effectofmoxa-burningheatstimulatingliangmen(st21)andzu- … · 2017-02-20 · ation ( xˉ± s)....

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JTCM |www. journaltcm. com June 15, 2016 |Volume 36 | Issue 3 |

Online Submissions: http://www.journaltcm.com J Tradit Chin Med 2016 June 15; 36(3): [email protected] ISSN 0255-2922

EXPERIMENTAL STUDY

Effect of moxa-burning heat stimulating Liangmen (ST 21) and Zu-sanli (ST 36) on proliferation and apoptosis signaling proteins inrats with stress-induced gastric ulcer

Peng Li, Wang Yadong, Chang Xiaorong, Wu Huangan, Liu Mi, Ma Fuqiang, Wang Hong, Chen Jiaolong, WangChao, Quan Renfu, Yang Zongbaoaa

Peng Li, Wang Yadong, Ma Fuqiang, Wang Hong, Chen Ji-aolong, Yang Zongbao, Medical College, Xiamen Universi-ty, Xiamen 361102, China.Chang Xiaorong, Liu Mi, Wang Chao, Acupuncture andMoxibustion college, Hunan University of TCM, Changsha410208, ChinaWu Huangan, Research Institute of Acupuncture and Moxi-bustion, Shanghai University of TCM, Shanghai 200030,ChinaQuan Renfu, Research Institute of Acupuncture and Moxi-bustion, Xiaoshan Traditional Chinese Medical Hospital,Zhejiang 311200, China.Supported by National Natural Science Foundation of Chi-na: To Study the Pre-protection Mechanism of Moxibus-tion Treatment of Acupoints of Stomach Meridian Regulat-ing Egfr Signaling Pathway in Chronic Atrophic GastritisPrecancerous Lesions (No. 81260556), National Natural Sci-ence Foundation of China: to Study Brain-stomach Re-sponse Mechanism of Electro-acupuncture Treatment ofAcupoints of Stomach Meridian Repairing Gastric MucosaDamage Based on Nuclear Magnetic Resonance Metabo-nomics Technology (No. 81473751), the National Key BasicResearch Development Plan (973 Plan): the Basic and Clini-cal Research of Moxibustion and Acupuncture Effect Char-acteristics and the Similarities and Differences of Their Ef-fect Response (2015cb554502), Scientific Research Founda-tion for the Returned Overseas Chinese Scholars, Ministryof Education of China: to Study the Mechanism of Com-patibility of Yiqi And Huoxue Traditional Chinese Herbs Se-lective Amplifying Regulatory T Cells to Inhibit Atheroscle-rosis (No. 2015-311)Correspondence to: Prof. Yang Zongbao, Medical College,Xiamen University, Xiamen 361102, China, [email protected]. Chang Xiaorong, Acupuncture and Moxibustion col-lege, Hunan University of TCM, Changsha 410208, China. [email protected]: +86-592-2183069Accepted: January 12, 2016

AbstractOBJECTIVE: To observe the effect of moxa-burningheat stimulating acupoints of Liangmen (ST 21)and Zusanli (ST 36) on the proliferation and apopto-sis signaling proteins in rats with stress-inducedgastric ulcer.

METHODS: Forty rats were randomly divided intofour groups: negative control (NC), ulcer control(UC), acupoints of stomach meridian (ASM), andacupoints control (AC). The acute gastric ulcer mod-el was established by bound and water immersion.Rats in NC and UC groups didn't receive anymoxa-burning heat stimulating treatment, whilerats in ASM and AC groups were treated with bur-ingmoxa heat stimulating the acupoints of Liang-men (ST 21) and Zusanli (ST 36) and their con-trolled points, respectively. Rats in all groups weresacrificed after 12 consecutive days treatment. Theulcer index was evaluated by using Guth's method.The expression of tumor necrosis factor-alpha(TNF-α), apoptotic protease activating facter-1(Apaf-1), Caspase-3, p21 activated kinase 1 (PAK1),extracellular regulated protein kinases 2 (ERK2),phosphorylated ERK2 (pERK2), phosphoinositide3-kinase (PI3K) and RAC-alpha serine/threo-nine-protein kinase (Akt) in gastric mucosa was de-tected by enzyme linked immunosorbent assay(ELISA).

