分子生物学知识拓展 (13).pdf

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1、Research ArticleBu-Zhong-Yi-Qi Granule Enhances Colonic Tight JunctionIntegrity via TLR4/NF-B/MLCK Signaling Pathway in UlcerativeColitis RatsXiuhong Kang,Mengdi Jia,Luqing Zhao,and Shengsheng ZhangDigestive Disease Center,Beijing Hospital of Traditional Chinese Medicine,Capital Medical University,B

2、eijing,ChinaCorrespondence should be addressed to Shengsheng Zhang;Received 22 October 2020;Revised 12 January 2021;Accepted 19 February 2021;Published 9 March 2021Academic Editor:Guang ChenCopyright2021XiuhongKangetal.TisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,whic

3、h permits unrestricted use,distribution,and reproduction in any medium,provided the original work is properly cited.Background.Bu-zhong-yi-qi granule(BZYQ),a sort of Chinese herbal medicine,has exhibited therapeutic effects on ulcerativecolitis(UC).However,the mechanism of BZYQ has not been fully cl

4、arified.Tis study was aimed at investigating the effects ofBZYQ on UC rats model and at exploring its potential mechanism.Methods.Te UC rats were established by enema of tri-nitrobenzene sulfonic acid(TNBS).Te therapeutic effects of BZYQ treatment were evaluated by disease activity index(DAI),colon

5、macroscopic damage index(CMDI)scores,and histological observation.Te mRNA levels of tumor necrosis factor-(TNF-),interleukin-1(IL-1),and interleukin-10(IL-10)were measured by quantitative real time-polymerase chain reaction(qPCR).Te expression of tight junction(TJ)proteins,occludin and claudin-1,in

6、the colon was determined by Western blot andimmunofluorescence.Te expression of toll-like receptors 4(TLR4),nuclear factor-kappa B(NF-B),p-NF-B,myosin lightchainkinase(MLCK),MLC,andp-MLClevelsincolonwasdeterminedbyWesternblotorqPCR.Results.TeresultsshowedthatBZYQcouldattenuateDAI,CMDI,andhistologica

7、linflammation.TJproteinsexpressionwasdecreasedinUCrats,buttreatmentwith BZYQ restored the expression of occludin and claudin-1.In addition,BZYQ administration ameliorated UC-associatedincreaseintheproductionofTNF-,IL-1,andtheexpressionofTLR4,NF-B,p-NF-B,MLCK,MLC,andp-MLC,whileBZYQadministration incr

8、eased the production of IL-10.Conclusions.Te therapeutic effect of BZYQ on UC is at least partially throughregulation of the secretion of some inflammatory cytokines and improvement of TJ integrity via TLR4/NF-B/MLCK pathway.1.IntroductionUlcerative colitis is a chronic idiopathic inflammatory bowel

9、disease characterized by persistent mucosal inflammationbeginning in the rectum and extending to the proximal end.Typical presenting symptoms include intermittent bloodydiarrhea,abdominal pain,tenesmus,and others 1.In thepast two decades,there has been an increasing tendency inincidence and prevalen

10、ce of UC 2.Multiple pathogeneses have been demonstrated in oc-currence and development of UC;intestinal barrier is apivotal pathogenic factor 3,4.Barrier dysfunction is themain driver of ulcerative colitis.Tis view is supported bythe fact that there is a decrease in goblet cells and a decreasein the

11、 permeability of the mucous barrier in patients withactive ulcerative colitis 5.Recent experimental evidenceindeedimplicatesacrucialfunctionofbarrierdysfunctioninthe onset of inflammatory bowel disease 6.IBD can becaused by the dysfunction of intestinal epithelial cell,whichis the internal molecular

12、 circuit to control the homeostasis,renewal,and repair of intestinal epithelial cells 7.As acrucial structural factor of this barrier,tight junction(TJ)proteins can maintain intestinal mucosal barrier,preventinvasion of bacterium and toxin,and thereby reduce theprogression of UC 8.Besides,TJ opening

13、 is driven bymyosin light chain(MLC)phosphorylation,which dependson myosin light chain kinase(MLCK)activation 9.Recentexperimental evidence also implicates that MLCK leads to amechanical tension-generated opening of the TJ barrier bycontracting the perijunctional actomyosin filaments 10,11.Experimen

