黄柏及其主要成分对细胞膜流动性的影响

黄柏及其主要成分对细胞膜流动性的影响 ? 156? [ArfideID】1000—4718(2006)01—0156—04 中国病理生理杂志ChineseJournalofPathophysiology2O06,22()156l59 Effectsofphellodendronanditsmaincomponentsonthe cellmembraneflm~ty LOYan—ning?,QIUQuan—ying,WANGYi,HAOYu (DepartmentofMicrobiologyandInmmnology,&UniversityofChineseMedicine andPharmacology,&100029,Ch/na) [ABSTRACT] 删:T0invest"gatetheeffectofphellodendronandthreekindsofitsmaincomponents,whichhavea suppressiveeffectontheinllnunesystem.onthemetI~ranefluidityofnormalmurinesplenocytes.M哐I1)DS:nlefluidityof meIIlbfarIehpidregionsofsplenocyteswasdeterminedbythefluorescencepolarizationtechniqueusing1,6一diphenyl一1,3,5 一 heatriene(DPH)asafluorescenceprobe.REsI】 I:I1leresultsshowedthatthewaterextractofphellodendronandoneofits maincomponents(palne)increasedthecellmembranefluidityintheinactivestate,buttheothertwocomponents,berberine andiatrorthizine,decreasedthecellmembranefluidity.AfteractivatedbyConA,allofthemcandecreasethecellnzmbraneflu- idity.CONCLUSION:Theseresultssuggestthattheir fluidity. functionmi出beduetodecreasingthecellmeI【Ib [KEYWORDS]Membranefluidity;Immunosuppression;Huorescencepolarization;Phell odendron [CLCIiillfltlll~r]R363[Docmnentcode]A Asplasmamembranesalecomplex,fluidandmosaic structures[?.d1emaintenanceofmembranefluidityisa prerequisiteforthefunction,viability,growthandrepro- ductionofcells.Moreover,therearevariousreceptorson thesurfaceofthelymphocytenlembranes,andthechanges ofmembranefluidityhaveinfluenceontheirfunctions.We haverecentlyidentifiedthatthewaterextractofphelloden— dronandthreekindsofitsmaincomponentshaveanim— rmmosuppressivefunction.Inthepresentstudy,weob— servedtheinfluenceofthesedrugsonthenormalmufine splenocyte~ranefluidity. MATERIAISAND?[田【(>DS 1Chemicalsandanimals ?1.1,6一diphenyl一1,3,5一heatriene(DPH) (Fhka,Switzerland). ?ConcanavalinA(COnA)(SigmaUSA). ?RPMI一1640(Gibco,GrandIsland,NewYork, USA). ?Newbombovineseruln(NBS)(Shanghai, China). ?Phellodendron(Phe)waspurchasedfromTon— rentangDrugStore,Beijing,China.Phe(20g)was boiledin200mLwaterfor20min.Thesupematantwas collected.Boiledinwateronceagain,allofthesuper- natantwasconcentratedto20mL.thatis100%ofthe phellodendronwaterextract. tan.storedat4?. ?Berbefine(Bet),jatrorrhizine(Jat)andpalma— tine(Pa1)arestandardreagents,whichwerepurchased fromNationalInstitutefortheControlofPharmaceutical andBiologicalProducts(Beijing,China). ?MaleBalb/cmice(6to8weeksold)wereob. minedfromChina—JapanFriendshipHospita1. 