastsrdquo American Journal of Physiology vol302 no 4 pp C686ndashC697 2012

[25] Y W Ma G T Bai and M X Zhang ldquoStudies on signal trans-duction pathway induced by fibronectin in human embryoniclung fibroblastsrdquo Shanxi Medical Journal vol 38 no 7 pp 587ndash589 2009

[26] J H Jang and C P Chung ldquoFibronectin-mediated adhesionrescues cell cycle arrest induced by fibroblast growth factor-1 bydecreased expression of P21CIPWAF in human chondrocytesrdquoIn Vitro Cellular and Developmental Biology vol 41 no 5-6 pp126ndash129 2005

[27] B Zhu Y J Wang C F Zhu et al ldquoTriptolide inhibitsextracellularmatrix protein synthesis by suppressing the Smad2

12 Evidence-Based Complementary and Alternative Medicine

but not the MAPK pathway in TGF-1205731-stimulated NRK-49Fcellsrdquo Nephrology Dialysis Transplantation vol 25 no 10 pp3180ndash3191 2010

[28] J Bai X S Liu Y J Xu M Xie and W Ni ldquoEffects ofextracellular signal regulated kinase signaling pathway on cellcycle of airway smooth muscle cells in asthmatic ratsrdquo ChineseJournal of Respiratory and Critical Care Medicine vol 9 no 1pp 23ndash27 2010

[29] S Matsusaka and I Wakabayashi ldquoEnhancement of vascu-lar smooth muscle cell migration by urotensin IIrdquo Naunyn-Schmiedebergrsquos Archives of Pharmacology vol 373 no 5 pp 381ndash386 2006

[30] Y Chen Z Dai Y M Liu et al ldquoInhibitory effects ofCGRP on vascular smooth muscle cell proliferation role ofcaveolaecaveolin-1ERK12 signal pathwayrdquo Progress in Bio-chemistry and Biophysics vol 40 no 5 pp 445ndash453 2013

[31] W L Wu A Q Zhang Q Yin and W H Gan ldquoEffectsof ERK signal pathway on extracellular matrix synthesis ofrat mesangial cells treated with PDGF-BBrdquo ACTA UniversityMedicinalis Nanjing vol 27 no 10 pp 1126ndash1129 2007

[32] L S Steelman W H Chappell S L Abrams et al ldquoRoles ofthe RafMEKERK and PI3KPTENAktmTOR pathways incontrolling growth and sensitivity to therapy-implications forcancer and agingrdquo Aging vol 3 no 3 pp 192ndash222 2011

[33] W Kolch ldquoMeaningful relationships the regulation of theRasRafMEKERK pathway by protein interactionsrdquo Biochem-ical Journal vol 351 no 2 pp 289ndash305 2000

[34] D Matallanas M Birtwistle D Romano et al ldquoRaf familykinases old dogs have learned new tricksrdquo Genes and Cancervol 2 no 3 pp 232ndash260 2011

[35] C S Mason C J Springer R G Cooper G Superti-Furga C JMarshall and RMarais ldquoSerine and tyrosine phosphorylationscooperate in Raf-1 but not B-Raf activationrdquo The EMBOJournal vol 18 no 8 pp 2137ndash2148 1999

[36] R J Fiddes P W Janes S P Sivertsen R L Sutherland EA Musgrove and R J Daly ldquoInhibition of the MAP kinasecascade blocks heregulin-induced cell cycle progression in T-47D human breast cancer cellsrdquo Oncogene vol 16 no 21 pp2803ndash2813 1998

[37] S T Eblen J K SlackM JWeber andA D Catling ldquoRac-PAKsignaling stimulates extracellular signal-regulated kinase (ERK)activation by regulating formation of MEK1-ERK complexesrdquoMolecular and Cellular Biology vol 22 no 17 pp 6023ndash60332002

[38] M Cargnello and P P Roux ldquoActivation and function ofthe MAPKs and their substrates the MAPK-activated proteinkinasesrdquo Microbiology and Molecular Biology Reviews vol 75no 1 pp 50ndash83 2011

[39] A V Khokhlatchev B Canagarajah J Wilsbacher et al ldquoPhos-phorylation of theMAP kinase ERK2 promotes its homodimer-ization and nuclear translocationrdquo Cell vol 93 no 4 pp 605ndash615 1998

