松果菊苷对TNFα诱导的SH2SY5Y细胞凋亡的保护作用

TNFSH2SY5Y1,1,2,2,1(1.,100083;2.,100083):2004-03-21,:2004-06-09:(No30070651):(1974-),,,E2mail:[e-mail];(1942-),,,,:,Tel:01026201769122651,Fax:[phone],E2mail:[e-mail]:R282171;R28411;R329125;R7301264:A:1001-1978(2005)02-0169-06TNFSH2SY5YMTT,DNA,,caspase23100gL-1TNF36h;,37%;caspase23;,/519701351,10100mgL-12h,;DNAladder;2519%,1813%812%;;;caspase23,TNFSH2SY5Y,,caspase23:;;TNF;SH2SY5Y;;;caspase23,,[1][2],,HerbaCistanches,,,[3,4],,[5,6],,,(echinacoside)(Cistanchetubulosa)(Fig1)[7],,,alpha(TNF),SH2SY5YFig1Chemicalstructureofechinacoside11.11.1.1alpha(TNF)(trypsin)42(MTT)Sigma;MEMGIBCO;(AnnexinV2FITC)(propidumiodid,PI)BoehringerMannheim;27-(2,72dichlorodihydrofluoresceindi2acetate,H2DCFDA)JC1MolecularProbes;DNAcaspase23Promega;,98%1.1.2SH2SY5Y(961ChinesePharmacologicalBulletin2005Feb;21(2):16974)1.1.3Waters600,2487-,Bruker300;:LEICA;CO2:SANYO;ELISA:BIO2RAD;:SPOT22CooledCCD,Metamorph,LEICA;:BD;:TCSSP2,LEICA;:WallacVictor2TM1.21.2.1SH2SY5Y10%()MEM,23d180%,010025,,13(1,10or100mgL-1)2h100gL-1TNF,36h100gL-1TNF,PBS1.2.2MTT1104SH2SY5Y96,24h100gL-1TNF(1,10or100mgL-1),36h,5gL-1MTT,4h,DMSO200l,,570nm(A570nm),1.2.3DNAPromegaDNADNADNA10l115%,60V1h1.2.4AnnexinV2FITC,,:36h,1106,PBS23,500rmin-15min200l,10lAnnexinV2FITC5lPI,,15min,300l,1.2.5[8]H2DCFDA,,,,,1104SH2SY5YConfocal,24h100gL-1TNF2h(1,10100mgL-1),36h,PBS2,10molL-1DCFH2DA100l,3730min,,488nm,530560nm1.2.6[9]JC21,,JC21,;,JC21,/SH2SY5YConfocal,36h,1molL-1JC2137310min,Hanks,490nm,590nm1.2.7caspase23[10]Apo2ONETMHomogeneousCaspase23(Promega),caspase23,:104/96,36h,PBS,caspase23100l,4h(WallacVictor2TM1420MultilabelCounter,USA)Z2DEVD2R110,498nm,521nm,relativefluores2cenceunits(RFU)1.2.8€xs,(ANOVA),Studentst,SPSS22.1TNFSH2SY5YMTTTNF36hSH2SY5Y4219%,2h(1,10100mgL-1)TNF,(32%,20%3%;Fig2)2.2TNFDNA,TNFSH2SY5Y36hDNA,10100mgL-1071ChinesePharmacologicalBulletin2005Feb;21(2)DNALad2der(Fig3)Fig2EffectofechinacosideonTNF2induceddecreaseinSH2SY5Ycellviability(€xs,n=8)CellviabilitywasdetectedbyMTTmethodasdescribedinMaterialsandMethods.##P0101vscontrolcells;3P0105,33P0101vsTNF.Fig3EffectofechinacosideonTNF2inducedDNAfragmentationinSH2SY5YcellsA:control;B:treatmentwith100gL-1TNF;C:treatmentwith100gL-1TNFand1mgL-1echinacoside;D:treatmentwith100gL-1TNFand10mgL-1echinacoside;E:treatmentwith100gL-1TNFand100mgL-1echinacoside.2.3TNFAn2nexinV2FITCPI,100gL-1TNFSH2SY5Y36h,37%;1,10100mgL-1,2519%,1813%812%(Fig4)2.4TNFSH2SY5Y100gL-1TNF36h,,1,10100mgL-12h,18%,53%82%(Fig5,Tab1)Tab1EffectofechinacosideonlevelofintracellularROSinTNF2inducedSH2SY5Ycells(€xs,n=8)GroupDCFfluorenscenceintensitycontrol9.81.3TNF89.612.5##TNF+1mgL-1echinacoside78.513.033TNF+10mgL-1echinacoside39.18.733TNF+100mgL-1echinacoside14.22.633##P0101vscontrolcells;33P0101vsTNF2.5TNFSH2SY5YSH2SY5Y,100gL-1TNF36h,,/51970121013501021,10100mgL-1,/11640109,4186010551770106(Fig6)2.6TNFSH2SY5Ycaspase23100gL-1TNF36hSH2SY5Ycaspase235,1,10100mgL-1,caspase23(Tab2),Tab2EffectofechinacosideonTNF2inducedincreaseofcaspase23activity(€xs,n=8)GroupCaspase23activity(RFU)control3076412TNF167251854##TNF+1mgL-1echinacoside12259153633TNF+10mgL-1echinacoside8162100233TNF+100mgL-1echinacoside629158633##P0101vscontrolcells;33P0101vsTNF3SH2SY5Y,TNF,MTT,DNA,An2nexinV-FITCPI,caspase23171ChinesePharmacologicalBulletin2005Feb;21(2)Fig4FlowcytometrichistogramsofcontrolSH2SY5YcellsandcellsexposedtoTNFaloneorwithechinacosideA:control;B:treatmentwith100gL-1TNF;C:treatmentwith100gL-1TNFand1mgL-1echinacoside;D:treatmentwith100gL-1TNFand10mgL-1echinacoside;E:treatmentwith100gL-1TNFand100mgL-1echinacoside.Normalcellsareinthebottomleftquadrant,apoptoticcellsinthebottomrightquadrant,andnecroticandlateapoptoticcellsintheupperright.Numbersineachquadrantarepercentageofcellstheycontain.Fig5LaserconfocalscanningmicroscopeimageofDCFfluorescenceincellsofvariousgruopA:controlcells;B:cellstreatedwith100gL-1TNF;C:cellstreatedwith100gL-1TNFand1mgL-1echinacoside;D:cellstreatedwith100gL-1TNFand10mgL-1echinacoside;E:cellstreatedwith100gL-1TNFand100mgL-1echinacoside.Fig6ConfocallaserscanningimagesofJC21fluorescenceinSH2SY5Ycellsafter36hexposuretoTNFintheabsenceorpresenceofechinacosideA:controlcells;B:treatmentwith100gL-1TNF;C:treatmentwith100gL-1TNFand1mgL-1echinacoside;D:treatmentwith100gL-1TNFand10mgL-1echinacoside;E:treatmentwith100gL-1TNFand100mgL-1echinacosideTNF,TNF,1100mgL-1,caspase23,,,,,,,[11,12],DNA[13]TNF,;,TNFCaspase,Caspase23,,,;,C,,Caspase,[14,15]Caspase23,ADP(PARP),DNA,271ChinesePharmacologicalBulletin2005Feb;21(2)[16]JC1Caspase23,TNF,Caspase23;TNF,Caspase23,TNFSH2SY5Y,:[1]MartinLJ.Neuronalcelldeathinnervoussystemdevelopmentdis2ease,andinjury[J].IntJMolMed,2001,7:455-78.[2]BrownDR,SchmidtB,KrelzschmarHA.EffectsofoxidativestressonprionproteininPC12cells[J].IntJDevlNeurosci,1997,15:961-72.[3],.[J].,2003,10:50-1.[4]LeiL,YangFQ,ZhangTY.Preparativeisolationandpurificationofacteosideand292acetylacteosidefromCistanchessalsa(C.A.Mey.)G.Beckbyhighspeedcounter2currentchromatography[J].JChromatogrA,2001,912:1815.[5]GaoJJ,IgalashiK,NukinaM.Radicalscavengingact