20mculture Thesplenocyteswerepreparedasthemo6tdescribed, erythrocytesweredestroiedbyaddedriffs—Clsolu— tim.Then,thecellswerewashedinPBSfortwiceby centrifugation,cellviabihty>95%(bydyeexcisionas— say).Thesplenocyteswereculturedunderstandardcondi- tionsinRPMI一1640mediumsupplementedwithglu— tamine(2mmol/L),HEPES(25mmol/L),Na—pyru— vate(1mmol/L),penicillin(1×105U/L),streptomycin (100mg/L),and10%heat—inactivatedNBSat1×10. cells/L. Dragsofdifferentconcentrationwereaddedrespec一 [Receiveddate】2OO4—10—27[Accepted】2OO5—01—18 ANowt~udyinginSchoolofBiologicalSciences,NanyangTechnologyUniversity,Singapo rea8aHIDstudent tively,withorwithoutConA(5mg/L).Afterincubated for2hoursat37oC,5%CO2,thecellswerewashed twiceinPBSandre—suspendedinPBSat1x10. cells/L. 3Fluorescencepolarizationanalysis[j ThefluorescenthydrocarbonDPHwasusedinthis studyasafluorescentprobeformonitoringdynamicchar- acterofcellularmelnbranelipids.AstocksolutionofDPH intetrahydrofuranataconcentrationof2x10一mol/L waskepttobeprotectedfromlightatFOOBtemperaturefor upto1month.FromtheDPHstocksolutionafreshdis- persionofDPHinPBSattheconcentrationof2x10一 moL/Lwaspreparedinjectionof0.1mLDPHstocksolu- tioninto100mLPBSthatwasbeingstirredvigorously. ThisDPH——PBSdispersionispracticallyclearandvoidof fluorescence.Forlabelingofcells.avolumeof1.5mL cellsuspension(10mcells/L)inPBSwasincubatedwith 1.5mLDPH—PBSdispersionfor30minat30oC.Under theseconditions,thefinalconcentrationofcellswas5x 109cells/L. andthefinalconcentrati0nofDPHwas10—6 moL/L.Thefluorescence—labeledcellsampleswerethen immediatelyusedforfluorescencepolarizationmeasure- ments.FluorescencepolarizationanalysisoftheDPH—la- beledcellsampleswascarriedoutwiththeFluorispec- trophotometerModelMPF一4.Forexcitation,thefluo- rimeterusedamonochromaticmemurylightsourcefiltered at432nInandpolarizedbyapolarizer.Thefluorescence emittedisthendetectedin2independentcross——polarized channelsequippedwithpolarizerafterpassingthrougha cutofffilterforwavelength362nm.Thefluorescencepo- larization(P)andfluorescenceanisotropy(r)canbede. terminedaccordingtothefollowingequations: P=(I?一Ij_)/(I?+Ij_). r=(I?一Ij_)/(I//+2I~). InwhichI?andIj_arethefluorescenceintensities polarizedparallelandperpendiculartothedirectionofPO- larizationofexcitationbeam.respectively.Themethod usedfortheevaluationofthedegreeoflipidfluidity (LFU)ofcellularmembranelipidisbasedontheEinstein equationforviscosityofsolutioncontainingridparticles: Ik-U=(Pm戕/P一1)/P InwhichPmaandParethemeasuredandlimiting valueofP,respectively.Thetheoreticallowerandupper fluorescencepolarizationale0and0.5,respectively, ? 157? thereforeP=0.5. 4Statistics Datawereexpressedasx_-4-5andassessedbystu- dent'st—test. RESULTS 1Effectofthedrugsonthenormalmurinespleno? cytemembranefluidity Thewaterextractofphellodendronatalowconcen- tration(6.25%)andoneofitsmaincomponents(paI眦- tine)increasedthecellmembranefluidityintheinactive state,buttheothertwocomponents(berberineandjatror- rhizine)decreasedthecellmembranefluidity(Tab1). Tab1EffectofthedrugsonthenormalnmdIlesplenocyte membranefluidity(-i-s.n=8) P<0.05.一P<0.01contro1. 