[40] R H Chen C Sarnecki and J Blenis ldquoNuclear localization andregulation of erk- and rsk-encoded protein kinasesrdquoMolecularand Cellular Biology vol 12 no 3 pp 915ndash927 1992

[41] S Traverse K Seedorf H Paterson C J Marshall P Cohenand A Ullrich ldquoEGF triggers neuronal differentiation of PC12cells that overexpress the EGF receptorrdquo Current Biology vol 4no 8 pp 694ndash701 1994

[42] T T Nguyen J C Scimeca C Filloux P Peraldi J L Car-pentier and E Van Obberghen ldquoCo-regulation of the mitogen-activated protein kinase extracellular signal-regulated kinase 1

and the 90-kDa ribosomal S6 kinase in PC12 cells Distincteffects of the neurotrophic factor nerve growth factor and themitogenic factor epidermal growth factorrdquo Journal of BiologicalChemistry vol 268 no 13 pp 9803ndash9810 1993

[43] I Dikic J Schlessinger and I Lax ldquoPC12 cells overexpressingthe insulin receptor undergo insulin-dependent neuronal dif-ferentiationrdquo Current Biology vol 4 no 8 pp 702ndash708 1994

[44] M H Shen Z R Li X R Xiang W M Niu and C B ZhangldquoEffect of electro-acupuncture on ERK protein expression ofMCAO ratsrdquo Journal of Clinical Acupuncture and Moxibustionvol 23 no 7 pp 62ndash63 2007

[45] M H Shen Z R Li X R Xiang W M Niu and C BZhang ldquoInvolvement of extracellular regulated protein kinasesignal transduction pathway in electroacupuncture-inducedimprovement of ischemic cerebral injury in rats with cerebralischemia reperfusionrdquo Acupuncture Research vol 32 no 6 pp368ndash372 2007

[46] Z B Yang J Yan and S X Yi ldquoUp-regulation of the ERK phos-phorylation in gastric mucosal cell by the serum of rats treatedwith electroacupuncture at stomach meridian acupointsrdquo Basicamp Clinical Medicine vol 29 no 2 pp 135ndash138 2009

[47] C L Wang S X Wang and W Gong ldquoChanges in phos-phatized ERK in the spinal dorsal horn in adjuvant arthritisrats during electroacupuncture of Jiaji points for analgesiardquoShanghai Journal of Acupuncture and Moxibustion vol 24 no12 pp 33ndash35 2005

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 11

RN6 Qihai acupointERK Extracellular signal-regulated kinaseMAPK Mitogen-activated protein kinaseTNBS 246-Trinitrobenzene sulfonic acid

Acknowledgments

The authors thank Professor Shuhui Jiang for technical assis-tance and the support of the National Natural Science Foun-dation of China (no 81173331) and National Basic ResearchProgram of China (973 program no 2009CB522900)Xiaomei Wang and Yuan Lu have the same right

References

[1] G Manning D B Whyte R Martinez T Hunter and SSudarsanam ldquoThe protein kinase complement of the humangenomerdquo Science vol 298 no 5600 pp 1912ndash1934 2002

[2] C Mowat A Cole A Windsor et al ldquoGuidelines for themanagement of inflammatory bowel disease in adultsrdquoGut vol60 no 5 pp 571ndash607 2011

[3] Inflammatory Bowel Disease Co-operation Group ChineseSociety of Gastroenterology ldquoChinese consensus on diagnosisand treatment standard of inflammatory bowel diseaserdquoChineseJournal of Gastroenterology vol 12 no 8 pp 490ndash494 2007

[4] J J Zheng X S Zhu Z Huangfu Z X Gao Z R Guo andZ Wang ldquoCrohnrsquos disease in mainland China a systematicanalysis of 50 years of researchrdquo Chinese Journal of DigestiveDiseases vol 6 no 4 pp 175ndash181 2005

[5] M Dai and B M Li ldquoResearch progress in mechanisms ofintestinal fibrosis in Crohnrsquos diseaserdquo Shijie Huaren XiaohuaZazhi vol 17 no 11 pp 1117ndash1121 2009

[6] O Grip J Malm B Veress A Bjartell S Lindgren and AEgesten ldquoIncreased presence of cells containing transforminggrowth factor alpha (TGF-120572) in ulcerative colitis both duringactive inflammation and in remissionrdquo European Journal ofGastroenterology and Hepatology vol 12 no 7 pp 761ndash7662000