14、ts have proved that increasing TJ permeabilitywas mediated by toll-like receptor 4(TLR4)/MyD88 signal-HindawiEvidence-Based Complementary and Alternative MedicineVolume 2021,Article ID 6657141,12 pageshttps:/doi.org/10.1155/2021/6657141transduction pathway upregulation of MLCK expression11.TLR4(toll

15、-like receptor 4)is a key pattern-recognitionreceptor for commensal recognition in gut innate immunityand it is overexpressed on the surface of inflamed colon12,13.Today,the pharmacologic management of UC mainlyrelies on 5-aminosalicylates,corticosteroids,and immuno-suppressants and biologic therapi

16、es 14,but the side effectsof these treatment still exist 15,16.In recent years,as analternative treatment modality for the treatment of UC,traditional Chinese medicine(TCM)has demonstratedobservablyclinicalefficacy17.TCMtreatmentofulcerativecolitis is based on syndrome differentiation,and TCM be-lie

17、ves that ulcerative colitis has the problem of spleen de-ficiency.Bu-zhong-yi-qi granule(BZYQ)is a classicalprescription for curing spleen deficiency in traditionalChinese medicine,which has been used for a long time inclinic,and it is often used in the treatment of ulcerativecolitis in China.It is

18、composed of 10 ingredients,includingAstragalus,Dangshen,Liquorice,Atractylodesmacro-cephala,Tangerine peel,Cohosh,Bupleurum,Angelica,Ginger,and Jujube.Studies have shown that BZYQ treat-ment has a protective effect on 5-FU-induced intestinalmucositis in mice,and concluded that BZYQ treatment mayinhi

19、bit the upregulation of inflammatory cytokines 18,andexperiments also have proved BZYQ could ameliorate pa-tients with chronic nephritis through TLRs/MyD88 sig-naling pathway 19.Active components of BZYQ,such asAstragalus polysaccharides,have been implicated to protectagainst DSS-induced colitis by

20、inhibiting NF-C activation20.However,the molecular mechanism by which BZYQinhibits ulcerative colitis is unclear.In this study,we aim tostudy the therapeutic efficacy of BZYQ treatment on UC.Inaddition,we analyzed the expression of tight junction-re-lated proteins to study the protective mechanism o

21、f BZYQsmucosal barrier.2.Materials and Methods2.1.Experimental Materials2.1.1.Animals.Tis animal experiment complies with theARRIVE guidelines and the National Institutes of HealthGuide for the Care and Use of Laboratory Animals(NIHPublications No.8023,revised 1978).Specific pathogen-freemale Wistar

22、 Rats(250g10g)were acquired from theChinese Peoples Liberation Army Military Medical Acad-emy Experimental Animal Center.Rats were housed in theInstitute of Basic Teory,China Academy of ChineseMedical Sciences(Te Approval Number is 2017-098).Allrats were kept in a standardized environment at a 12-ho

23、urlight/dark cycle(lights on at 8:00 a.m.)with a normativetemperature(2123C)and humidity(50%5%).All therats had free access to food and water.Te study was ap-proved by the Animal Care and Use Committee of ChinaAcademy of Traditional Chinese Medicine.2.1.2.Induction of UC in Rats.Intracolonic injecti

24、on withTNBS which induces UC in rats is an acknowledgedmodeling method 21.Briefly,rats were fasted for 36 hoursbut allowed free access to water.Te rats were deeplyanesthetizedbyinhalingUlan(epinpharmaceutical,c002160902);then,TNBS solution(sigma,P2297)was in-jected into the colon of the rats(100mg/k

25、g,dissolved in 50%ethanol according to the proportion of 1:1)by inserting a8cm medical-grade tube into the anus.After injection of theTNBS,the ratswerekept in ahead-downposition for 30minto prevent leakage 22.2.1.3.Drugs and Administration.Tey were as follows:Bu-zhong-yi-qi granule(BZYQ)(batch numbe

26、r:Z20040120,Beijing Han Dian Pharmaceutical Co.,Ltd.,Beijing,China);SulfasalazineEnteric-coatedTablets(SASP)(batch number:H31020557,Shanghai SINE Pharmaceu-tical Co.,Ltd,Shanghai,China).According to theequivalent dose-ratio table of human and animal bodysurfacearea,thedosageoftheratsXmg/kg70kg0.018/

27、0.2kg(X is the adult clinical dosage)23.Twenty-four hours after induction of UC,treatment beganand was continued for seven days.Te normal control(no handling)rats were classified ascontrol group;UC rats were divided into three groups:model group,the positive control group:SASP group(0.315g/kg),and B

28、ZYQ group(1.89g/kg).After establish-ment of the model,the rats in positive control group and theBZYQ group were given intragastric administration withtreatment,respectively;the control group and model groupwere given distilled water.All groups were treated for 7consecutive days.After being administe

29、red by gastric gavagefor 7 days,all rats were killed with the method of cervicaldislocation and the colon was excised for the followingexperiments.2.1.4.Ultra Performance Liquid Chromatography-Tandem MassSpectrometry(UPLC-MS/MS)Analysis.Chromatographicseparation was performed by Termo Scientific u30

30、00 fastliquid chromatography.A Rapid Separation LC spectrom-eter and hybrid quadrupole Orbitrap mass spectrometerwere employed.Te voltage of positive and negative ionsource was 3.7kv and 3.5kv,respectively.Te capillaryheating temperature was 320C.Te warping pressure was30psi and the auxiliary gas pr

31、essure was 10psi.Volumeheating evaporation temperature was 300C.Te warpinggas and auxiliary gas were both nitrogen.Te collision gaswas nitrogen and the pressure was 1.5 mtorr.Te liquidquality system was controlled by Xcalibur 2.2 SP1.48 soft-ware.Data acquisition and targeted metabolite quantitative

32、processing were operated by the software.2.2.DAI,CMDI,and Histological Evaluation2.2.1.Disease Activity Index(DAI).A disease activity index(DAI)score was used for the evaluation of the severity ofcolonic inflammation in rats after 7 days of the treatment:DAI(score of weight loss+stool consistency+de

33、gree ofhematochezia)/3 24.Te details are shown in Table 1.2Evidence-Based Complementary and Alternative Medicine2.2.2.Colon Macroscopic Damage Index(CMDI).After 7days of the treatment,the distal colon(8cm from the anus)was immediately obtained and longitudinally incised frommesentery.Ten,the opened

34、sample of colon was washedout thoroughly by cold normal saline and measured by blindevaluation via colon macroscopic damage index(CMDI)involving severity of inflammation and existence of ulcer2527.Te scoring criteria were described as follows:0:noulcer,no inflammation;1:no ulcer,local hyperemia;2:ul

35、cerwithout hyperemia;3:ulcer and inflammation at one siteonly;4:two or more sites of ulcer and inflammation;and 5:ulcer extending more than 2cm.2.2.3.Histological Evaluation.Four samples of rat for eachgroup were selected by random choice,and then the samplesof colonic tissue from each rat were fixe

36、d in 4%bufferedformaldehyde and embedded in paraffin.Sections weresliced to a thickness of 5m and stained with hematoxylin-eosin.Te sections were detected under microscope(NanoZoomer S60,Japanese)by a researcher unaware of thetreatment.2.3.Quantitative Real-Time Polymerase Chain Reaction(qPCR).Te to

37、tal RNA was extracted from colon sampleswith Trizol reagent(Termo Fisher Scientific,USA)and 5ugof total RNA was reversely transcribed to cDNA accordingto the reagent instructions.Te expression levels of TLR4,MLCK,NF-B,TNF-,IL-1,and IL-10 were detected bySYBR Master Mix(Promega,USA)and the CFX 96 Rea

38、l-Time PCR System(BIO-RAD,USA).RT-PCR was based onour previous report.Te primers are shown in Table 2.2.4.Western Blot.Te colonic tissue was homogenized byapplying ice-cold RIPA buffer containing 1mM PMSF(Solarbio,China).TeproteinconcentrationwasassessedbyBCA assay.Te equal amount of protein(80g)of

39、eachsample was separated by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis(SDS-PAGE)using 10%polyacrylamide gels and electrophoretically transferred topolyvinylidene fluoride membranes after separation.Temembrane was then incubated with the following primaryantibodies:anti-occludin(1:400