RESULTS: Compared with UC group, the ulcer in-dex of ASM and AC groups decreased, and the in-jured gastric mucosa was improved, the expressionof TNF-α, Apaf-1 and Caspase-3 in gastric mucosawas significantly reduced (P < 0.05), while the ex-

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This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).© 2016 JTCM.

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Peng L et al. / Experimental Study

pression of PAK1, ERK2, pERK2, PI3K and Akt in gas-tric mucosa was significantly increased (P < 0.05).And ASM showed better effect than AC group (P <0.05).

CONCLUSION: Moxa-burning Heat stimulating ofLiangmen (ST 21) and Zusanli (ST 36) could pro-mote the recovery of gastric mucosal lesion proba-bly by inhibiting cell apoptosis and promoting cellproliferation in stress-induced gastric ulcer.

© 2016 JTCM. All rights reserved.

Key words: Moxibustion; Point ST 21 (Liangmen);Point ST 36 (Zusanli); Stomach ulcer; Apoptosis; Cellproliferation

INTRODUCTIONPeptic ulcer is one of the common and frequent occur-ring gastrointestinal diseases, which can be induced bymany factors including cold stimulation, ethanol in-take, emotional stress, high-dose use of non-steroidalanti-inflammatory drugs, etc.1 Erosion, ulceration andhemorrhage following gastric mucosal protection bro-ken are the major pathological foundation of stress-in-duced gastric ulcer.2 Now, the treatment strategy of thisdisease include two approaches, one is to inhibit the se-cretion of gastric acid, the other one it to strength thegastric mucosal protection.3 However, the present medi-cations can not cure the ulcers, and have some side ef-fects.4 Considering its lethal complication and the de-fects of present drug treatment, it is necessary to seek anew and more effective therapeutic mean for stress-in-duced gastric ulcer.At present, the detailed mechanism of stress-inducedgastric ulcer is not clear. It is reported that gastric mu-cosal cell apoptosis, mucosal ischemia, mucus-mucosalbarrier damage and inflammatory cytokines over-ex-pression are believed to be involved in its mechanism.2

Moxa-burning heat stimulating acupoints of Zusanli(ST 36) and Zhongwan (CV 12) could alleviate gastricmucosal lesion by over-expressing HSP60 and HSP70,down-regulating Smac and inhibiting mucosal cellapoptosis, the selected two acupoints located on stom-ach meridian.5 Our preliminary results showed thatmoxa-burning heat stimulating acupoints of stomachmeridian-Liangmen (ST 21) and Zusanli (ST 36)could regulate phosphorylation level of lots of signal-ing proteins, promote the recovery of injured gastricmucosa in rat model of stress gastric ulcers.6 But westill don't know which signaling pathway can be target-ed by moxa-burning heat stimulating treatment. Thisstudy focused on the effect of moxa-burning heat stim-ulating acupoints on cell apoptosis and proliferationduring the repair of gastric mucosa.

MATERIALS AND METHODSAnimalsAll experiments were carried out using specific patho-gen free level healthy Sprague-Dawley rats (200-220 g,10-11 weeks old, half male and half female) with freeaccess to food and water and in a 12 h dark-light cycleat 20-22 ℃ and 65%-70% relative humidity. All theserats were provided by Xiamen University LaboratoryAnimal Center [Certificate number: SCXK (Min)2008-0001] and housed in an air-conditioned atmo-sphere. The rats were handled in accordance with theregulations for the administration of affairs concerningexperimental animals (Approved by People's Republicof China State Council on October 31, 1988 and pro-mulgated by Decree No. 2 of the State Science andTechnology Commission on November 14, 1988). In-ternational rules were strictly followed when dealt withanimals and the animal protocol has been previouslyapproved by the ethics committee for administration ofexperimental animals in Medical College of XiamenUniversity.