[7] I E Koutroubakis E Petinaki P Dimoulios et al ldquoSerumlaminin and collagen IV in inflammatory bowel diseaserdquoJournal of Clinical Pathology vol 56 no 11 pp 817ndash820 2003

[8] S Speca I Giusti F Rieder and G Latella ldquoCellular andmolecular mechanisms of intestinal fibrosisrdquo World Journal ofGastroenterology vol 18 no 28 pp 3635ndash3661 2012

[9] Y F Song J H Hong and L M Xu ldquoTGF-120573 1ERK pathwaysand its inhibitors and fibrosisrdquo Chinese Journal of ClinicalHepatology vol 21 no 2 pp 125ndash127 2005

[10] H C Huang M Yang J Z Li and H Y Wang ldquoConnectivetissue growth factor promotes the proliferation ofmyofibroblastthrough Erk-12 signaling pathwayrdquoNational Medical Journal ofChina vol 85 no 19 pp 1322ndash1326 2005

[11] L P Guo H C Huang and J Z Li ldquoHypoxia inducesmyofibroblast formation and stimulates production of collagenI in myofibroblasts through ERK12 pathwayrdquo Chinese Journalof Pathophysiology vol 24 no 12 pp 2428ndash2432 2008

[12] H R Liu X P Ma T P Zhao and H G Wu ldquoThe adjustmentof acupuncture on colon collagen synthesis and TGF-120573mRNAexpression in Crohnrsquos disease ratrdquo Journal of Anhui TraditionalChinese Medical College vol 24 no 4 pp 25ndash30 2005

[13] X P Ma C P An H G Wu et al ldquoHerbs-partitionedmoxibustion and electro-acupuncture effects on transforming

growth factor-beta 1 connective tissue growth factor collagentype I and fibronectin expression in the colon of Crohnrsquo sdisease ratsrdquo Journal of Clinical Rehabilitative Tissue EngineeringResearch vol 12 no 20 pp 3853ndash3858 2008

[14] G P Morris P L Beck M S Herridge W T Depew M RSzewczuk and J LWallace ldquoHapten-inducedmodel of chronicinflammation and ulceration in the rat colonrdquoGastroenterologyvol 96 no 3 pp 795ndash803 1989

[15] TheMinistry of Science andTechnology of the Peoplersquos Republicof China Guidance Suggestions for the Care and Use ofLaboratory Animals 2006

[16] J D Butzner R Parmar C J Bell andVDalal ldquoButyrate enematherapy stimulates mucosal repair in experimental colitis in theratrdquo Gut vol 38 no 4 pp 568ndash573 1996

[17] X M Wang Y Lu L Y Wu et al ldquoMoxibustion inhibitsinterleukin-12 and tumor necrosis factor alpha and modulatesintestinal flora in rat with ulcerative colitisrdquo World Journal ofGastroenterology vol 18 no 46 pp 6826ndash6835 2012

[18] J B Pucilowska K K McNaughton N K Mohapatra et alldquoIGF-I and procollagen 1205721(I) are coexpressed in a subset ofmesenchymal cells in active Crohnrsquos diseaserdquo American Journalof Physiology vol 279 no 6 pp G1307ndashG1322 2000

[19] M Gharaee-Kermani B Hu S H Phan and M R GyetkoldquoRecent advances in molecular targets and treatment of idio-pathic pulmonary fibrosis focus on TGF120573 signaling and themyofibroblastrdquo Current Medicinal Chemistry vol 16 no 11 pp1400ndash1417 2009

[20] E M Zimmermann L Li Y T Hou M Cannon G MChristman andKN Bitar ldquoIGF-I induces collagen and IGFBP-5 mRNA in rat intestinal smooth musclerdquo American Journal ofPhysiology vol 273 no 4 pp G875ndashG882 1997

[21] L J ZhangQ Li P Chen et al ldquoMediating effect of extracellularsignal-regulated kinase 12 in PDGF-induced proliferation andcollagen synthesis of cultured rat cardiac fibroblastsrdquo ChineseJournal of Anatomy vol 13 no 4 p 223 2007

[22] M Tang W Zhang H Lin H Jiang H Dai and Y ZhangldquoHigh glucose promotes the production of collagen types Iand III by cardiac fibroblasts through a pathway dependenton extracellular-signal-regulated kinase 12rdquo Molecular andCellular Biochemistry vol 301 no 1-2 pp 109ndash114 2007