2EffectofthedrugsontheConA--stimulated murinesplenocytemembranefluidity AfteractivatedbyConA,allofthedrugsdescribed abovedecreasedthecellmembranefluidity(Tab2).AU oftheseeffectshavenoobviousdose—dependentrelation. Tab2EffectofthedrugsontheCortA—stimulatednme splenocytemembranefluidity(-i-s.n=8) P<0.05.P<0.01control ? 158? ItisknownthatthemembFanefluidityplaysanlm— portantroleinthecellularfunctionsuchasthecellactiva- tion,signaltransductionandreceptorsexpression. Yasumibaeta1.foundthattheimmunosuppressive functionofcyclosporinAmaybegainedthroughreducing thecanalicularmembFanefluidityl.Accordingtothe studiesofFraseretall,toachieveaninhibitoryeffect uponlymphocyteproliferationinvitrousingthepredomi— nantcirculatingFFAs,itwasnecessarytoalterthebal— anceoffattyacidstowardseitheranexcessofunsaturated orsaturatedfattyacids.Similarfindingshavealsobeenre— portedinconcanavalinA——stimulatedratlymphnodelym?- phocytesl6J.Theinhibitoryeffectsmayoccursecondaryto changesinmembFanefluidityinducedbyanexcessofei— therunsaturatedfattyacidsorsaturatedfattyacids.The fluidityandflexibilityofthecellmembFaneplayacentral roleinbothreceptorexpressionandproteinmovementin themen】【bIane[7].b0th0fwhich刮intimatelvinvolvedin T—cellactivation.Purasirieta1.demonstratethatEFAs (GLA,EPA,DHA)havethepotentialtoinhibitsignifi— cantlyvariousaspectsofhumanlymphocytecell——mediated andhum0ralim咖ner.eactivities[8j. AlltheseI?sultsindi— catethatcellmembFanefluiditychangecaninfluencethe functionoflylnphocytes.LENGeta1.[]foundthat berberinecanmodulatelipidsbyinhibitinglipidperoxida— tion.Berberinehasananti—lipogenesisactivity;/nvitro studyshowedthatberberinesuppressedlipogenesisbyre— ducingthesynthesisoftriglyceridesfromfreefatty acids[01.andthisfunctionmaybeattributestotheeffect ofthesedragsthatinfluencesthecellmembranefluidityby meansofchangingthelipidcompositionofplasmamem— brahe. Inthepresentstudy,berberineandjatrorrhizine couldobviouslydecreasethecellmembFanefluidityinboth inactivestateandactivatedstateofsplenocytes.However, thephellodendronwaterextractandpalmatinehadopposite effectsonthemembranefluidity.Theseresultsmaybere— latedt0thecellstateorthedoseused.A1lofthesewi11be detectedfurther. Intherecentstudies,wehavefoundthatthewater extractsofphellodendronandthreekindsofitsmaincom— ponents(berberine,jatrorrhizineandpalmatine)havean immunosuppressivefunction.Forexample,theycanin— hibitthemurinelymphocyteproliferationinv&o,decrease theinte:rleukin一1(IL一1)andinterleukin2(IL一2) production,decreasetheintensityofthemurine'sdelayed typehypersensitivity(I),IH)reactioninducedbyDNFB. Basedonourpreviousreports,wesupposethatthesedrugs mayinfluencethecellularfunctionbymeansofchanging thecellmembFanefluidity.butthemechanismremainsto befurtherinvestigated. [REFERENCT~] [1]HollanS.Membranefluidityofbloodcells[J].Haematologia (Budap),1996,27(3):109—127. [2]BlanchardN,HivrozC.Theimmunologicalsynapse:themoi'e youlookthelessyouknow[J].BiolCell,2002,94(6):345 — 354. [3]DiomedeL,SozzaniS,njW,eta1.Activationeffectsofa prionproteinfragment[PrP一(106—126)]onhumanleuco— cytes[J].Biochem,1996,320(1):563—570.