Reagents and instrumentsMicroplate reader (type 352, LabSystems MultiskanMS) and plate washer (type AC8, LabSystems Multi-skan MS) were purchased from Thermo Fisher Scientif-ic Inc. (Waltham, MA, USA); Homothermal incubator(type GNP-9080) was purchased from Shanghai JingHong Laboratory Instrument Co., Ltd. (Shanghai, Chi-na); Anti-rat ELISA kits, including tumor necrosis fac-tor-alpha (TNF-α), apoptotic protease activating fact-er-1 (Apaf-1), Caspase-3, p21 activated kinase 1(PAK1), extracellular regulated protein kinases 2(ERK2), phosphorylated ERK2 (pERK2), phos-phoinositide 3-kinase (PI3K), RAC-alpha serine/threo-nine-protein kinase (Akt) were bought from Wuhan Nu-olitai BiologicalTechnologyCo., Ltd. (Wuhan,China).

Modeling and groupingForty rats were divided randomly into 4 groups accord-ing to the random number table method: negative con-trol (NC), ulcer control (UC), acupoints of stomachmeridian (ASM) and acupoints control (AC) (10 ratsfor each group). Briefly, each group need at least 8 ratsaccording to our preliminary results, here we use 10 ratsin each group. Next, all 40 rats were labeled with 1-40number and put these numbers in random number ta-ble, we chose the first 10 numbers as NC group, thenext 10 numbers as UC group, the next 10 numbers asASM group and last 10 numbers as AC group. Model-ing method of stress-induced gastric ulcer as follow:the four limbs of all rats were bound on the boards for20 min. Except the rats in negative control, the otherrats were dipped into thermostatic water tank (20 ±1) ℃ vertically for 10 h (the level of the water reachedthe sternum xiphoid). Rats were taken out of waterand the acute gastric ulcer model was established suc-cessfully by the pathological diagnosis.

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Localization of acupoints and controlled pointsAcupoints of Liangmen (ST 21) and Zusanli (ST 36)were pinpointed following the methods described inExperimental Acupuncture & Moxibustion, a textbookfor National Higher Institutions of Traditional ChineseMedicine in the New Century.7 Their controlled pointswere 0.5 cm away from them.

TreatmentRats in ASM and UC groups were treated withmoxa-burning heat stimulating acupoints of Liangmen(ST 21) and Zusanli (ST 36) and their controlledpoints, respectively after the gastric ulcer model was es-tablished. Rats in the other two groups received nosuch treatment. Rats in ASM and AC groups werefixed on the boards. Hair was clipped around treatedacupoints and the controlled points. Moxa-burningsticks were fixed with hand to make sure that the litends were 2 cm away from the skin. Each acupoint wastreated for 20 min per day for 12 consecutive days. Ev-ery treatment use unilateral acupoints for one time andthe other lateral acupoints for next time.

Specimen collectionFasting for 24 h after the treatment, rats were anesthe-tized intraperitoneally with 10% chloral hydrate, andthen were sacrificed and their stomachs were dissected.Each stomach was opened along the greater curvatureand washed with ice-cold saline. Injured gastric tissuewith the size of 1 cm × 0.5 cm was cut out and homog-enized in saline. The homogenate was put in refrigera-tor with the temperature of － 20℃ . Cell membranewas damaged by repeated freezing and thawing twiceand the homogenate was centrifuged for 5 min (5000rpm/min, 2-8 ℃ ), and the supernatant fluid was usedfor protein test. In addition, representative stomachspecimens were taken from each group for histopatho-logical examination.

Ulcer index of gastric mucosaThe ulcer index was recorded and calculated accordingto the method of Guth's.8 Ulcer length ≤ 1mm (includ-ing erosion foci) was sored 1; 1 mm < ulcer length ≤2 mm scored 2; 2 mm < ulcer length ≤ 3 mm scored 3;3 mm < ulcer length ≤ 4 mm scored 4; ulcer length >4 mmscored 5; ulcerwidth > 2mm, scorewas doubled.

Pathological morphological analysisThe ulcer area of 0.5 cm × 0.5 cm was collected fromgastric mucosa and immobilized for 24 h in 10% neu-tralized formaldehyde solution. After paraffin was em-bedded, the specimens were ready for cut. Paraffin sec-tions (5 μm) were stained with hematoxylin-eosin(HE) and observed under the microscope magnified100 times or 400 times respectively.

Enzyme linked immunosorbent assay (ELISA)Signaling molecules of cell apoptosis and proliferationin stress-induced gastric ulcer were assessed by measur-

ing the levels of TNF-alpha, Caspase-3, Apaf-1 andPAK1, ERK2, pERK2, PI3K, Akt in the gastric tissueof different groups respectively. The steps were carriedout according to the manufacturer's instructions of an-ti-rat ELISA kits. Briefly, Pipette 50 μL of standards,controls, and diluted samples to the appropriate mi-crotiter wells. Cover wells with plate cover and incu-bate for 30 min at 37 ℃ . Thoroughly aspirate or de-cant solution from wells and discard the liquid. Washwells 4 times. Pipette 50 μL Biotin-conjugated detec-tion antibody was put into each well except the chro-mogen blank. Cover wells with plate cover and incu-bate 30 min at 37 ℃. Thoroughly aspirate or decant so-lution from wells and discard the liquid. Wash wells4 times. Pipette 50 μL streptavidin-HRP solution wasadded in each well except the chromogen blank. Coverwells with the plate cover and incubate for 30 min at37 ℃ . Thoroughly aspirate or decant solution fromwells and discard the liquid. Wash wells 4 times. Pi-pette 50 μL of stabilized chromogen to each well. Theliquid in the wells will begin to turn blue. Incubate for15 min at 37 ℃ and in the dark. Pipette 50 μL of stopsolution was added in each well. The solution in thewells should change from blue to yellow. Read the ab-sorbance of each well at 450 nm (OD values) within2 h after adding the stop solution.

Statistical analysisParameter data were expressed as mean ± standard devi-ation ( x̄ ± s). Analysis of variance were performed totest the differences between the groups) with SPSS forWindows (SPSS Inc., V ersion 11.5, Chicago, IL, USA).P < 0.05 was considered to be statistically significant.

RESULTSStress-induced gastric lesionsThe ulcerated area in gastric mucosa has been mea-sured and the ulcer index was used to evaluate injuryin all groups (Figure 1). Ulcer index for rats in negative

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GroupsFigure 1 Effect of moxa-burning heat stimulating acupointson ulcer index in ulcerated ratsThe figure show ulcer index of four groups. Rats in NC andUC groups didn't receive any treatment. Acupoints of stom-ach meridian-Liangmen (ST 21) and Zusanli (ST 36) in ASMgroup were treated with moxa-burning heat stimulating.Their control points in AC group were treated withmoxa-burning heat stimulating. NC: negative control; UC: ul-cer control; ASM: acupoints of stomach meridian; AC: acu-points control. Data are expressed as mean ± standard devi-ation (n = 10). aP < 0.05, compared with NC group; bP < 0.05,compared with UC group; cP < 0.05, compared with ACgroup.

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control group was very low. Rats received stress injurygot a serious gastric mucosal injury and their ulcer in-dex increased about 59-fold compared with negativecontrol. Moxa-burning heat stimulating acupoints de-creased the ulcer index, especially selecting the acu-points of stomach meridian, which suggested that mox-ibustion of acupoints of stomach meridian could pro-mote gastric mucosa recovery.

Gastric mucosal pathologyFigure 2 showed the histopathological alterations instomach specimens of different groups. Gastric mucosafrom the negative control showed that epithelium struc-ture was intact and cell had no hyperaemia and edema,submucosa and muscularis contained no inflammatorycells. The stress-induced gastric ulcer from ulcer con-trol showed that the structure of gastric mucosa hadbeen damaged totally, gastric gland cell necrosis, cellcontained hyperaemia and edema, lots of inflammato-ry cells infiltrated in mucosa, submucosa as well asmuscularis. The stomach specimen of moxa-burningheat stimulating acupoints in stomach meridianshowed slight injury, epithelium structure was intactand no inflammatory cells infiltrating in it. Whilemoxa-burning heat stimulating the control acupointsshowed unintact epithelium structure, there was noblood capillary proliferation and hyperaemia, only lit-tle inflammatory infiltration in gastric wall. This resultsuggested that moxa-burning heat stimulating the acu-points of stomach meridian could reduce gastric muco-sa injury induced by stress.

Cell apoptosis-related factorsSignaling molecules, such as TNF-α, Apaf-1 and Cas-pase-3, are the markers of cell apoptosis. The normallevel of TNF-α, Apaf-1 and Caspase-3 in gastric muco-sa were 168.13, 295.75 and 5.93 ng/mL respectively.Stress-induced gastric ulcer could increase to 1.24,

1.22, 1.36-fold in gastric mucosa. Moxa-burning heatstimulating the acupoints significantly attenuated thestress-induced increase in these signaling molecules byabout 15%, 12% and 20% respectively, which were bet-ter effective than moxa-burning heat stimulating on thecontrolled points (Figure 3). These findings suggestedthat moxa-burning heat stimulating the acupoints couldreduce cell apoptosis in stress-induced gastric ulcer.

Cell proliferation-related signaling proteinsThe effect of moxa-burning heat stimulating acupointson the intracellular signaling pathway was showed inFigure 4, including PAK1/ERK2 pathway and PI3K/Akt pathway. These targets were significantly increasedin stress-induced gastric ulcer compared to negativecontrol. Moxa-burning heat stimulating the acupointsor the control points could increase their expression sig-nificantly in gastric mucosa compared to ulcer control.When compared to AC group,moxa-burning heat stimu-lating the acupoints was more effective in increasingtheir expression. These results implied that moxa- burn-ing heat stimulating the acupoints in stomach meridi-an activated PAK1/ERK2 and PI3K/Akt signaling path-ways and following proliferation signal transduction.

DISCUSSIONPeptic ulcer is a common digestive disease. Modernmedical treatment, including the eradication of H. py-lori, inhibition of gastric acid secretion and protectionof gastric mucosa, do not cure this disease, and accom-panied with many side effects.9 Previous studies haveshown that moxa-burning heat stimulating the acu-points of stomach meridian can reduce the gastric mu-cosal injury, increase the gastric protection mechanism,and so can be used as an effective treatment of gastroin-testinal diseases.10,11 Bound and water dipping inducedsevere gastric mucosal injury, which was confirmed by

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Figure 2 Histological evaluation of stomach sections of rats from different groups (HE staining)A-D: the low amplification (× 100) of hematoxylin-eosin staining of representative gastric mucosa section from four groups: A:negative control; B: ulcer control; C: acupoints of stomach meridian; D: acupoints control. E-H: the high amplification (× 400) fromA-D groups respectively. Rats in negative control and ulcer control groups didn't receive any treatment. Acupoints of stomach me-ridian-Liangmen (ST 21) and Zusanli (ST 36) in ASM group were treated with moxa-burning heat stimulating. Their control pointsin AC group were treated with moxa-burning heat stimulating. HE: hemotoxylin and eosin.

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the histopathological examination, including cell hyper-aemia, edema and necrosis, inflammatory cells infiltra-tion, and the damage of stomach mucosa structure.The ulcer recording showed a significant high ulcer in-dex in ulcer control group (about 59-fold compared

with negative control). In contrast, the ulcer index wassignificantly decreased by moxa-burning heat stimulat-ing the acupoints, especially better effect when chosethe acupoints of stomach meridian than the controlpoints. Moxa-burning heat stimulating acupoints also

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Groupswere treated with moxa-burning heat stimulating. Their control points in AC group were treated with moxa-burning heat stimu-lating. Data are expressed as mean ± standard deviation (n = 10). aP < 0.05, compared with NC group; bP < 0.05, compared with UCgroup; cP < 0.05, compared with AC group.

Figure 3 Effect of moxa-burning heat stimulating acupoints on cell apopto-sis-related factors in gastric mucosaA: the expression of TNF-α in gastric mucosa from four groups; B: the proteinlevel of Apaf-1 in gastic mucosa from four groups; C: the comparison of Cas-pase-3 in gastric mucosa between four groups. All these protein were detect-ed by ELISA method after 12 consecutive days moxa-burning heat stimulat-ing treatment. TNF-α: tumor necrosis factor-alpha; Apaf-1: apoptotic proteaseactivating facter-1; ELISA: enzyme linked immunosorbent assay; NC: negativecontrol; UC: ulcer control; ASM: acupoints of stomach meridian; AC: acupointscontrol. Rats in NC and UC groups didn't receive any treatment. Acupoints ofstomach meridian-Liangmen (ST 21) and Zusanli (ST 36) in ASM group

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GroupsFigure 4 Effect of moxa-burning heat stimulating acupoints on cell prolif-eration-related signaling proteins in gastric mucosaA: the protein level of PAK1 in gastric mucosa from four groups; B and C:the expression of ERK2 and pERK2 in gastric mucosa from four groups; Dand E: the protein expression of PI3K and Akt in gastric mucosa from fourgroups. All these protein were detected by ELISA method after 12 consec-utive days' moxa-burning heat stimulating treatment. PAK1: p21 activat-ed kinase 1; ERK2: extracellular regulated protein kinases 2; pERK2: phos-phorylated ERK2; PI3K: phosphoinositide 3-kinase; Akt: RAC-alpha serine/

threonine-protein kinase; ELISA: enzyme linked immunosorbent assay; NC: negative control; UC: ulcer control; ASM: acupoints ofstomach meridian; AC: acupoints control. Rats in NC and UC groups didn't receive any treatment. Acupoints of stomach meridi-an-Liangmen (ST 21) and Zusanli (ST 36) in ASM group were treated with moxa-burning heat stimulating. Their control points inAC group were treated with moxa-burning heat stimulating. Data are expressed as mean ± standard deviation (n = 10). aP < 0.05,compared with NC group; bP < 0.05, compared with UC group; cP < 0.05, compared with AC group.

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absolutely attenuated stress-induced acute stomach mu-cosa injury by protecting stomach mucosal structureand decreasing inflammatory cells infiltration in sub-mucosa and muscularis. Based on the above results,moxa-burning heat stimulating the acupoints of stom-ach meridian showed a significant protective role instress-induced gastric ulcer.A lot of evidence showed that the induction of apopto-sis was involved in the mechanism of acute gastric ul-cer in both human and rats, the signaling molecules in-cluding TNF-α and caspase-3 were positive related togastric mucosal damage.5,12,13 The role of TNF-α is toactivate NF-κB and MAPK signaling pathway andstart caspase-3-mediated apoptotic program, andApaf-1 play an important role in Akt1-mediated cellapoptosis.14-16 In this study, TNF-α, Apaf-1 and cas-pase-3 were significantly increased in ulcer controlgroup. Moxa-burning heat stimulating the acupointsdecreased their content in ulcerated stomach and thebetter effect of acupoints of stomach meridian hadbeen observed. This suggested that moxa-burning heatstimulating the acupoints ameliorated stress-inducedgastric ulcer via anti-apoptotic activity.Previous studies have showed that the balance betweencell apoptosis and cell proliferation play an importantrole in maintaining gastric mucosa integrity.17 Bothapoptosis and cell proliferation were augmented inchronic ulcers induced by acetic acid, which suggesteda complex mechanism in the restore homeostatic stateof gastric mucosa.18 Our previous study have foundthat moxa-burning heat stimulating the acupoints ofstomach meridian had regulated lots of protein phos-phorylation by using genomics technology, includingdown-regultaing TRAF2, Stat1, p53, Mkp-3 andNf-κB, up-regulating MEK, Ras, P38MAPK, PI3K,PAK, B-Raf and ERK2, suggesting a balance regulatingrole of moxa-burning heat stimulating the acupointson apoptosis and cell proliferation.6 The activation ofPAK1/ERK2 pathway and PI3K/Akt pathway can in-duce cell proliferation and plays a pivotal role in gastricmucosal healing.19,20 The present study showed thatboth PAK1/ERK2 pathway and PI3K/Akt pathwayhad been up-regulated by stress induction. Moxa-burn-ing heat stimulating the acupoints of stomach meridi-an had further up-regulated these two pathways, sug-gesting a healing effect for ulcerated mucosa recoveryvia cell proliferation.In conclusion, the moxa-burning heat stimulating Li-angmen (ST 21) and Zusanli (ST 36) had a significantgastroprotective effect on bound and water immer-sion-induced gastric ulcer, and the underlying mecha-nism might involve inhibiting cell apoptosis and pro-moting cell proliferation.

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