[23] H Y Dai Z M Ge and Y H Li ldquoEffect of urotensin II on pro-liferative potential and phosphorylation of extracellular signal-regulated kinase 12 adventitial fibroblasts from spontaneouslyhypertensive ratrdquoActaAcademiaeMedicinae Sinicae vol 28 no6 pp 776ndash780 2006

[24] I Fuentes-Calvo A M Blazquez-Medela N Eleno E SantosJ M Lopez-Novoa and C Martınez-Salgado ldquoH-Ras isoformmodulates extracellular matrix synthesis proliferation andmigration in fibroblastsrdquo American Journal of Physiology vol302 no 4 pp C686ndashC697 2012

[25] Y W Ma G T Bai and M X Zhang ldquoStudies on signal trans-duction pathway induced by fibronectin in human embryoniclung fibroblastsrdquo Shanxi Medical Journal vol 38 no 7 pp 587ndash589 2009

[26] J H Jang and C P Chung ldquoFibronectin-mediated adhesionrescues cell cycle arrest induced by fibroblast growth factor-1 bydecreased expression of P21CIPWAF in human chondrocytesrdquoIn Vitro Cellular and Developmental Biology vol 41 no 5-6 pp126ndash129 2005

[27] B Zhu Y J Wang C F Zhu et al ldquoTriptolide inhibitsextracellularmatrix protein synthesis by suppressing the Smad2

12 Evidence-Based Complementary and Alternative Medicine

but not the MAPK pathway in TGF-1205731-stimulated NRK-49Fcellsrdquo Nephrology Dialysis Transplantation vol 25 no 10 pp3180ndash3191 2010

[28] J Bai X S Liu Y J Xu M Xie and W Ni ldquoEffects ofextracellular signal regulated kinase signaling pathway on cellcycle of airway smooth muscle cells in asthmatic ratsrdquo ChineseJournal of Respiratory and Critical Care Medicine vol 9 no 1pp 23ndash27 2010

[29] S Matsusaka and I Wakabayashi ldquoEnhancement of vascu-lar smooth muscle cell migration by urotensin IIrdquo Naunyn-Schmiedebergrsquos Archives of Pharmacology vol 373 no 5 pp 381ndash386 2006

[30] Y Chen Z Dai Y M Liu et al ldquoInhibitory effects ofCGRP on vascular smooth muscle cell proliferation role ofcaveolaecaveolin-1ERK12 signal pathwayrdquo Progress in Bio-chemistry and Biophysics vol 40 no 5 pp 445ndash453 2013

[31] W L Wu A Q Zhang Q Yin and W H Gan ldquoEffectsof ERK signal pathway on extracellular matrix synthesis ofrat mesangial cells treated with PDGF-BBrdquo ACTA UniversityMedicinalis Nanjing vol 27 no 10 pp 1126ndash1129 2007

[32] L S Steelman W H Chappell S L Abrams et al ldquoRoles ofthe RafMEKERK and PI3KPTENAktmTOR pathways incontrolling growth and sensitivity to therapy-implications forcancer and agingrdquo Aging vol 3 no 3 pp 192ndash222 2011

[33] W Kolch ldquoMeaningful relationships the regulation of theRasRafMEKERK pathway by protein interactionsrdquo Biochem-ical Journal vol 351 no 2 pp 289ndash305 2000

[34] D Matallanas M Birtwistle D Romano et al ldquoRaf familykinases old dogs have learned new tricksrdquo Genes and Cancervol 2 no 3 pp 232ndash260 2011

[35] C S Mason C J Springer R G Cooper G Superti-Furga C JMarshall and RMarais ldquoSerine and tyrosine phosphorylationscooperate in Raf-1 but not B-Raf activationrdquo The EMBOJournal vol 18 no 8 pp 2137ndash2148 1999

[36] R J Fiddes P W Janes S P Sivertsen R L Sutherland EA Musgrove and R J Daly ldquoInhibition of the MAP kinasecascade blocks heregulin-induced cell cycle progression in T-47D human breast cancer cellsrdquo Oncogene vol 16 no 21 pp2803ndash2813 1998

[37] S T Eblen J K SlackM JWeber andA D Catling ldquoRac-PAKsignaling stimulates extracellular signal-regulated kinase (ERK)activation by regulating formation of MEK1-ERK complexesrdquoMolecular and Cellular Biology vol 22 no 17 pp 6023ndash60332002