40、,SantaCruzBiotechnology),anti-claudin-1(1:1500,Abcam)and anti-actin(1:1500,ZSGB-BIO),anti-NF-B(1:1000,YM3111,Immunoway,USA),anti-p-NF-B(1:1500,YP0193,Immunoway,USA),anti-TLR4(1:1000 dilution,Proteintech),anti-MLC(1:400,Abcam),and anti-p-MLC(1:400,CSL)at 4C overnight.Blots were washed three times by

41、TBST(Solarbio,China);then,themembranewasincubatedwithmatchingsecondaryantibodies:DyLight 680-labeled goat anti-rabbit or goatanti-mouse(both 1:10000,KPL).Te specific protein bandsonthemembranewerevisualizedandtheproteinlevelswereanalyzed with Image-ProPlus.2.5.Immunofluorescence.Te colonic sections

42、were fixed inOCTand then sliced at6 m thickness byfreezing microtome(TermoFisher,USA).Sections were incubated with anti-occludin(1:50,Santa Cruz Biotechnology)or anti-claudin-1(1:40,Abcam)at 4C overnight.After three times repeatedTNBS washing,and then followed by incubation with rabbitanti-goat or g

43、oat anti-rabbit(1:100,ZSGB-BIO)2nd anti-body for 30min at room temperature,respectively,the sec-tions were mounted with DAPI(ZSGB-BIO)and detected byfluorescence microscope(Pannoramic MIDI,USA).2.6.Statistical Methods.Te statistical analyses were exam-ined byapplying SPSS17.0 software(SPSS,Chicago,I

44、L,USA)and each value was expressed as means SE.All the originaldata in the study were distributed normallyand conformed tohomogeneity of variance.Te differences among the differentgroups were analyzed using one-way analysis of variance(ANOVA)followed by least-significant difference(LSD)testto compar

45、e the differences between every two groups.P15Liquid stoolGrossbloodTable 2:Primers used for RT-PCR.TargetgenePrimer sequence(5-3)-ActinForward:5-AGAGGGAAATCGTGCGTGAC-3Reverse:5-CCATACCCAGGAAGGAAGGCT-3TLR4Forward:5-CCAGAGCCGTTGGTGTATCT-3Reverse:5-TCAAGGCTTTTCCATCCAAC-3MLCKForward:5-GACGTGTTCACCCTGGT

46、TCT-3Reverse:5-TTTGTGCAGCATCAGTGACA-3NF-bForward:5-GGCAGCACTCCTTATCAA-3Reverse:5-GGTG TCGTCCCATCGTAG-3TNF-Forward:5-AGAACTCCAGGCGGTGTCT-3Reverse:5-TCCCTCAGGGGTGTCCTTAG-3IL-1Forward:5-ATAGCAGCTTTCGACAGTGAG-3Reverse:5-GTCAACTATGTCCCGACCATT-3IL-10Forward:5-GCAGGACTTTAAGGGTTACTTGG-3Reverse:5-TCATTCTTCAC

47、CTGCTCCACTG-3Evidence-Based Complementary and Alternative Medicine3(Figure 1(a)and negative(Figure 1(b)ion chromatogramsof BZYQ granule demonstrated the composition of all in-gredients(Figure 1(a).3.2.BZYQ Treatment Alleviated TNBS-Induced Colitis.Firstly,we observed the clinical features,weight los

48、s,andloose stool with mucus and/or blood in model group.InSASP and BZYQ on the 7th day,bloody stools were alle-viated,characteristics of stools were better formed,andweight loss was relieved.Te length and gross appearance ofcolon were evaluated as shown in Figures 2(b)and 2(c);thecolonfrommodelgroup

49、showedobviousedema,hyperemia,inflammation,and ulcer.After treatment with SASP orBZYQ,the above damaged signs of colon were significantlyalleviated.DAI can reflect the severity of UC through three aspects,which include weight loss,stool consistency,and hema-tochezia.CMDI is known to be a macroscopic

50、evaluation ofinflammation.As shown in Table 3,compared with thecontrol rats,UC rats showed a significant increase in DAIand CMDI(3.170.39 vs 0.000.00,P0.01;3.670.49 vsRT:0.00 16.01012345678910111213141516Time(min)05101520253035404550556065707580859095100Relative abundance5.740.850.904.056.084.019.20