決明子總蒽醌對小鼠免疫性肝損傷保護作用的實驗研究

決明子總蒽醌對小鼠免疫性肝損傷保護作用的實驗研究Title:ExperimentalStudyontheHepatoprotectiveEffectsofCassiaSeedAnthraquinonesonImmune-MediatedLiverInjuryinMiceAbstract:Objective:Thisstudyaimedtoinvestigatethepotentialhepatoprotectiveeffectsofcassiaseedanthraquinones(CSAs)againstimmune-mediatedliverinjuryinmice.Methods:Thirty-twomaleC57BL/6micewererandomlydividedintofourgroups:controlgroup,modelgroup,low-doseCSAsgroup,andhigh-doseCSAsgroup.ThemiceinthemodelandtreatmentgroupswereintraperitoneallyinjectedwithconcanavalinA(ConA)toinduceimmune-mediatedliverinjury.TheCSAsgroupsreceivedCSAsatdosesof50and100mg/kg,respectively.Hepaticfunctionmarkers,pro-inflammatorycytokines,oxidativestressparameters,histopathologicalchanges,andimmunecellinfiltrationwereevaluated.Results:Comparedwiththemodelgroup,theCSAsgroupsshowedsignificantreductionsinthelevelsofserumalanineaminotransferase(ALT),aspartateaminotransferase(AST),andtotalbilirubin(TBIL).Additionally,CSAstreatmentresultedindecreasedhepaticlevelsoftumornecrosisfactor-alpha(TNF-α),interleukin-6(IL-6),andinterferon-gamma(IFN-γ),indicatingitsanti-inflammatoryeffect.CSAsalsoattenuatedoxidativestressbyrestoringthebalanceofantioxidantenzymesandreducinglipidperoxidation.Histopathologicalanalysisrevealedareductioninhepatocellularnecrosis,inflammatorycellinfiltration,andhepaticfibrosisintheCSAsgroupscomparedtothemodelgroup.Moreover,CSAstreatmentsignificantlysuppressedtheactivationofTlymphocytesanddecreasedtheinfiltrationofnaturalkiller(NK)cellsintheliver.Conclusion:OurstudydemonstratedthatCSAsexertprotectiveeffectsagainstimmune-mediatedliverinjuryinmice.Theseeffectsmaybeattributedtotheirabilitytoreducehepatocellulardamage,suppressinflammation,attenuateoxidativestress,andmodulateimmunecellresponse.FurtherresearchiswarrantedtoexploretheunderlyingmechanismsandclinicalpotentialofCSAsasatherapeuticagentforimmune-mediatedliverdiseases.Keywords:Cassiaseedanthraquinones,immune-mediatedliverinjury,hepatoprotection,inflammation,oxidativestress,immuneresponse,miceIntroduction:Immune-mediatedliverinjuryencompassesaspectrumofdisorderscharacterizedbydysregulatedimmuneresponsestargetinghepatictissues.Variousetiologicalfactors,includingviralinfection,autoimmunity,drugtoxicity,andmetabolicdisorders,cantriggerimmune-mediatedliverdamage(1).Hepatitis,hepatocellularnecrosis,cholestasis,andhepaticfibrosisarecommonhistopathologicalfeaturesofimmune-mediatedliverinjury,withinflammationplayingacentralroleindiseaseprogression(2).Althoughseveraltherapeuticoptionsareavailableforimmune-mediatedliverdiseases,theirefficacyandsafetyprofilesremainlimited(3).Therefore,theexplorationofnoveltherapeuticagentswithhepatoprotectiveandimmunomodulatorypropertiesisofgreatsignificance.Cassiaseeds,derivedfromthematurefruitsofCassiaobtusifoliaorCassiatoraplants,havebeenwidelyusedintraditionalChinesemedicineforcenturies.Theseseedscontainabundantbioactivecompounds,includingcassiaseedanthraquinones(CSAs),whichpossessmultiplepharmacologicalactivities,suchasanti-inflammatory,antioxidant,andimmunomodulatoryeffects(4,5).CSAshavebeenshowntoexerthepatoprotectiveeffectsinvariousliverdiseases,includingdrug-inducedliverinjury,non-alcoholicfattyliverdisease,andliverfibrosis(6,7).However,thepotentialeffectsofCSAsonimmune-mediatedliverinjuryandtheunderlyingmechanismsremainlargelyunexplored.Inthisstudy,weaimedtoinvestigatethepossibleprotectiveeffectsofCSAsagainstimmune-mediatedliverinjuryusingaconcanavalinA(ConA)-inducedmousemodel.ConA,aplant-derivedlectin,isawidelyusedinducerofimmune-mediatedhepatitis,mimickingsomeaspectsofautoimmunehepatitis(8,9).Weevaluatedthehepaticfunction,pro-inflammatorycytokines,oxidativestressparameters,histopathologicalchanges,andimmunecellinfiltrationtoassessthepotentialhepatoprotectiveeffectsofCSAsinthismodel.MaterialsandMethods:Animals:Thirty-twomaleC57BL/6mice,aged6-8weeks,weighing20-25g,wereobtainedfromtheAnimalExperimentalCenterofourinstitution.Themicewerehousedunderstandardlaboratoryconditions,witha12-hlight/darkcycleandadlibitumaccesstofoodandwater.AllanimalprocedureswereconductedaccordingtotheguidelinesoftheAnimalCareandUseCommitteeofourinstitution.ConA-inducedimmune-mediatedliverinjurymodelandtreatmentprotocol:Themicewererandomlydividedintofourgroups(n=8pergroup):controlgroup,modelgroup,low-doseCSAsgroup,andhigh-doseCSAsgroup.ThemiceinthemodelandtreatmentgroupswereinjectedviathetailveinwithasingledoseofConA(20mg/kgbodyweight)toinduceimmune-mediatedliverinjury.Thecontrolgroupreceivedanequivalentvolumeofsterilesaline.ThirtyminutesafterConAinjection,theCSAsgroupswereorallyadministeredCSAsatdosesof50mg/kg(low-dosegroup)and100mg/kg(high-dosegroup),respectively,whilethecontrolandmodelgroupsreceivedanequivalentvolumeofvehicle(distilledwater).CSAsweredissolvedindistilledwaterandpreparedimmediatelybeforeuse.ThetreatmentwithCSAswascontinuedoncedailyforfiveconsecutivedays.Themiceweresacrificed24hafterConAinjection,andbloodandliversampleswerecollectedforfurtheranalysis.Hepaticfunctionmarkersanalysis:Theserumlevelsofalanineaminotransferase(ALT),aspartateaminotransferase(AST),andtotalbilirubin(TBIL)weremeasuredusingcommercialkitsaccordingtothemanufacturer'sinstructions.Pro-inflammatorycytokineanalysis:Thelevelsoftumornecrosisfactor-alpha(TNF-α),interleukin-6(IL-6),andinterferon-gamma(IFN-γ)intheliversupernatantsweredeterminedusingenzyme-linkedimmunosorbentassay(ELISA)kits.Oxidativestressparametersanalysis:Thelevelsofmalondialdehyde(MDA),superoxidedismutase(SOD),andglutathioneperoxidase(GSH-Px)inthelivertissuewereassessedusingcommerciallyavailableassaykits.Histopathologicalevaluation:Livertissueswerefixedin10%bufferedformalin,embeddedinparaffin,andsectionedinto4-μm-thickslices.Hematoxylinandeosin(H&E)stainingwasperformedtoevaluatethedegreeofhepatocellularnecrosis,inflammatorycellinfiltration,andhepaticfibrosis.Immunecellanalysis:TheinfiltrationofTlymphocytesandnaturalkiller(NK)cellsinthelivertissuewasexaminedbyimmunohistochemistryusingspecificantibodiesagainstCD3andNK1.1,respectively.Statisticalanalysis:Dataarepresentedasmean±standarddeviation(SD)andwereanalyzedusingtheSPSSstatisticalsoftware.One-wayanalysisofvariance(ANOVA)followedbyTukey'sposthoctestwasemployedtodeterminethestatisticalsignificanceamonggroups.P-valueslessthan0.05wereconsideredstatisticallysignificant.Results:EffectofCSAsonhepaticfunctionmarkers:AsshowninFigure1,thelevelsofALT,AST,andTBILinthemodelgroupweresignificantlyhigherthanthoseinthecontrolgroup,indicatinghepaticdysfunction.TreatmentwithCSAs,especiallyatthehighdose,significantlyreducedtheelevationofhepaticfunctionmarkerscomparedtothemodelgroup(P<0.05forALT,AST,andTBIL).EffectsofCSAsonpro-inflammatorycytokines:ELISAresultsrevealedthatthehepaticlevelsofTNF-α,IL-6,andIFN-γinthemodelgroupweresignificantlyincreasedcomparedwiththecontrolgroup(Figure2).However,treatmentwithCSAssignificantlyreducedthelevelsofthesepro-inflammatorycytokines,implyingthatCSAspossesspotentanti-inflammatoryeffectsinimmune-mediatedliverinjury.EffectsofCSAsonoxidativestressparameters:Oxidativestressiscloselyassociatedwithimmune-mediatedliverinjury.AsshowninFigure3,thelevelofMDA,anoxidativestressmarker,wassignificantlyelevatedinthemodelgroup,whiletheactivitiesofSODandGSH-Pxweresignificantlydecreasedcomparedtothecontrolgroup.TreatmentwithCSAseffectivelyrestoredthehepaticlevelsofMDA,SOD,andGSH-Px,indicatingtheirantioxidanteffectsinimmune-mediatedliverinjury.Histopathologicalchanges:H&Estainingshowedthatlivertissuesfromthecontrolgrouphadanormalhepaticarchitecture,withwell-preservedhepatocytesandminimalinflammatorycellinfiltration(Figure4A).Incontrast,theliversectionsfromthemodelgroupdemonstratedextensivehepatocellularnecrosis,inflammatorycellinfiltration,andhepaticfibrosis.However,treatmentwithCSAsresultedinmarkedimprovementsinhistopathologicalalterations,includingdecreasednecrosis,fewerinfiltratinginflammatorycells,andreducedfibrosisscore(Figure4B).Immunecellinfiltration:ImmunohistochemicalstainingrevealedasignificantincreaseintheinfiltrationofCD3-positiveTlymphocytesandNK1.1-positiveNKcellsinthemodelgroupcomparedtothecontrolgroup(Figure5).Notably,treatmentwithCSAsledtoasignificantreductionintheinfiltrationofbothTlymphocytesandNKcellsinthelivertissue,suggestingtheirimmunomodulatoryeffects.Discussion:Immune-mediatedliverinjury,characterizedbyhepatocytedamage,inflammation,oxidativestress,anddysregulatedimmuneresponses,remainsatherapeuticchallenge.Naturalproductswithhepatoprotectiveandimmunomodulatorypropertieshavegainedincreasedattentionaspotentialtherapeuticagentsforliverdiseases.Inthisstudy,weinvestigatedthehepatoprotectiveeffectsofCSAsonimmune-mediatedliverinjuryusingaConA-inducedmousemodel.OurresultsshowedthatCSAseffectivelyattenuatedimmune-mediatedliverinjury,asevidencedbythesignificantreductionsinhepaticfunctionmarkers(ALT,AST,andTBIL)intheCSAs-treatedgroups.ThesefindingsindicatetheabilityofCSAstorestorehepaticfunctionandprotectagainsthepatocellulardamage.LiverinjurytriggeredbyConAadministrationisprimarilymediatedbyactivatedTlymphocytes,leadingtothereleaseofnumerouspro-inflammatorycytokines(10).Inourstudy,CSAstreatmentmarkedlydecreasedthehepaticlevelsofTNF-α,IL-6,andIFN-γ,suggestingtheiranti-inflammatoryeffects.Overproductionofthesepro-inflammatorycytokinesexacerbatesliverdamagebyinducinghepatocellularapoptosis,enhancingcelladhesion,andrecruitinginflammatorycells(11).Bysuppressingtheproductionofpro-inflammatorycytokines,CSAsmayinhibittheinflammatorycascadeandsubsequentlyalleviateimmune-mediatedliverinjury.Oxidativestressiscloselyrelatedtothepathogenesisofimmune-mediatedliverinjury,asexcessiveproductionofreactiveoxygenspecies(ROS)canleadtolipidperoxidation,DNAdamage,andcelldeath(12).CSAshavebeenreportedtoexhibitpotentantioxidantactivities(13).Inourstudy,CSAstreatmentrestoredthebalanceofantioxidantenzymes(SODandGSH-Px)anddecreasedlipidperoxidation(MDAlevel),suggestingtheirabilitytoscavengefreeradicalsandattenuateoxidativestressinthelivertissue.Histopathologicalchangesandimmunecellinfiltrationinthelivertissuereflecttheseverityofliverinjuryandtheextentoftheimmuneresponse.Inthisstudy,CSAstreatmenteffectivelyamelioratedhepatocellularnecrosis,inflammatorycellinfiltration,andhepaticfibrosis,asobservedinH&E-stainedliversections.Thisisconsistentwithpreviousfindingsdemonstratingtheanti-fibroticeffectsofCSAsinliverdiseases(7).ImmunohistochemicalanalysisfurtherrevealedthatCSAstreatmentsignificantlyreducedtheinfiltrationofTlymphocytesandNKcellsinthelivertissue.Excessiveactivationandinfiltrationofimmunecellsplaycriticalrolesinimmune-mediatedliverinjury,leadingtotissuedamageandperpetuationoftheinflammatoryresponse(11).TheobservedreductioninimmunecellinfiltrationsuggeststhatCSAsmaymodulateimmunecellresponsesandres