[38] M Cargnello and P P Roux ldquoActivation and function ofthe MAPKs and their substrates the MAPK-activated proteinkinasesrdquo Microbiology and Molecular Biology Reviews vol 75no 1 pp 50ndash83 2011

[39] A V Khokhlatchev B Canagarajah J Wilsbacher et al ldquoPhos-phorylation of theMAP kinase ERK2 promotes its homodimer-ization and nuclear translocationrdquo Cell vol 93 no 4 pp 605ndash615 1998

[40] R H Chen C Sarnecki and J Blenis ldquoNuclear localization andregulation of erk- and rsk-encoded protein kinasesrdquoMolecularand Cellular Biology vol 12 no 3 pp 915ndash927 1992

[41] S Traverse K Seedorf H Paterson C J Marshall P Cohenand A Ullrich ldquoEGF triggers neuronal differentiation of PC12cells that overexpress the EGF receptorrdquo Current Biology vol 4no 8 pp 694ndash701 1994

[42] T T Nguyen J C Scimeca C Filloux P Peraldi J L Car-pentier and E Van Obberghen ldquoCo-regulation of the mitogen-activated protein kinase extracellular signal-regulated kinase 1

and the 90-kDa ribosomal S6 kinase in PC12 cells Distincteffects of the neurotrophic factor nerve growth factor and themitogenic factor epidermal growth factorrdquo Journal of BiologicalChemistry vol 268 no 13 pp 9803ndash9810 1993

[43] I Dikic J Schlessinger and I Lax ldquoPC12 cells overexpressingthe insulin receptor undergo insulin-dependent neuronal dif-ferentiationrdquo Current Biology vol 4 no 8 pp 702ndash708 1994

[44] M H Shen Z R Li X R Xiang W M Niu and C B ZhangldquoEffect of electro-acupuncture on ERK protein expression ofMCAO ratsrdquo Journal of Clinical Acupuncture and Moxibustionvol 23 no 7 pp 62ndash63 2007

[45] M H Shen Z R Li X R Xiang W M Niu and C BZhang ldquoInvolvement of extracellular regulated protein kinasesignal transduction pathway in electroacupuncture-inducedimprovement of ischemic cerebral injury in rats with cerebralischemia reperfusionrdquo Acupuncture Research vol 32 no 6 pp368ndash372 2007

[46] Z B Yang J Yan and S X Yi ldquoUp-regulation of the ERK phos-phorylation in gastric mucosal cell by the serum of rats treatedwith electroacupuncture at stomach meridian acupointsrdquo Basicamp Clinical Medicine vol 29 no 2 pp 135ndash138 2009

[47] C L Wang S X Wang and W Gong ldquoChanges in phos-phatized ERK in the spinal dorsal horn in adjuvant arthritisrats during electroacupuncture of Jiaji points for analgesiardquoShanghai Journal of Acupuncture and Moxibustion vol 24 no12 pp 33ndash35 2005

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

12 Evidence-Based Complementary and Alternative Medicine

but not the MAPK pathway in TGF-1205731-stimulated NRK-49Fcellsrdquo Nephrology Dialysis Transplantation vol 25 no 10 pp3180ndash3191 2010

[28] J Bai X S Liu Y J Xu M Xie and W Ni ldquoEffects ofextracellular signal regulated kinase signaling pathway on cellcycle of airway smooth muscle cells in asthmatic ratsrdquo ChineseJournal of Respiratory and Critical Care Medicine vol 9 no 1pp 23ndash27 2010

[29] S Matsusaka and I Wakabayashi ldquoEnhancement of vascu-lar smooth muscle cell migration by urotensin IIrdquo Naunyn-Schmiedebergrsquos Archives of Pharmacology vol 373 no 5 pp 381ndash386 2006

[30] Y Chen Z Dai Y M Liu et al ldquoInhibitory effects ofCGRP on vascular smooth muscle cell proliferation role ofcaveolaecaveolin-1ERK12 signal pathwayrdquo Progress in Bio-chemistry and Biophysics vol 40 no 5 pp 445ndash453 2013

[31] W L Wu A Q Zhang Q Yin and W H Gan ldquoEffectsof ERK signal pathway on extracellular matrix synthesis ofrat mesangial cells treated with PDGF-BBrdquo ACTA UniversityMedicinalis Nanjing vol 27 no 10 pp 1126ndash1129 2007

