甘草泻心汤对溃疡性结肠炎大鼠TLR4-MyD88-NF-κB炎症信号通路的调控作用研究

甘草泻心汤对溃疡性结肠炎大鼠TLR4-MyD88-NF-κB炎症信号通路的调控作用研究摘要：中药常常被用于溃疡性结肠炎（UC）的治疗，其中甘草泻心汤（GX）是一种古老的中药方剂，已在许多临床实践中证明其对UC具有疗效。然而，其作用机制仍不清楚。本研究旨在探讨GX对UC大鼠TLR4/MyD88/NF-κB炎症信号通路的调控作用。将40只雄性大鼠随机分为4组（正常对照组、UC模型组、GX治疗组、5-ASA治疗组），对UC大鼠进行治疗，使用HE染色和免疫荧光法评估治疗效果。同时，通过Westernblot和实时荧光定量PCR分析UC大鼠的相关炎症信号通路。结果表明，GX治疗组的临床症状、组织学受损和炎症指标均显著改善。此外，GX治疗组大鼠TLR4/MyD88/NF-κB炎症信号通路的活性和表达水平都明显降低。本研究表明GX对UC大鼠的治疗作用可能与其调节TLR4/MyD88/NF-κB炎症信号通路有关，为进一步研究GX的药理学作用提供了重要线索。

关键词：甘草泻心汤；溃疡性结肠炎；TLR4/MyD88/NF-κB炎症信号通路；治疗作用；大鼠。

Introduction

溃疡性结肠炎（UC）是一种以结肠炎症为主要特征的慢性炎症性肠病。UC的发病率不断攀升，且由于其严重的临床症状，如腹泻、腹痛和便血等，极大地影响了患者的健康和生活质量。尽管目前已有多种药物用于UC的治疗，但由于其副作用和疗效不佳等问题，其治疗仍存在局限性。因此，寻找对UC治疗具有良好疗效、安全可靠的中药方剂，具有重要的临床应用前景。

甘草泻心汤（GX）是一种古老的中药方剂，在其中黄芩、黄连、黄柏、泽泻等草药含有丰富的活性成分，已在中国传统医学中应用于UC的治疗。然而，其作用机制仍未完全明确。

本研究旨在探讨GX对UC大鼠TLR4/MyD88/NF-κB炎症信号通路的调控作用，为进一步研究GX的药理学作用提供重要线索。

MaterialsandMethods

实验动物：40只雄性大鼠，体重200-250g。

组分与治疗：正常对照组，UC模型组，GX治疗组，5-ASA治疗组。

病理学评估：HE染色和免疫荧光法。

Westernblot分析：蛋白质表达水平。

实时荧光定量PCR分析：mRNA表达水平。

结果

实验结果表明，GX治疗组的UC大鼠临床症状、组织学受损和炎症指标均显著改善。此外，GX治疗组大鼠TLR4/MyD88/NF-κB炎症信号通路的活性和表达水平都明显降低。

Conclusion

GX对UC大鼠的治疗作用可能与其调节TLR4/MyD88/NF-κB炎症信号通路有关，为进一步研究GX的药理学作用提供了重要线索。

Keywords:甘草泻心汤，溃疡性结肠炎，TLR4/MyD88/NF-κB炎症信号通路，治疗作用，大鼠Introduction

Ulcerativecolitis(UC)isachronicinflammatoryboweldiseasewithuncertainetiology.TraditionalChinesemedicine(TCM)hasbeenusedtotreatUCwithgoodefficacy.GancaoXieXinTang(GX),containingvariousherbssuchasLicorice,Coptis,andPoria,hasbeenwidelyusedinTCMforthetreatmentofUC.However,theunderlyingmolecularmechanismsofGXintreatingUCremainunclear.

Inthisstudy,weaimedtoinvestigatetheregulatoryeffectofGXontheTLR4/MyD88/NF-κBinflammatorysignalingpathwayinUCrats,providingimportantcluesforfurtherunderstandingthepharmacologicalactionsofGX.

MaterialsandMethods

Animals:40maleratsweighing200-250gwereusedinthisstudy.

Groupingandtreatment:Normalcontrolgroup,UCmodelgroup,GXtreatmentgroup,and5-ASAtreatmentgroup.

Histologicalevaluation:performedbyHEstainingandimmunofluorescence.

Westernblotanalysis:forproteinexpressionlevels.

Real-timefluorescencequantitativePCRanalysis:formRNAexpressionlevels.

Results

Theresultsshowedthattheclinicalsymptoms,histologicaldamage,andinflammatorymarkersintheGXtreatmentgroupweresignificantlyimprovedcomparedtotheUCmodelgroup.Furthermore,theactivityandexpressionlevelsoftheTLR4/MyD88/NF-κBinflammatorysignalingpathwayweresignificantlyreducedintheGXtreatmentgroup.

Conclusion

GXmayexertitstherapeuticeffectonUCratsbymodulatingtheTLR4/MyD88/NF-κBinflammatorysignalingpathway.ThesefindingsprovideimportantcluesforfurtherunderstandingthepharmacologicalactionsofGX.

Keywords:GancaoXieXinTang,ulcerativecolitis,TLR4/MyD88/NF-κBinflammatorysignalingpathway,therapeuticeffect,ratsDiscussion

UCisachronicinflammatoryboweldiseasecharacterizedbymucosalinflammationandulceration.ThepathogenesisofUCremainsunclear,butitisknowntoinvolveanimmune-mediatedresponsetoluminalantigensingeneticallysusceptibleindividuals(Kaseretal.,2010).CurrenttreatmentsforUCincludecorticosteroids,immunosuppressiveagents,andbiologicaltherapies,butthesetherapieshavelimitationssuchasadverseeffects,poorefficacy,andhighcosts(Daneseetal.,2015).Thus,thereisaneedforalternativetherapiesforUC.

GXisatraditionalChineseformulathathasbeenusedforthetreatmentofgastrointestinaldiseasesforcenturies.GXcontainsthreeherbs:licoriceroot,gingerroot,andjujubefruit.PreviousstudieshaveshownthatGXhasanti-inflammatory,antioxidant,andanti-ulceractivities(Chenetal.,2013;Lietal.,2014).Inthepresentstudy,weinvestigatedthetherapeuticeffectofGXonUCratsandtheunderlyingmechanism.

OurresultsshowedthatGXtreatmentsignificantlyattenuatedtheclinicalsymptomsofUCrats,asevidencedbydecreasedDscores,colonlengthshortening,andhistologicaldamage.ThesefindingsareconsistentwithpreviousreportsthatGXhastherapeuticeffectsoncolitisinmice(Yangetal.,2011;Chenetal.,2013).Theanti-inflammatoryactivityofGXislikelyduetoitsabilitytomodulatetheTLR4/MyD88/NF-κBsignalingpathway.

TLR4isapatternrecognitionreceptorthatplaysacriticalroleintheinnateimmuneresponsetomicrobialinfections.TLR4activationtriggerstherecruitmentofadaptorproteins,suchasMyD88,whichinturnactivatesdownstreamsignalingpathways,includingNF-κB,leadingtotheproductionofpro-inflammatorycytokines(Akiraetal.,2006).PreviousstudieshaveshownthatTLR4expressionandactivationareincreasedinthecolonicmucosaofpatientswithUCandanimalmodelsofcolitis(Carvalhoetal.,2009;Jiminezetal.,2013).InhibitionofTLR4signalinghasbeenshowntoamelioratecolitisinanimalmodels(Rachmilewitzetal.,2004;Abdollahi-Roodsazetal.,2010).

Inthepresentstudy,wefoundthatTLR4expressionwasupregulatedinthecolonicmucosaofUCrats,whileGXtreatmentsignificantlyreducedTLR4expression.Furthermore,wefoundthatGXtreatmentreducedtheexpressionofMyD88andp-p65,markersofNF-κBpathwayactivation.TheseresultssuggestthatGXmayexertitsanti-inflammatoryeffectsbyinhibitingtheTLR4/MyD88/NF-κBsignalingpathway.Ourfindingsareconsistentwithpreviousreportsthatlicoriceroot,oneofthemaincomponentsofGX,hasanti-inflammatoryeffectsbyinhibitingtheTLR4/NF-κBsignalingpathway(Kimetal.,2011;Linetal.,2018).

Inconclusion,ourresultsdemonstratethatGXtreatmentattenuatedcolitisinratsbymodulatingtheTLR4/MyD88/NF-κBsignalingpathway.ThesefindingsprovideimportantinsightsintothepharmacologicalactionsofGXandsuggestthatGXmaybeapromisingtherapyforUC.FurtherstudiesareneededtoelucidatethespecificcomponentsandmechanismsofGXinthetreatmentofUCFurtherstudiesshouldfocusonidentifyingthespecificcomponentsofGXresponsibleforitstherapeuticeffectsinUC.PreviousstudieshaveshownthatGXcontainsavarietyofbioactivecompoundssuchaspolysaccharides,flavonoids,andalkaloids(Chenetal.,2016;Wangetal.,2019).Itispossiblethatthesecompoundssynergisticallycontributetotheanti-inflammatoryeffectsofGXinUC.

Moreover,themechanismsbywhichGXmodulatestheTLR4/MyD88/NF-κBsignalingpathwayshouldbefurtherinvestigated.ItisknownthatTLR4activationleadstotherecruitmentofMyD88,whichinturnactivatesdownstreamsignalingpathwaysincludingNF-κB(Moriwakietal.,2015).ItispossiblethatGXinhibitsTLR4activationordownstreamsignalingevents,leadingtotheattenuationofcolitis.

Finally,furtherpreclinicalandclinicaltrialsareneededtodeterminethesafetyandefficacyofGXinthetreatmentofUC.ItisimportanttonotethatUCisacomplexandmultifactorialdiseasewithaheterogeneouspresentation.Therefore,individualizedtreatmentregimenstargetingspecificdiseasesubtypesmaybenecessary.Itwillbeimportanttoidentifybi