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[]100723949(2005)132022012920421,,,(,050017)[];;Western;2;1;B;[]B21Western1P65,212.52550mol/L2mRNA35.85%42.46%53.46%;19.77%40.87%46.83%BP65,,P65B21[]R96[]AEffectsof12o2AcetylbritannilactoneonCyclo2Oxygenase22andIntercellularAdhesionMolecule21ExpressioninHumanVascularEndothelialCellsCHENYing2Zhu,WENJin2Kun,HANMei,andZHANGDi2Qun(LaboratoryofMedicalBiotechnology,InstituteofBasicMedicalScience,HebeiMedicalUniversity,Shijiazhuang050017,China)KEYWORDS12o2Acetylbritannilactone;VascularEndothelialCells;Cyclo2Oxygenase22;IntercellularAdhesionMolecule21;NuclearFactor2B;Lipopolysaccharide;WesternBlottingABSTRACTAimToinvestigatetheeffectsof12o2acetylbritannilactone(ABL)isolatedfromInulaBritannica2Fonacti2vationofnuclearfactor2B(NF2B)andcyclo2oxygenase22(COX22)andintercellularadhesionmolecule21(ICAM21)expressioninhumanvascularendothelialcells(ECV304)treatedwithlipopolysaccharide(LPS).MethodsICAM21andnuclearP65inproteinextractsfromECV304cellsweredetectedbyWesternblotting.COX22mRNAwasassayedbyreversetranscription2polymerasechainreaction(RT2PCR).ResultsAfterECV304cellswereincubatedwith12.5,25,50mol/LABLfor1h,COX22mRNAlevelinECV304cellstreatedwithLPSwasreducedby35.85%,42.46%and53.46%,respectively.Expres2sionofICAM21wasdecreasedby9.77%,40.87%and46.83%,respectively,comparedwithcontrolgroup.ABLcouldan2tagonizethenucleartranslocationofP65,andtheincreaseinnuclearP65inducedbyLPSnolongeroccurredfollowingtreatmentwithABL.ConclusionsABLinhibitsCOX22andICAM21expressioninvascularendothelialcellsbyinhibitingNF2Bacti2vation.[]2004210227[]2005202215[](30472167)[],,,,,,,,031126265563,E2mailWJK@hebmu.edu.cn,,,,(InulaBritannica2F),,(12o2acetylbritanni2lactone,ABL),,/,ABLB(nuclearfactor2B,NF2B),2(cyclo2oxygenase22,COX22)(inducednitricoxidesynthase,iNOS)2(prostaglandin2,PGE2)(Nitrogenmonoxide,NO),12,ABL/1,ABLCOX221(intercellularad2hesionmolecule21,ICAM21)B921CN4321262/R2005132,11.1,1(Figure1)ECV304M199GIBCO;P65ICAM21SantaCruz;Sigma;(polymerasechainreaction,PCR);1.Figure1.Chemicalstructureof12o2acetylbritannilactone1.2ECV30410%M199375%CO2,70%,2%,24h012.52550mol/LABL1h,100g/L61824h,RNA1.3MTT96ECV304ABL1h,100g/L24h20L5g/LMTT,374h,,100L10min,490nm(8)1.4WesternECV3041,RI2PA(50mmol/LTris2HCl,pH7.5,150mmol/LNaCl,1%NP40,0.1%SDS,1mmol/LED2TA,1mmol/LNaVO4,1mmol/LPMSF),,Lowry60g,SDS2PAGE,PVDF,5%,ICAM21(1200)(110000),ECV304140gSDS2PAGE,P65(1200)(110000),1.5RNA3ABLRNA,Oligo(dT)16,PETaqman,COX22PCRCOX2252ATTCTTTGCCCAGCACTTCA23,52TCTTTGACTGTGGGAGGATA23,207bp,GAPDH1.6xs,(OnewayANOVA),SPSS10.022.1ECV30412.52550mol/LECV30424h,MTT,,,ABLECV304(1,Table1),ABL1.ECV304(xs)Table1.EffectsofABLoncellsviabilityofECV(mol/L)(100g/L)A49000.1790.02101000.1740.02312.51000.1750.02925.01000.1800.03350.01000.1830.018031ISSN100723949ChinJArterioscler,Vol13,No22.22mRNA(100g/L)ECV304061824h,COX22mRNA2(Figure2),COX22,18h(140%),24h,18hABLCOX22mRNA,2(Figure2),12.52550mol/LABLCOX22mRNA,3ABLCOX22mRNA,35.85%42.46%53.46%50mol/LABLCOX22mRNA2.ECV3042mRNAFigure2.Effectsof12o2acetylbritannilactoneonCOX22mRNAexpressioninECV304treatedbyLPS2.31Western,(100g/L)ECV3046h,ICAM21,18h,6.33,24h,(3,Figure3)12.52550mol/LABLICAM21,3ABL,ICAM21(),9.77%40.87%46.83%50mol/LABL(4,Figure4)2.4B5(Figure5),ECV304P65;1h,P65,4.2450mol/LABL1h,P65,P65ABLB3.ECV3041Figure3.EffectsofLPSonintercellularadhesionmolecule21proteinexpressioninECV3044.ECV3041Figure4.Effectsof12o2acetylbritannilactoneonintercellularadhesionmolecule21expressioninECV304treatedwithLPS5.ECV304BFigure5.Effectsof12o2acetylbritannilactoneontheactivationofNF2BinECV304stimulatedwithLPS3,RAW264.7NOPGE2,BiNOSCOX221131CN4321262/R2005132,,/4,5ICAM215,COX22,COX226ABL/,ECV304,,BCOX22ICAM21,ABLCOX22ICAM21B,(12.550mol/L),ABLCOX22ICAM21,ABL50mol/L,50%BCOX22ICAM21,P50P65,BI2B,7,I2BB,8B,9I2B2,10ABLCOX22ICAM21B,ABLECV304,P65,ABLP65,B,ABL/,BCOX22ICAM21iNOSBICAM21COX22,ABL,ABL[]1HanM,WenJK,ZhengB,ZhangDQ.AcetylbritannilatonesuppressesNOandPGE2synthesisinRAW264.7macrophagesthroughtheinhibitionofiNOSandCOX22geneexpression.LifeSciences,2004,75:67526842ChomczynskiP,SacchiN.Single2stepmethodofRNAisolationbyacidguani2diniumthiocyanate2phenol2chloroformextraction.AnalBiochem,1987,162:15621593RossR.Thepathogenesisofatherosclerosis:aperspectiveforthe1990s.Nature,1993,362:80128094BeltonO,ByrneD,KearneyD,LeahyA,FitzgeraldDJ.Cyclooxygenase21and222dependentprostacyclinformationinpatientswithatherosclerosis.Circulation,2000,102:84028455OBrienKD,McDonaldTO,ChaitA,AllenMD,AlpersCE.Neovascularex2pressionofE2selectin,intercellularadhesionmolecule21,andvascularcelladhe2sionmolecule21inhumanatherosclerosisandtheirrelationtointimalleukocytecontent.Circulation,1996,93:67226826,,,,,,..,2004,12:53325367BaeuerlePA,BaltimoreD.I2B:aspecificinhibitoroftheNF2Btranscrip2tionfactor.Scinece,1988,242:54025468ChenCC,RosenbloomCL,AndersonDC,ManningAM.SelectiveinhibitionofE2selectin,vascularcelladhesionmolecule21,andintercellularadhesionmolecule21expressionbyinhibitorsofI2B2phosphorylation.JImmunol,1995,155:353825459CollinsT.Endothelialnuclearfactor2Bandtheinitiationoftheatheroscleroticlesion.LabInvest,