[32] L S Steelman W H Chappell S L Abrams et al ldquoRoles ofthe RafMEKERK and PI3KPTENAktmTOR pathways incontrolling growth and sensitivity to therapy-implications forcancer and agingrdquo Aging vol 3 no 3 pp 192ndash222 2011

[33] W Kolch ldquoMeaningful relationships the regulation of theRasRafMEKERK pathway by protein interactionsrdquo Biochem-ical Journal vol 351 no 2 pp 289ndash305 2000

[34] D Matallanas M Birtwistle D Romano et al ldquoRaf familykinases old dogs have learned new tricksrdquo Genes and Cancervol 2 no 3 pp 232ndash260 2011

[35] C S Mason C J Springer R G Cooper G Superti-Furga C JMarshall and RMarais ldquoSerine and tyrosine phosphorylationscooperate in Raf-1 but not B-Raf activationrdquo The EMBOJournal vol 18 no 8 pp 2137ndash2148 1999

[36] R J Fiddes P W Janes S P Sivertsen R L Sutherland EA Musgrove and R J Daly ldquoInhibition of the MAP kinasecascade blocks heregulin-induced cell cycle progression in T-47D human breast cancer cellsrdquo Oncogene vol 16 no 21 pp2803ndash2813 1998

[37] S T Eblen J K SlackM JWeber andA D Catling ldquoRac-PAKsignaling stimulates extracellular signal-regulated kinase (ERK)activation by regulating formation of MEK1-ERK complexesrdquoMolecular and Cellular Biology vol 22 no 17 pp 6023ndash60332002

[38] M Cargnello and P P Roux ldquoActivation and function ofthe MAPKs and their substrates the MAPK-activated proteinkinasesrdquo Microbiology and Molecular Biology Reviews vol 75no 1 pp 50ndash83 2011

[39] A V Khokhlatchev B Canagarajah J Wilsbacher et al ldquoPhos-phorylation of theMAP kinase ERK2 promotes its homodimer-ization and nuclear translocationrdquo Cell vol 93 no 4 pp 605ndash615 1998

[40] R H Chen C Sarnecki and J Blenis ldquoNuclear localization andregulation of erk- and rsk-encoded protein kinasesrdquoMolecularand Cellular Biology vol 12 no 3 pp 915ndash927 1992

[41] S Traverse K Seedorf H Paterson C J Marshall P Cohenand A Ullrich ldquoEGF triggers neuronal differentiation of PC12cells that overexpress the EGF receptorrdquo Current Biology vol 4no 8 pp 694ndash701 1994

[42] T T Nguyen J C Scimeca C Filloux P Peraldi J L Car-pentier and E Van Obberghen ldquoCo-regulation of the mitogen-activated protein kinase extracellular signal-regulated kinase 1

and the 90-kDa ribosomal S6 kinase in PC12 cells Distincteffects of the neurotrophic factor nerve growth factor and themitogenic factor epidermal growth factorrdquo Journal of BiologicalChemistry vol 268 no 13 pp 9803ndash9810 1993

[43] I Dikic J Schlessinger and I Lax ldquoPC12 cells overexpressingthe insulin receptor undergo insulin-dependent neuronal dif-ferentiationrdquo Current Biology vol 4 no 8 pp 702ndash708 1994

[44] M H Shen Z R Li X R Xiang W M Niu and C B ZhangldquoEffect of electro-acupuncture on ERK protein expression ofMCAO ratsrdquo Journal of Clinical Acupuncture and Moxibustionvol 23 no 7 pp 62ndash63 2007

[45] M H Shen Z R Li X R Xiang W M Niu and C BZhang ldquoInvolvement of extracellular regulated protein kinasesignal transduction pathway in electroacupuncture-inducedimprovement of ischemic cerebral injury in rats with cerebralischemia reperfusionrdquo Acupuncture Research vol 32 no 6 pp368ndash372 2007

[46] Z B Yang J Yan and S X Yi ldquoUp-regulation of the ERK phos-phorylation in gastric mucosal cell by the serum of rats treatedwith electroacupuncture at stomach meridian acupointsrdquo Basicamp Clinical Medicine vol 29 no 2 pp 135ndash138 2009

[47] C L Wang S X Wang and W Gong ldquoChanges in phos-phatized ERK in the spinal dorsal horn in adjuvant arthritisrats during electroacupuncture of Jiaji points for analgesiardquoShanghai Journal of Acupuncture and Moxibustion vol 24 no12 pp 33ndash35 2005

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom