qingchang wenzhong decoction ameliorates dextran sulphate … · 2016-09-15 ·...

Research ArticleQingchang Wenzhong Decoction AmelioratesDextran Sulphate Sodium-Induced Ulcerative Colitis inRats by Downregulating the IP10CXCR3Axis-Mediated Inflammatory Response

Tang-you Mao12 Rui Shi2 Wei-han Zhao12 Yi Guo12 Kang-li Gao12

Chen Chen12 Tian-hong Xie12 and Jun-xiang Li2

1Beijing University of Chinese Medicine No 11 North Third Ring East Road Beijing 100029 China2Gastroenterology Department Dongfang Hospital Beijing University of Chinese Medicine No 61st Section Fangxingyuan Fangzhuang Beijing 100078 China

Correspondence should be addressed to Jun-xiang Li lijunxiang1226163com

Received 30 January 2016 Revised 12 May 2016 Accepted 30 May 2016

Academic Editor Ying-Ju Lin

Copyright copy 2016 Tang-you Mao 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

Qingchang Wenzhong Decoction (QCWZD) is an effective traditional Chinese medicine prescription Our previous studies haveshown that QCWZDhas significant efficacy in patients withmild-to-moderate ulcerative colitis (UC) and in colonic mucosa repairin UC rat models However the exact underlying mechanism remains unknown Thus this study was conducted to determineQCWZDrsquos efficacy and mechanism in dextran sulphate sodium- (DSS-) induced UC rat models which were established by 7-dayadministration of 45 DSS solution QCWZD was administered daily for 7 days after which the rats were euthanized Diseaseactivity index (DAI) histological score (HS) and myeloperoxidase (MPO) level were determined to evaluate UC severity Seruminterferon gamma-induced protein 10 (IP10) levels were determined using ELISA kits Western blotting and real-time polymerasechain reaction were respectively used to determine colonic protein and gene expression of IP10 chemokine (cys-x-cys motif)receptor (CXCR)3 and nuclear factor- (NF-) 120581B p65 Intragastric QCWZD administration ameliorated DSS-induced UC asevidenced by decreased DAI HS and MPO levels Furthermore QCWZD decreased the protein and gene expression of IP10CXCR3 and NF-120581B p65 Overall these results suggest that QCWZD ameliorates DSS-induced UC in rats by downregulating theIP10CXCR3 axis-mediated inflammatory response and may be a novel UC therapy

1 Introduction

Ulcerative colitis (UC) a major phenotype of inflammatorybowel diseases (IBD) is characterized by chronic nonspecificinflammation of the colorectal mucosa and a relapsing-remitting course [1 2] The number of patients with UC inAsia is increasing annually and the incidence of UC hasincreased more than 3 times in the last 10 years in China [3]Although the exact pathogenesis of UC remains unclear it iswell known that intestinalmucosal immune systemdisordersintestinal mucosal barrier defects persistent intestinal infec-tions and genetic and environmental factors are involved inthe development of UC [4 5] Persistent intestinal infections

in particular are a major cause of UC [6] and alleviation ofinfection has become the main approach in the treatment ofUC

Interferon gamma- (IFN-120574-) induced protein 10 (IP10) isan endogenous chemokine belonging to the CXC subfamilyPrevious studies have demonstrated that the amounts ofIP10 in the serum and colon tissue of UC patients weresignificantly higher than the corresponding amounts inhealthy individuals [7] Thus inhibiting IP10 expression caneffectively resolve the clinical symptoms of UC Furthermoreblocking of IP10 expression was shown to alleviate sponta-neous colitis induced by IL-10 knockout in mice [8 9] IP10expression was shown to increase in multiple cells such as

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 4312538 10 pageshttpdxdoiorg10115520164312538

2 Evidence-Based Complementary and Alternative Medicine

Table 1 DAI scoring criteria

Types 0 1 2 3 4Percent weight loss 0 1ndash5 5ndash10 10ndash15 gt15Stool consistency Normal Mushy DiarrhoeaHaematochezia level Negative Positive Blood traces visible in stool

monocytes natural killer (NK) cells T helper (Th)1 cells andendothelial cells (ECs) under stimulation by intestinal IFN-120574and tumour necrosis factor- (TNF-) 120572 [10]

Chemokine (cys-x-cys motif) receptor 3 (CXCR3) is thespecific binding protein of IP10 and it is mainly expressedby epithelial and endothelial cells as well as lymphoid cellssuch as memory T cells NK cells B cells neutrophilsand monocytes Upon binding to IP10 CXCR3 is activatedwhich increases the chemotactic activity of CXCR3-positivecells and contributes to its transfer to local inflammatorylesions [11] Finally the intestinal mucosa produces largeamounts of inflammatory cytokines [12 13] which not onlyfurther stimulate IP10 thus recruitingmore immune cells andforming a vicious cycle but also directly or indirectly damagethe intestinal mucosal barrier and the intestinal mucosa[14 15] and promote the occurrence and development ofUC [16]Therefore the IP10CXCR3-mediated inflammatoryresponse plays an important role in the pathogenesis ofUC and inhibiting the IP10CXCR3 axis to block excessiveinflammation may be a new approach for treatment ofUC

At present an effective treatment for UC is lackingAmino salicylic acid steroid hormones and immunosup-pressive drugs are the main treatments for UC and theprimary goals of these treatments are to induce remissionand prevent a relapse [17 18] However these treatments areassociated with numerous problems such as drug intoleranceside effects requirement of a prolonged treatment course andhigh recurrence rate Thus there is an urgent need for thedevelopment of novel and effective drugs for treatment of UC[19]

QingchangWenzhongDecoction (QCWZD) is a new andeffective traditional Chinese medicine prescription formu-lated by Li Jun-Xiang a professor at the Beijing Universityof Chinese Medicine Our previous studies have shownsignificant clinical efficacy of QCWZD in patients with mild-to-moderate UC [20 21] Furthermore in rat models of UCQCWZD reduced damage to colonic epithelial cells repairedthe colonic mucosa and downregulated proinflammatorycytokines [22 23] However the exact mechanism of actionis yet to be elucidated Therefore the present study soughtto further explore the molecular mechanism underlyingQCWZDrsquos protective effects in rat models of UC

2 Materials and Methods

21 Preparation of QCWZD and Mesalazine QCWZDgranules were purchased from the Pharmacy Depart-ment of Dongfang Hospital Beijing University of ChineseMedicine (Beijing China) The QCWZD granules contained

Qingchang Wenzhong ingredients in equal weights Huan-glian (coptis) 6 g Pao Jiang (ginger) 10 g Kushen (matrine)15 g Qingdai (indigo) 6 g Diyutan (sanguisorba carbon)30 g Muxiang (wood) 6 g Sanqi (pseudoginseng) 6 g andGancao (licorice) 6 g Mesalazine was purchased from LosanPharma GmbH Germany

22 Experimental Animals All the experimental procedureswere approved by the Animal Ethics Committee of BeijingUniversity of Chinese Medicine in accordance with guide-lines issued by Regulations of Beijing Laboratory AnimalManagement Fiftymale Sprague-Dawley rats (weighing 180ndash220 g SPF Biological Technology Co Ltd Beijing China)were housed in a specific pathogen-free animal room withtemperaturemaintained at 20ndash24∘C 50ndash60 humidity and alight-controlled environment (1212 h lightdark cycle) withfree access to food and sterile tap water All animals wereallowed to adapt for 7 days before the experiments werestarted

23 Induction of Colitis by DSS and Experimental ProcedureColitis was induced using 45 (wv) DSS (MW 36ndash50KDaMP Biomedical California USA) added to distilled waterFresh DSS solution was administered every day The rats inthe DSS (DSS 119899 = 10) low-dose QCWZD (low 03 gkgdaypo 119899 = 10) high-dose QCWZD (high 12 gkgday po 119899 =10) and mesalazine (mesalazine 003 gkgday po 119899 = 10)groups were administered 45 DSS solution from day 1 today 7 The control group rats received only distilled waterAll rats were killed after deep anaesthesia with 10 chlorinehydrate solution (35mLkg ip) and blood samples werecollected from the abdominal aorta Colon samples (8 cmin length) were removed and cut into 6 segments eachmeasuring 05 cm starting from the anus and cut at 1 cmintervals One of every two sections was fixed in 10 neutralbuffered formalin for histological analysis and the other onewas placed into a freezer tube and stored at minus80∘C until used

24 Analysis of Disease Activity Index All rats were checkeddaily for colitis by evaluating their weight faecal occultblood and stool consistencyThe disease activity index (DAI)was evaluated daily in a blinded manner by nonprojectteam members (Table 1) as previously described [24] DAIwas calculated using the following formula DAI = (percentweight loss score + stool consistency score + haematochezialevel score)3

25 Histological Analysis After being fixed with 10 neutralbuffered formalin for 24 h the tissues were embedded inparaffin blocks and sectioned to obtain a thickness of 6 120583m

Evidence-Based Complementary and Alternative Medicine 3

Table 2 Histological score criteria

Histological changes 0 1 2 3Inflammatory cell infiltration No Mild Moderate SevereGranuloma No Mild Moderate SevereLesion depth Mucosa Submucosa Muscular layer Serosa

Thereafter the sections were stained with haematoxylin andeosin (HE) and the histological score (HS) was determinedusing an optical microscope (Table 2) Each sample wasrandomly selected from 3 perspectives and the average scorewas calculated as previously described [25] The analysis wasperformed by nonproject team members under the guidanceof a pathologist

26 Assay forMyeloperoxidase (MPO)Activity Frozen colonsthat were dissected 1 cm above the anus were homogenizedin phosphate-buffered saline and centrifuged at 20000timesgThen myeloperoxidase (MPO) activity in the colonic tissuewas detected using chemical colorimetry (NANODROP2000 Thermo USA) as previously described [26]

27 Measurement of Serum IP10 Level Using ELISA Afterblood samples were collected from the abdominal aorta ofrats IP10 levels in the serum were tested using rat ELISA kits(MULTISKANMK3 Thermo USA)

28 Western Blotting for Detection of IP10 CXCR3 and NF-120581B p65 Expression in the Colon Western blot analysis wasperformed as described previously [27] Proteins were iso-lated from ice-cold colon tissues and protein concentrationswere determined using the bicinchoninic acid (BCA) assay(Cwbiotech Beijing China) The proteins were then sepa-rated using 10 SDS-PAGE for 15 h before being transferredto polyvinylidene fluoride (PVDF) membranes The mem-branes were probed with IP10 (1 5000) CXCR3 (1 5000)NF-120581B p65 (1 2000) and GAPDH (1 1000) antibodies (TDYBiotech Beijing China) Each membrane was washed threetimes for 10min and incubated with goat polyclonal sec-ondary antibody to rabbit antibodies (111-035-003 JacksonLaboratory USA) Finally densitometry was performed toquantitate protein band intensities by using the Gel ImageSystem ver 400 (Tanon China)

29 Real-Time Polymerase Chain Reaction for IP10 CXCR3and NF-120581B p65 mRNA Expression Evaluation of mRNAexpression was performed by using real-time polymerasechain reaction as previously described [28] Total RNA wasextracted from the colon tissue samples by using extractionkits (CWbio Beijing China) After reverse transcriptionPCR amplification was performed using the TRIzol method(TRIzol reagent Invitrogen Life Technologies Carlsbad CAUSA) The real-time PCR primer sequences for target geneswere as follows 51015840-GCGGCTAGTCCTAACTGTCC-3101584051015840-GAATTGGGAAGCCTTGCTGC-31015840 for IP10 51015840-TCACTT-CCTCTGTTCACGGC-3101584051015840-AGGAGGCTGTAGAGGAC-TGG-31015840 for CXCR3 51015840-CAGACACCTTTGCACTTGGC-3101584051015840-CTTGAGTAGGACCCCGAGGA-31015840 for NF-120581B p65

and 51015840-CCCATCTATGAGGGTTACGC-3101584051015840-TTTAATGT-CACGCACGATTTC-31015840 for GAPDH (CWbio BeijingChina) For real-time PCR the cycling conditions were 95∘Cfor 10min and 45 times (95∘C for 10 s 59∘C for 60 s) followedby a melting curve analysis-based assay with conditions of95∘C for 15 s and 72∘C for 15 s and increase in temperature to95∘C for 15 s Relative expression was assessed by calculatingthe expression relative to that of GAPDH by using the 2minusΔΔCtmethod

210 Statistical Analysis All data are expressed as meanplusmn standard deviation (SD) values SPSS v180 (IBM CorpArmonk NY USA) was used for statistical analysesThe datawere compared between groups using one-way analysis ofvariance (ANOVA) followed by Studentrsquos 119905-tests 119875 lt 005was considered statistically significant

3 Results

31 Effect of QCWZD on Body Weight and DAI in UC RatsAll animals tolerated the entire experiment well and nodeaths occurred After free access to 45 DSS for 4 daysbody weight started to decrease in the DSS group By the8th day the weight loss was significantly higher than that inthe control group (DSS group versus control group 22170 plusmn1035 g versus 31000 plusmn 1021 g 119875 lt 001) Both the low-dose(25690plusmn1096 g) and high-dose QCWZD (24670plusmn10140 g)groups showed significant attenuation of body weight loss(119875 lt 005 resp) similar to the mesalazine group (26910 plusmn12260 g versus 22170 plusmn 1035 g 119875 lt 005) (Figure 1(a))There were no significant differences between low-dose andhigh-dose QCWZD (119875 gt 005) possibly because of the smallsample size

On the 8th day the DSS group showed a significantlyincreased DAI score (213 plusmn 042) compared to that of thecontrol group (010 plusmn 032 119875 lt 001 Figure 1(b)) The DAIscores in the low-dose group (147 plusmn 088) high-dose group(127 plusmn 096) and mesalazine group (128 plusmn 078) weresignificantly lower than that in the DSS group (119875 lt 005 119875 lt001 resp Figure 1(b))

Subsequently we separately analysed the categorical dataof DAI according to each category As shown in Figure 1(c)the haematochezia level (300plusmn141) stool consistency (260plusmn097) and percent weight loss (080 plusmn 103) in the DSS groupwere significantly higher than those in the control group(020 plusmn 063 000 plusmn 000 and 010 plusmn 032 119875 lt 005 and119875 lt 001 resp Figure 1(c)) The haematochezia level in thelow-dose group (220 plusmn 116) high-dose group (220 plusmn 116)and mesalazine group (180 plusmn 114) was significantly lowerthan that in the DSS group (119875 lt 005 119875 lt 001 respFigure 1(c))The stool consistency (220plusmn175 in the low-dose


Body

wei

ght (

g)

300

250

200

t (d)

lowast

lowast

lowast

0 1 2 3 4 5 6 7 8

ControlDSSLow

HighMesalazine

(a)

lowast

lowastlowast

lowastlowast

Control DSS Low High Mesalazine

Dise

ase a

ctiv

ity in

dex

scor

e

3

2

1

0

(b)

lowast

lowastlowast

lowast

lowastlowastlowastlowast

lowastlowast

lowast

ControlDSSLow

HighMesalazine

Cate

goric

al d

ata o

f DA

I sco

re

5

4

3

2

1

0

Haematochezia Stool Weight loss

(c)

Figure 1 Effect of QCWZD on body weight (a) DAI score (b) and categorical data of DAI (c) in UC rats Control blank control groupDSS DSS-treated group low low-dose QCWZD group high high-dose QCWZD group mesalazine mesalazine group haematocheziahaematochezia level stool stool consistency and weight percent weight loss Data are presented as the mean plusmn SD 119875 lt 001 119875 lt 005versus the control group lowastlowast119875 lt 001 lowast119875 lt 005 versus the DSS group (119899 = 10 per group)

group 140 plusmn 165 in the high-dose group and 180 plusmn 148in the mesalazine group) and percent weight loss (000 plusmn000 in the low-dose high-dose and mesalazine groups)showed the same tendency (119875 lt 005 and 119875 lt 001 respFigure 1(c))

32 QCWZD Improved Histopathology in Rats Comparedwith the control group (Figure 2(a)) the DSS group showedseverely damaged crypts and epithelial integrity and remark-able inflammatory cell infiltration in the mucosa (Fig-ure 2(b)) After intrarectal administration of QCWZD andmesalazine mild infiltration of inflammatory cells cryptregeneration and epithelial restoration were observed (Fig-ures 2(c) 2(d) and 2(e)) Similarly as shown in Figure 2(f)the histological score reached a significantly higher value

in the DSS group (813 plusmn 037) than in the control group((215plusmn023)119875 lt 001) In contrast to theDSS group the low-and high-dose QCWZD groups and the mesalazine groupshowed protection against histological damage (464 plusmn 060371 plusmn 046 and 397 plusmn 040 resp 119875 lt 005) (Figure 2(f))

33 QCWZDDecreasedColonicMPOActivity inDSS-InducedUC Rats To determine the degree of infiltration by neu-trophil granulocytes in the colonic tissue the activity ofMPO was detected by chemical colorimetry Compared withthe control group (098 plusmn 006) the DSS group showedsignificantly higher MPO activity (143 plusmn 016 119875 lt 005)As expected the difference was significantly less when thegroups given QCWZD (119 plusmn 022 in the low-dose group107 plusmn 011 in the high-dose group 119875 lt 005 119875 lt 001 resp)


(a) (b) (c)

(d) (e)

lowast

lowastlowast

Control DSS Low

10

Hist

olog

ical

scor

e

8

6

4

2

0High Mesalazine

(f)

Figure 2 QCWZD ameliorated histological damage (andashe) and reduced the histological score (f) in rats (a) Control blank control group (b)DSS DSS group (c) low low-dose QCWZD group (d) high high-dose QCWZD group and (e) mesalazine mesalazine group 119875 lt 001versus the control group lowast119875 lt 005 versus the DSS group (119899 = 10 per group)

and mesalazine (103 plusmn 009 119875 lt 001) were compared to theDSS group (Figure 3)

34 Effects of QCWZD on Serum IP10 Level The serum IP10level was determined in the control rats andDSS-inducedUCrats treated with distilled water low- and high-dose QCWZDgroups and mesalazine group As shown in Figure 4 DSSsignificantly elevated IP10 levels in serum compared withthose in the control group (5528 plusmn 1585 ngmL in the DSSgroup versus 3678 plusmn 4430 ngmL 119875 lt 001) The low-dose QCWZD group (3130 plusmn 7269 ngmL) the high-doseQCWZD group (2667 plusmn 8999 ngmL) and the mesalazinegroup (2700 plusmn 9834 ngmL) showed significant inhibition of

DSS-induced elevation of serum IP10 level (versus the DSSgroup all 119875 lt 001) (Figure 4)

35 QCWZD Regulated Colonic IP10 and IP10 mRNA Expres-sion in DSS-Induced UC Rats We also investigated whetheror not QCWZD had a regulatory effect on IP10 expression inthe colon IP10 levels in the colon were significantly increasedin the DSS group (119875 lt 001 versus the control group)Additionally high-doseQCWZDandmesalazine produced adramatically reduced effect on IP10 expression (Figure 5(a))

Furthermore we measured IP10 gene expression to con-firm the effects of QCWZD on the colon As shown inFigure 5(b) induction of colitis significantly elevated colonic

6 Evidence-Based Complementary and Alternative MedicineM

PO ac

tivity

(OD

tota

l pro

tein

)

20

15

10

05

00

lowast


lowast


Figure 3 QCWZD decreased colonic MPO activity in rats withDSS-induced UC Control blank control group DSS DSS grouplow low-dose QCWZD group high high-dose QCWZD groupmesalazine mesalazine group 119875 lt 001 versus the control grouplowastlowast

119875 lt 001 lowast119875 lt 005 versus the DSS group (119899 = 10 per group)

lowastlowastlowastlowastlowastlowast


Seru

m IP10

(ng

mL)

800

600

400

200

0

Figure 4 Effects of QCWZD on serum IP10 level Control blankcontrol groupDSSDSS group low low-doseQCWZDgroup highhigh-dose QCWZD group mesalazine mesalazine group

119875 lt

001 versus the control group lowastlowast119875 lt 005 versus DSS group (119899 = 10per group)

IP10 gene expression compared with that in the control group(119875 lt 001) Treatment with QCWZD and mesalazine for 7days decreased IP10 gene expression in a dose-dependentmanner (119875 lt 005 119875 lt 001 resp) compared to that in theDSS group

36 QCWZD Decreased Colonic CXCR3 and CXCR3 mRNAExpression in Rats with DSS-Induced UC Because CXCR3specifically binds to IP10 its levels were analysed by westernblot Compared to the control group the DSS group showedincreased CXCR3 expression (119875 lt 001) the QCWZD andmesalazine groups showed alleviation of the effects of DSS onCXCR3 expression in rats with DSS-induced UC (versus theDSS group 119875 lt 005 and 119875 lt 001 resp Figure 6(a)) Theexpression of the CXCR3 gene was measured as describedabove Gene expression in the DSS group was increasedcompared to that in the control group (119875 lt 001 Figure 6(b))Remarkably QCWZDandmesalazine both greatly decreased

CXCR3 gene expression (versus the DSS group 119875 lt 005 and119875 lt 001 resp Figure 6(b))

37 QCWZD Suppressed the Increase in Colonic NF-120581B p65and NF-120581B p65 mRNA Expression in Rats with DSS-InducedUC Because the cytokines TNF-120572 and IL-6 produced byCXCR3-positive cells contribute to the activation of NF-120581Bwe examined the effect of CXCR3 on NF-120581B p65 activationWe found distinctly increased NF-120581B p65 and NF-120581B p65mRNA expression in the DSS group compared to that in thecontrol group (119875 lt 001 Figures 7(a) and 7(b)) NeverthelessNF-120581B p65 and NF-120581B p65 mRNA expression was signifi-cantly reduced inDSS-inducedUC rats treatedwithQCWZDand mesalazine (119875 lt 005 resp Figures 7(a) and 7(b))

4 Discussion

In the present study to observe the anti-inflammatory effectsof QCWZD we generated an ulcerative colitis model in ratsby allowing them to drink 45 DSS freely for 7 days thismodel is widely used because of its similarities to humanUCDSS is a polysaccharide extracted from sucrose synthesisAlthough the exact mechanism by which it induces UCremains unclear it may be related to the negative chargeof DSS which may affect the synthesis of colonic epithelialDNA inhibit the proliferation of epithelial cells and damagethe intestinal mucosal barrier [29]

In our study faecal occult blood began to appear on the2nd day and the animalsrsquo body weight started to decreaseon the 4th day in the DSS group With time gross bloodbegan to appear in the stool and the body weight continuedto decrease and the DAI score increased and reached themaximum value on the 8th day In the histological analysiswe found that the number of inflammatory cells (mainlyneutrophils) was increased in the colonic tissue of rats thatreceived DSS and that the crypts and epithelial integrity wereseverely damaged Correspondingly the histological scoreincreased to a significantly higher value in theDSS group thanin the control group After administration of QCWZD andmesalazine the body weight loss DAI score and histologicalscore were significantly decreased and improved (Figures 1and 2) These results suggest that QCWZD has prominenttherapeutic effects on rats with DSS-induced ulcerative col-itis

After observing a definite effect we further explored thetherapeuticmechanismofQCWZD in ratswithDSS-inducedUC Persistent intestinal infection plays an important role inthe pathogenesis and development of UC [30 31] and theIP10CXCR3 axis that regulates the infiltration of immunecells to sites of inflammatory injury plays an essential rolein the inflammatory response Previous studies showed thatboth IP-10 expression in colonic tissue and IP10 levels inserum were higher in patients with UC than in healthycontrols [16 32] and that anti-IP-10 antibodies alleviatedacute colitis and enhanced crypt cell survival inmice withUCinduced by DSS [8] Thus anti-IP-10 therapy may provide animportant direction for the treatment of UC A recent phase


IP10

GAPDH


Control DSS Low High MesalazineIP10

GA

PDH

06

04

02

00

lowast lowast

(a)

lowastlowast

lowast

lowast


IP10

mRN

A ex

pres

sion

5

4

3

2

1

0

(b)

Figure 5 QCWZD regulated colonic IP10 (a) and IP10 mRNA (b) expression in DSS-induced UC rats Control blank control group DSSDSS group low low-dose QCWZD group high high-dose QCWZD group and mesalazine mesalazine group 119875 lt 001 versus controlgroup lowastlowast119875 lt 001 lowast119875 lt 005 versus DSS group (119899 = 10 per group)

lowastlowast

lowastlowast

lowast



CXCR

3G

APD

H

CXCR3

GAPDH

06

04

02

00

(a)

lowastlowast

lowastlowast


CXCR

3m

RNA

expr

essio

n

4

3

2

1

0

(b)

Figure 6 QCWZD inhibited colonic CXCR3 (a) and CXCR3 mRNA (b) expression in rats with DSS-induced UC Control blank controlgroup DSS DSS group low low-doseQCWZDgroup high high-doseQCWZDgroup andmesalazine mesalazine group 119875 lt 001 versuscontrol group lowastlowast119875 lt 001 lowast119875 lt 005 versus DSS group (119899 = 10 per group)




NF-120581

B p65

GA

PDH

08

06

04

02

00

NF-120581B p65

GAPDH

lowastlowast

lowast

(a)

Control DSS Low High MesalazineN

F-120581

B p65

mRN

A ex

pres

sion

25

20

15

10

05

00

lowast

lowast

(b)

Figure 7 QCWZD suppressed the increase in colonic NF-120581B p65 (a) andNF-120581B p65mRNA (b) levels in rats with DSS-inducedUC Controlblank control group DSS DSS group low low-dose QCWZD group high high-dose QCWZD group and mesalazine mesalazine group119875 lt 001 versus control group lowastlowast119875 lt 001 lowast119875 lt 005 versus DSS group (119899 = 10 per group)

II study for UC [7] showed that anti-IP-10 therapy with BMS-936557 effectively relieved clinical symptoms and increasedhistological improvement in patients with moderate-to-severe UC Although robust efficacy was demonstratedthe drug dose response and safety profile require furtherresearch Therefore it is necessary to identify a new methodto evaluate the therapeutic value of targeting the IPCXCR3axis for UC In this regard traditional Chinese medicinehas been proven to have a curative effect in the treatmentof ulcerative colitis [5 33] Previous studies have shownsignificant clinical efficacy of QCWZD in patients with UCbut the specific mechanism is not clear Thus we designedthe present study to determine whether or not QCWZDexerts an anti-inflammatory effect through IPCXCR3 axisIn the present study abnormally high expression of IP10 andCXCR3was observed in the colonicmucosa of rats with DSS-induced UC and treatment with QCWZD and mesalazinedose-dependently decreased IP10 and CXCR3 expression(Figures 4 5 and 6)

An increase in neutrophils is a key feature in the patho-genesis of UC [34 35] Coupling of IP10 to CXCR3 in thecolon leads to activation of CXCR3 which promotes recruit-ment of neutrophils to the inflammatory site whereasCXCR3blockade limits neutrophil accumulation in the inflamed siteTherefore we evaluated the expression of MPO in colontissues which reflects the degree of mucosal neutrophilinfiltration [36 37] As expected DSS significantly increasedMPO activity in the colon tissue and QCWZD treatmentpartially reversed this effect (Figure 3)

In addition to promoting the recruitment of neutrophilsthe IPCXCR3 axis increases the chemotactic activity ofCXCR3-positive cells which are directly and indirectlyinvolved in the generation and recruitment of inflammatorycytokines [38] such as TNF-120572 and MCP-1 thereby damagingthe intestinalmucosal barrier and colonmucosa [39 40] NF-120581B is an upstream regulator for TNF-120572 and MCP-1 whichcan in turn activate NF-120581B [41] NF-120581B activation is involvedin inflammatory recruitment [42] therefore we investigatedwhether or not the IP10CXCR3 axis plays a role in NF-120581B activation in the development of UC Overexpression ofNF-120581B p65 has been identified in the colonic mucosa andis positively correlated with the increase in IP10CXCR3In the present study QCWZD and mesalazine played amajor role in decreasing NF-120581B p65 expression (Figure 7)Although other signalling pathways may also activate NF-120581B or proinflammatory cytokine genes the proinflammatoryeffect of IP10CXCR3 axis inUC is at least partly related to theinduction of NF-120581B NF-120581B activation is an important waythat the IP10CXCR3 axis exerts its proinflammatory effect

In the present study using a DSS-induced rat colonicmucosal injury as a model we found that QCWZD sig-nificantly improved the body weight DAI score HS andMPO activity in DSS-induced UC rats The protective effectsof QCWZD may be attributed to its significant regulationof the IP10CXCR3 axis-mediated inflammatory responseQCWZD can reverse the dysregulated expression of serumand colonic IP10 and IP10mRNA reduce colonic CXCR3 andCXCR3 mRNA production and thereby inhibit activation


of the IP10CXCR3 axis in turn decreasing the generationand recruitment of inflammatory cytokines and promotingepithelial cell proliferation and repair of injured mucosa

5 Conclusions

Our results show that intragastric administration ofQCWZDcan ameliorate DSS-induced UC in rats The protectivemechanism may be attributed to significant downregulationof the IP10CXCR3 axis-mediated inflammatory responseOur results suggest that QCWZDmay be a novel therapeuticoption for UC

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contributions

Tang-you Mao and Rui Shi contributed equally to this work

Acknowledgments

This studywas supported by theNational Science Foundationof China (Grant no 81403369) and the self-selected topic ofthe Beijing University of Chinese Medicine (Grant no 2015-JYB-XS172)

References

[1] S Danese and C Fiocchi ldquoUlcerative colitisrdquoThe New EnglandJournal of Medicine vol 365 no 18 pp 1713ndash1725 2011

[2] H M Zhao X Y Huang F Zhou et al ldquoSi Shen Waninhibits mRNA expression of apoptosis-related molecules inp38MAPK signal pathway inmice with colitisrdquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID432097 8 pages 2013

[3] APDW 2004 Chinese IBD Working Group ldquoRetrospectiveanalysis of 515 cases of Crohnrsquos disease hospitalization in Chinanationwide study from 1990 to 2003rdquo Journal of Gastroenterol-ogy and Hepatology vol 21 no 6 pp 1009ndash1015 2006

[4] A Kaser S Zeissig and R S Blumberg ldquoInflammatory boweldiseaserdquo Annual Review of Immunology vol 28 pp 573ndash6212010

[5] X P Li Y J Xu C L Zhang et al ldquoProtective effect of CalculusBovis Sativus on dextran sulphate sodium-induced ulcerativecolitis in micerdquo Evidence-Based Complementary and AlternativeMedicine vol 2015 Article ID 469506 8 pages 2015

[6] HMatsuoka H Ikeuchi M Uchino et al ldquoClinicopathologicalfeatures of ulcerative colitis-associated colorectal cancer point-ing to efficiency of surveillance colonoscopy in a large retro-spective Japanese cohortrdquo International Journal of ColorectalDisease vol 28 no 6 pp 829ndash834 2013

[7] LMayerW J Sandborn Y Stepanov et al ldquoAnti-IP-10 antibody(BMS-936557) for ulcerative colitis a phase II randomisedstudyrdquo Gut vol 63 no 3 pp 442ndash450 2014

[8] S Sasaki H Yoneyama K Suzuki et al ldquoBlockade of CXCL10protects mice from acute colitis and enhances crypt cell sur-vivalrdquo European Journal of Immunology vol 32 no 11 pp 3197ndash3205 2002

[9] U P Singh S Singh R Singh Y Cong D D Taub and JW Lillard Jr ldquoCXCL10-producing mucosal CD4+ T cells NKcells and NKT cells are associated with chronic colitis in IL-10minusminus mice which can be abrogated by anti-CXCL10 antibodyinhibitionrdquo Journal of Interferon and Cytokine Research vol 28no 1 pp 31ndash43 2008

[10] K Van Raemdonck P E Van den Steen S Liekens J VanDamme and S Struyf ldquoCXCR3 ligands in disease and therapyrdquoCytokine amp Growth Factor Reviews vol 26 no 3 pp 311ndash3272015

[11] M Liu S Guo J M Hibbert et al ldquoCXCL10IP-10 in infectiousdiseases pathogenesis and potential therapeutic implicationsrdquoCytokine amp Growth Factor Reviews vol 22 no 3 pp 121ndash1302011

[12] J-M Xu and G-P Shi ldquoEmerging role of mast cells andmacrophages in cardiovascular and metabolic diseasesrdquoEndocrine Reviews vol 33 no 1 pp 71ndash108 2012

[13] B-G Xiao W-H Zhu and C-Z Lu ldquoThe presence of GM-CSF and IL-4 interferes with effect of TGF-1205731 on antigenpresenting cells in patients with multiple sclerosis and in ratswith experimental autoimmune encephalomyelitisrdquo CellularImmunology vol 249 no 1 pp 30ndash36 2007

[14] T Y Ma G K Iwamoto N T Hoa et al ldquoTNF-120572-inducedincrease in intestinal epithelial tight junction permeabilityrequires NF-120581B activationrdquo American Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 286 no 3 pp G367ndashG376 2004

[15] P K Kim and C S Deutschman ldquoInflammatory responses andmediatorsrdquo Surgical Clinics of North America vol 80 no 3 pp885ndash894 2000

[16] A E Ostvik A V B Granlund M Bugge et al ldquoEnhancedexpression of CXCL10 in inflammatory bowel disease potentialrole of mucosal toll-like receptor 3 stimulationrdquo InflammatoryBowel Diseases vol 19 no 2 pp 265ndash274 2013

[17] A Kornbluth D B Sachar and Practice Parameters Committeeof the American College of Gastroenterology ldquoUlcerative colitispractice guidelines in adults American college of gastroen-terology practice parameters committeerdquo American Journal ofGastroenterology vol 105 no 3 pp 501ndash523 2010

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

[19] D Laharie A Bourreille J Branche et al ldquoCiclosporin versusinfliximab in patients with severe ulcerative colitis refractoryto intravenous steroids a parallel open-label randomised con-trolled trialrdquoThe Lancet vol 380 no 9857 pp 1909ndash1915 2012

[20] S B Wei Clinical Research on the Treatment of UlcerativeColitis with Qingchang Wenzhong Decoction Beijing Universityof Chinese Medicine Beijing China 2014 (Chinese)

[21] J W Chen Clinical Effect of Qingchang Wenzhong on theTreatment of Ulcerative Colitis Beijing University of ChineseMedicine Beijing China 2015 (Chinese)

[22] L J Liu Y L Wang R Shi et al ldquoEffect of QingchangWenzhong decoction on colonic mucosa of TNBS-inducedcolitis ratsrdquo Global Traditional Chinese Medicine vol 7 no 3pp 187ndash191 2014

[23] L J Liu Y LWang S BWei M J Yang and J X Li ldquoEffects ofQingchang Wenzhong decoction on intestinal mucosa barrier


in colitis rats induced by TNBSrdquo China Journal of TraditionalChinese Medicine and Pharmacy vol 30 no 9 pp 3266ndash32692015

[24] S Sasaki I Hirata KMaemura et al ldquoProstaglandin E2

inhibitslesion formation in dextran sodium sulphate-induced colitis inrats and reduces the levels of mucosal inflammatory cytokinesrdquoScandinavian Journal of Immunology vol 51 no 1 pp 23ndash282000

[25] L A Dieleman M J H J Palmen H Akol et al ldquoChronicexperimental colitis induced by dextran sulphate sodium (DSS)is characterized by Th1 and Th2 cytokinesrdquo Clinical and Exper-imental Immunology vol 114 no 3 pp 385ndash391 1998

[26] AMallakin LW Kutcher S AMcDowell et al ldquoGene expres-sion profiles of Mst1r-deficient mice during nickel-inducedacute lung injuryrdquo American Journal of Respiratory Cell andMolecular Biology vol 34 no 1 pp 15ndash27 2006

[27] F Huang C Y Kao S Wachi P Thai J Ryu and R WuldquoRequirement for both JAK-mediated PI3K signaling andACT1TRAF6TAK1-dependent NF-120581B activation by IL-17Ain enhancing cytokine expression in human airway epithelialcellsrdquo Journal of Immunology vol 179 no 10 pp 6504ndash65132007

[28] S A Bustin ldquoAbsolute quantification of mrna using real-timereverse transcription polymerase chain reaction assaysrdquo Journalof Molecular Endocrinology vol 25 no 2 pp 169ndash193 2000

[29] Y Araki K I Mukaisyo H Sugihara Y Fujiyama and THattori ldquoIncreased apoptosis and decreased proliferation ofcolonic epithelium in dextran sulfate sodium-induced colitis inmicerdquo Oncology Reports vol 24 no 4 pp 869ndash874 2010

[30] S Watanabe M Yamakawa T Hiroaki S Kawata and OKimura ldquoCorrelation of dendritic cell infiltration with activecrypt inflammation in ulcerative colitisrdquo Clinical Immunologyvol 122 no 3 pp 288ndash297 2007

[31] M Zhao and F Gao ldquoNew progress in the pathogenesis ofulcerative colitisrdquo Progress in Modern Biomedicine vol 10 no16 pp 3160ndash3165 2010

[32] S Schroepf R Kappler S Brand et al ldquoStrong overexpressionof CXCR3 axis components in childhood inflammatory boweldiseaserdquo Inflammatory Bowel Diseases vol 16 no 11 pp 1882ndash1890 2010

[33] Y Hao K Nagase K Hori et al ldquoXilei San ameliorates exper-imental colitis in rats by selectively degrading proinflamma-tory mediators and promoting mucosal repairrdquo Evidence-BasedComplementary and Alternative Medicine vol 2014 Article ID569587 10 pages 2014

[34] L A Boxer ldquoRole of neutrophils in genetic disorders ofphagocyte function leading to IBDrdquo Journal of Pediatric Gas-troenterology and Nutrition vol 46 supplement 1 p E17 2008

[35] Y Xu N H Hunt and S Bao ldquoThe role of granulo-cyte macrophage-colony-stimulating factor in acute intestinalinflammationrdquoCell Research vol 18 no 12 pp 1220ndash1229 2008

[36] J Zhao T Hong M Dong Y Meng and J Mu ldquoProtectiveeffect of myricetin in dextran sulphate sodium-induced murineulcerative colitisrdquo Molecular Medicine Reports vol 7 no 2 pp565ndash570 2013

[37] D Lau H Mollnau J P Eiserich et al ldquoMyeloperoxidasemediates neutrophil activation by associationwithCD11bCD18integrinsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 102 no 2 pp 431ndash436 2005

[38] M F Neurath S Finotto and L H Glimcher ldquoThe role ofTH1TH2 polarization in mucosal immunityrdquoNature Medicinevol 8 no 6 pp 567ndash573 2002

[39] S Molesworth-Kenyon A Mates R Yin R Strieter J Oakesand R Lausch ldquoCXCR3 IP-10 and Mig are required for CD4+T cell recruitment during the DTH response to HSV-1 yet areindependent of the mechanism for viral clearancerdquo Virologyvol 333 no 1 pp 1ndash9 2005

[40] P Romagnani F Annunziato L Lasagni et al ldquoCell cycle-dependent expression of CXC chemokine receptor 3 byendothelial cells mediates angiostatic activityrdquo Journal of Clini-cal Investigation vol 107 no 1 pp 53ndash63 2001

[41] X Zhang J Han KMan et al ldquoCXC chemokine receptor 3 pro-motes steatohepatitis in mice through mediating inflammatorycytokines macrophages and autophagyrdquo Journal of Hepatologyvol 64 no 1 pp 160ndash170 2016

[42] G C Farrell D van Rooyen L Gan and S Chitturi ldquoNASHis an inflammatory disorder pathogenic prognostic and ther-apeutic implicationsrdquo Gut and Liver vol 6 no 2 pp 149ndash1712012


Table 1 DAI scoring criteria

Types 0 1 2 3 4Percent weight loss 0 1ndash5 5ndash10 10ndash15 gt15Stool consistency Normal Mushy DiarrhoeaHaematochezia level Negative Positive Blood traces visible in stool

monocytes natural killer (NK) cells T helper (Th)1 cells andendothelial cells (ECs) under stimulation by intestinal IFN-120574and tumour necrosis factor- (TNF-) 120572 [10]

Chemokine (cys-x-cys motif) receptor 3 (CXCR3) is thespecific binding protein of IP10 and it is mainly expressedby epithelial and endothelial cells as well as lymphoid cellssuch as memory T cells NK cells B cells neutrophilsand monocytes Upon binding to IP10 CXCR3 is activatedwhich increases the chemotactic activity of CXCR3-positivecells and contributes to its transfer to local inflammatorylesions [11] Finally the intestinal mucosa produces largeamounts of inflammatory cytokines [12 13] which not onlyfurther stimulate IP10 thus recruitingmore immune cells andforming a vicious cycle but also directly or indirectly damagethe intestinal mucosal barrier and the intestinal mucosa[14 15] and promote the occurrence and development ofUC [16]Therefore the IP10CXCR3-mediated inflammatoryresponse plays an important role in the pathogenesis ofUC and inhibiting the IP10CXCR3 axis to block excessiveinflammation may be a new approach for treatment ofUC

At present an effective treatment for UC is lackingAmino salicylic acid steroid hormones and immunosup-pressive drugs are the main treatments for UC and theprimary goals of these treatments are to induce remissionand prevent a relapse [17 18] However these treatments areassociated with numerous problems such as drug intoleranceside effects requirement of a prolonged treatment course andhigh recurrence rate Thus there is an urgent need for thedevelopment of novel and effective drugs for treatment of UC[19]

QingchangWenzhongDecoction (QCWZD) is a new andeffective traditional Chinese medicine prescription formu-lated by Li Jun-Xiang a professor at the Beijing Universityof Chinese Medicine Our previous studies have shownsignificant clinical efficacy of QCWZD in patients with mild-to-moderate UC [20 21] Furthermore in rat models of UCQCWZD reduced damage to colonic epithelial cells repairedthe colonic mucosa and downregulated proinflammatorycytokines [22 23] However the exact mechanism of actionis yet to be elucidated Therefore the present study soughtto further explore the molecular mechanism underlyingQCWZDrsquos protective effects in rat models of UC

2 Materials and Methods

21 Preparation of QCWZD and Mesalazine QCWZDgranules were purchased from the Pharmacy Depart-ment of Dongfang Hospital Beijing University of ChineseMedicine (Beijing China) The QCWZD granules contained

Qingchang Wenzhong ingredients in equal weights Huan-glian (coptis) 6 g Pao Jiang (ginger) 10 g Kushen (matrine)15 g Qingdai (indigo) 6 g Diyutan (sanguisorba carbon)30 g Muxiang (wood) 6 g Sanqi (pseudoginseng) 6 g andGancao (licorice) 6 g Mesalazine was purchased from LosanPharma GmbH Germany

22 Experimental Animals All the experimental procedureswere approved by the Animal Ethics Committee of BeijingUniversity of Chinese Medicine in accordance with guide-lines issued by Regulations of Beijing Laboratory AnimalManagement Fiftymale Sprague-Dawley rats (weighing 180ndash220 g SPF Biological Technology Co Ltd Beijing China)were housed in a specific pathogen-free animal room withtemperaturemaintained at 20ndash24∘C 50ndash60 humidity and alight-controlled environment (1212 h lightdark cycle) withfree access to food and sterile tap water All animals wereallowed to adapt for 7 days before the experiments werestarted

23 Induction of Colitis by DSS and Experimental ProcedureColitis was induced using 45 (wv) DSS (MW 36ndash50KDaMP Biomedical California USA) added to distilled waterFresh DSS solution was administered every day The rats inthe DSS (DSS 119899 = 10) low-dose QCWZD (low 03 gkgdaypo 119899 = 10) high-dose QCWZD (high 12 gkgday po 119899 =10) and mesalazine (mesalazine 003 gkgday po 119899 = 10)groups were administered 45 DSS solution from day 1 today 7 The control group rats received only distilled waterAll rats were killed after deep anaesthesia with 10 chlorinehydrate solution (35mLkg ip) and blood samples werecollected from the abdominal aorta Colon samples (8 cmin length) were removed and cut into 6 segments eachmeasuring 05 cm starting from the anus and cut at 1 cmintervals One of every two sections was fixed in 10 neutralbuffered formalin for histological analysis and the other onewas placed into a freezer tube and stored at minus80∘C until used

24 Analysis of Disease Activity Index All rats were checkeddaily for colitis by evaluating their weight faecal occultblood and stool consistencyThe disease activity index (DAI)was evaluated daily in a blinded manner by nonprojectteam members (Table 1) as previously described [24] DAIwas calculated using the following formula DAI = (percentweight loss score + stool consistency score + haematochezialevel score)3

25 Histological Analysis After being fixed with 10 neutralbuffered formalin for 24 h the tissues were embedded inparaffin blocks and sectioned to obtain a thickness of 6 120583m











3 Results





Body

wei

ght (

g)

300

250

200

t (d)

lowast

lowast

lowast

0 1 2 3 4 5 6 7 8

ControlDSSLow

HighMesalazine

(a)

lowast

lowastlowast

lowastlowast


Dise

ase a

ctiv

ity in

dex

scor

e

3

2

1

0

(b)

lowast

lowastlowast

lowast


lowastlowast

lowast

ControlDSSLow

HighMesalazine

Cate

goric

al d

ata o

f DA

I sco

re

5

4

3

2

1

0


(c)







(a) (b) (c)

(d) (e)

lowast

lowastlowast

Control DSS Low

10

Hist

olog

ical

scor

e

8

6

4

2

0High Mesalazine

(f)








PO ac

tivity

(OD

tota

l pro

tein

)

20

15

10

05

00

lowast


lowast






Seru

m IP10

(ng

mL)

800

600

400

200

0


119875 lt






4 Discussion





IP10

GAPDH



GA

PDH

06

04

02

00

lowast lowast

(a)

lowastlowast

lowast

lowast


IP10

mRN

A ex

pres

sion

5

4

3

2

1

0

(b)


lowastlowast

lowastlowast

lowast



CXCR

3G

APD

H

CXCR3

GAPDH

06

04

02

00

(a)

lowastlowast

lowastlowast


CXCR

3m

RNA

expr

essio

n

4

3

2

1

0

(b)





NF-120581

B p65

GA

PDH

08

06

04

02

00

NF-120581B p65

GAPDH

lowastlowast

lowast

(a)


F-120581

B p65

mRN

A ex

pres

sion

25

20

15

10

05

00

lowast

lowast

(b)








5 Conclusions


Competing Interests




Acknowledgments


References
























































3 Results





Body

wei

ght (

g)

300

250

200

t (d)

lowast

lowast

lowast

0 1 2 3 4 5 6 7 8

ControlDSSLow

HighMesalazine

(a)

lowast

lowastlowast

lowastlowast


Dise

ase a

ctiv

ity in

dex

scor

e

3

2

1

0

(b)

lowast

lowastlowast

lowast


lowastlowast

lowast

ControlDSSLow

HighMesalazine

Cate

goric

al d

ata o

f DA

I sco

re

5

4

3

2

1

0


(c)







(a) (b) (c)

(d) (e)

lowast

lowastlowast

Control DSS Low

10

Hist

olog

ical

scor

e

8

6

4

2

0High Mesalazine

(f)








PO ac

tivity

(OD

tota

l pro

tein

)

20

15

10

05

00

lowast


lowast






Seru

m IP10

(ng

mL)

800

600

400

200

0


119875 lt






4 Discussion





IP10

GAPDH



GA

PDH

06

04

02

00

lowast lowast

(a)

lowastlowast

lowast

lowast


IP10

mRN

A ex

pres

sion

5

4

3

2

1

0

(b)


lowastlowast

lowastlowast

lowast



CXCR

3G

APD

H

CXCR3

GAPDH

06

04

02

00

(a)

lowastlowast

lowastlowast


CXCR

3m

RNA

expr

essio

n

4

3

2

1

0

(b)





NF-120581

B p65

GA

PDH

08

06

04

02

00

NF-120581B p65

GAPDH

lowastlowast

lowast

(a)


F-120581

B p65

mRN

A ex

pres

sion

25

20

15

10

05

00

lowast

lowast

(b)








5 Conclusions


Competing Interests




Acknowledgments


References















































Body

wei

ght (

g)

300

250

200

t (d)

lowast

lowast

lowast

0 1 2 3 4 5 6 7 8

ControlDSSLow

HighMesalazine

(a)

lowast

lowastlowast

lowastlowast


Dise

ase a

ctiv

ity in

dex

scor

e

3

2

1

0

(b)

lowast

lowastlowast

lowast


lowastlowast

lowast

ControlDSSLow

HighMesalazine

Cate

goric

al d

ata o

f DA

I sco

re

5

4

3

2

1

0


(c)







(a) (b) (c)

(d) (e)

lowast

lowastlowast

Control DSS Low

10

Hist

olog

ical

scor

e

8

6

4

2

0High Mesalazine

(f)








PO ac

tivity

(OD

tota

l pro

tein

)

20

15

10

05

00

lowast


lowast






Seru

m IP10

(ng

mL)

800

600

400

200

0


119875 lt






4 Discussion





IP10

GAPDH



GA

PDH

06

04

02

00

lowast lowast

(a)

lowastlowast

lowast

lowast


IP10

mRN

A ex

pres

sion

5

4

3

2

1

0

(b)


lowastlowast

lowastlowast

lowast



CXCR

3G

APD

H

CXCR3

GAPDH

06

04

02

00

(a)

lowastlowast

lowastlowast


CXCR

3m

RNA

expr

essio

n

4

3

2

1

0

(b)





NF-120581

B p65

GA

PDH

08

06

04

02

00

NF-120581B p65

GAPDH

lowastlowast

lowast

(a)


F-120581

B p65

mRN

A ex

pres

sion

25

20

15

10

05

00

lowast

lowast

(b)








5 Conclusions


Competing Interests




Acknowledgments


References















































(a) (b) (c)

(d) (e)

lowast

lowastlowast

Control DSS Low

10

Hist

olog

ical

scor

e

8

6

4

2

0High Mesalazine

(f)








PO ac

tivity

(OD

tota

l pro

tein

)

20

15

10

05

00

lowast


lowast






Seru

m IP10

(ng

mL)

800

600

400

200

0


119875 lt






4 Discussion





IP10

GAPDH



GA

PDH

06

04

02

00

lowast lowast

(a)

lowastlowast

lowast

lowast


IP10

mRN

A ex

pres

sion

5

4

3

2

1

0

(b)


lowastlowast

lowastlowast

lowast



CXCR

3G

APD

H

CXCR3

GAPDH

06

04

02

00

(a)

lowastlowast

lowastlowast


CXCR

3m

RNA

expr

essio

n

4

3

2

1

0

(b)





NF-120581

B p65

GA

PDH

08

06

04

02

00

NF-120581B p65

GAPDH

lowastlowast

lowast

(a)


F-120581

B p65

mRN

A ex

pres

sion

25

20

15

10

05

00

lowast

lowast

(b)








5 Conclusions


Competing Interests




Acknowledgments


References















































PO ac

tivity

(OD

tota

l pro

tein

)

20

15

10

05

00

lowast


lowast






Seru

m IP10

(ng

mL)

800

600

400

200

0


119875 lt






4 Discussion





IP10

GAPDH



GA

PDH

06

04

02

00

lowast lowast

(a)

lowastlowast

lowast

lowast


IP10

mRN

A ex

pres

sion

5

4

3

2

1

0

(b)


lowastlowast

lowastlowast

lowast



CXCR

3G

APD

H

CXCR3

GAPDH

06

04

02

00

(a)

lowastlowast

lowastlowast


CXCR

3m

RNA

expr

essio

n

4

3

2

1

0

(b)





NF-120581

B p65

GA

PDH

08

06

04

02

00

NF-120581B p65

GAPDH

lowastlowast

lowast

(a)


F-120581

B p65

mRN

A ex

pres

sion

25

20

15

10

05

00

lowast

lowast

(b)








5 Conclusions


Competing Interests




Acknowledgments


References















































IP10

GAPDH



GA

PDH

06

04

02

00

lowast lowast

(a)

lowastlowast

lowast

lowast


IP10

mRN

A ex

pres

sion

5

4

3

2

1

0

(b)


lowastlowast

lowastlowast

lowast



CXCR

3G

APD

H

CXCR3

GAPDH

06

04

02

00

(a)

lowastlowast

lowastlowast


CXCR

3m

RNA

expr

essio

n

4

3

2

1

0

(b)





NF-120581

B p65

GA

PDH

08

06

04

02

00

NF-120581B p65

GAPDH

lowastlowast

lowast

(a)


F-120581

B p65

mRN

A ex

pres

sion

25

20

15

10

05

00

lowast

lowast

(b)








5 Conclusions


Competing Interests




Acknowledgments


References

















































NF-120581

B p65

GA

PDH

08

06

04

02

00

NF-120581B p65

GAPDH

lowastlowast

lowast

(a)


F-120581

B p65

mRN

A ex

pres

sion

25

20

15

10

05

00

lowast

lowast

(b)








5 Conclusions


Competing Interests




Acknowledgments


References
















































5 Conclusions


Competing Interests




Acknowledgments


References














































qingchang wenzhong decoction ameliorates dextran sulphate … · 2016-09-15 ·...

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