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  • 简介:AIM:Toelucidatethemechanism(s)bywhichS-adenosyl-L-methionine(SAM)decreaseshepatitisCvirus(HCV)expression.METHODS:WeexaminedtheeffectsofSAMonviralexpressionusinganHCVsubgenomicrepliconcellculturesystem.Huh7HCV-repliconcellsweretreatedwith1mmol/LSAMfordifferenttimes(24-72h),thentotalRNAandproteinswereisolated.cDNAwassynthesizedandrealtime-PCRwasachievedtoquantifyHCV-RNA,superoxidedismutase1and2(SOD-1,SOD-2)catalase,thioredoxin1,methionineadenosyltransferase1Aand2A(MAT1A,MAT2A)expression,andGAPDHandRPS18asendogenousgenes.Expressionofcellularandviralproteinwasevaluatedbywestern-blotanalysisusingantibodiesvsHCV-NS5A,SOD-1,SOD-2,catalase,thioredoxin-1,MAT1A,MAT2A,GAPDHandactin.TotalglutathionelevelsweremeasuredatdifferenttimesbyEllman’srecyclingmethod(0-24h).Reactiveoxidativespecies(ROS)levelswerequantifiedbythedichlorofluoresceinassay(0-48h);Pyrrolidindithiocarbamate(PDTC)wastestedasanantioxidantcontrolandH2O2asapositiveoxidantagent.RESULTS:SAMexpositiondecreasedHCV-RNAlevels50%-70%comparedtonon-treatedcontrols(24-72h).SAMinducedasynergicantiviraleffectwithstandardIFNtreatmentbutitwasindependentofIFNsignaling.Inaddition,1mmol/LSAMexpositiondidnotmodifyviralRNAstability,butitneedscellulartranslationmachineryinordertodecreaseHCVexpression.TotalglutathionelevelsincreaseduponSAMtreatmentinHCV-repliconcells.Transcriptionalantioxidantenzymeexpression(SOD-1,SOD-2andthioredoxin-1)wasincreasedatdifferenttimesbutinterestingly,therewasnosignificantchangeinROSlevelsuponSAMtreatment,contrarytowhatwasdetectedwithPDTCtreatment,whereanaverage40%reductionwasobservedinexposedcells.TherewasaturnoverfromMAT1A/MAT2A,sinceMAT1Aexpressionwasincreased(2.5fold-timesat48h)andMAT2Awasdiminished(from24h)uponSAMtreatmentatboththetranscriptionalandtranslationall

  • 标签: Hepatitis C virus S-ADENOSYL-L-METHIONINE Superoxide DISMUTASE