(Printedin GreatBritain). [4]YasumibaS,TazumaS,OchiH,eta1.CyclesporinAre- ducescanalicularmembl'anefluidityandregulatestransporter functioninrats[J].Biochem,2001,354(1ot3):591—596. [5]FraserDA,ThoenJ,RnstanAC,eta1.Changesinplasma freefattyacidconcentrationsinrheumatoidarthritispatients duringfastingandtheireffectsuponT—-lymphocytepmlifem-- tion[J].Rheumatology,1999,38(10):948—952. [6]CalderP,BondJ,BevanS,eta1.Effectoffattyacidsonthe proliferationofconcanavalinA—stimulatedratlymphnode lymphocytes[J].IntJBiochem,1991,23(5—6):579— 588. [7]McMurchieE.Dietarylipidsandtheregulationofmembl'ane fluidityandfunction.In:AloiaRC,ed.PhysiologicalreSul~一 tionofmembranefluidity[M].NewYork:Liss,1988.189— 2o4. [8]PurasifiP,MckechineA,HeysSD,eta1.Modulationinvit— roofhumannaturalcytotoxicity,lymphocyteproliferativere- sponsetomitogensandcytokineproductionbyessentialfatty acids[J].Immunology,1997,92(2):166—172. [9]LENGSan—hua,LUFu—er,XULi—jun.Therapouticef- fectsofberbefineiniIl1paireducosetoleranceratsanditsin- fluenceoninsulinsecretion[J].ActaPharmacolSin,2OO4, 25(4):496—502. [10]SekiT,MorohashiM.Effectofsomealkaloids,flavonoids andtriterpenoids,contentsofJatmnese—Chinesetraditional herbalmedicines.onthelipogenesisofsebaceousgt~dsEJJ. SkinPharmaco1.1993.6(1):56—60. 黄柏及其主要成分对细胞膜流动性的影响 吕燕宁,邱全瑛,王毅,郝钰 (北京中医药大学基础医学院微生物与免疫学教研室,北京100029) ? 159? [摘要]目的:观察具有免疫抑制作用的药物黄柏及其3种主要成分:小檗碱,药根碱与巴马汀对正常小鼠 脾细胞膜流动性的影响.方法:以1,6一二苯基,1,3,5一己三烯(DPH)作为荧光探针,利用荧光偏振法测定脾细胞膜 脂质区的流动性.结果:在无ConA刺激时,黄柏的水提物可提高小鼠脾细胞膜的流动性,6.25%的浓度与对照组相 比有极显着性差异;小檗碱与药根碱可降低脾细胞膜的流动性,与对照组相比均有显着性差异;巴马汀可提高脾细 胞膜的流动性,与对照组相比有极显着性差异.经ConA刺激后,黄柏的水提物,小檗碱,药根碱及巴马汀均可降低 脾细胞膜流动性,与对照组相比有显着性差异.结论:上述结果提示这些药物的免疫抑制作用有可能是通过降低淋 巴细胞膜的流动性来实现的. [关键词]膜流动性;免疫抑制;荧光偏振;黄柏 [中图分类号]R363[文献标识码]A 中国病理生理杂志ChineseJoumalofPathophysiology2006,22(1):159,181 [文章编号]1000—4718(2006)01—0159—02 氨甲酰胆碱与去甲肾上腺素对心室肌细胞 钠钙交换电流的联合作用* 孟华千,崔香丽,刘磊,吴博威 (山西省卫生厅卫生监督所,山西医科大学,山西太原030045) 交感和副交感神经在心脏活动的调节中起重要作用. 交感神经末梢释放的去甲肾上腺素(NE)对心脏起正性肌力 作用,而副交感神经末梢释放的乙酰胆碱(Ach)则对心脏起 负性肌力作用.两者的作用相互拮抗.氨甲酰胆碱(CCh)是 一 种胆碱受体激动药,与胆碱受体结合后,产生与递质Ach 相似的作用,而且它性质稳定,作用持久,常用作工具药代替 Ach来研究胆碱受体激动后的效应.新近的研究明高浓度 的CCIl可以起正性肌力作用.其机制还不清楚.那么当Ccll 与NE联合作用时,CCh是发挥激动作用还是抑制作用还有 待观察.一般来说,心肌收缩的加强是细胞内游离钙离子浓 度升高所致.而细胞外钙离子进入细胞内主要通过两条途 径,即L型钙通道或反向钠钙交换.心肌细胞膜上的钠钙交 换体是一种双向的,生电性的离子交换系统(3Na:1C), 对于维持心肌细胞钙稳态非常重要.前向钠钙交换(Na内 流,Ca2外排)参与静息期C外排,促进心肌的舒张功能. 而一些实验结果表明反向钠钙交换可通过钙诱导钙释放(CI. CR)机制参与兴奋一收缩耦联而影响心肌的收缩功能.而且 [收稿日期]2OO4—07—12[修回日期]2004—10—18 *[基金项目]山西省自然科学基金资助项目(No.20031101) rd:0351—7239645 有报道认为CCh对豚鼠心肌的正性肌力作用可能与钠钙交 换有关.因此本研究观察了NE与CCh合用对单个心肌细胞 反向钠钙交换电流的影响,以探讨两者的联合作用效应. 材料和方法 1单个大鼠心室肌细胞的分离 实验选用Wistar大鼠,体重240—300g,雌雄不拘.猛击 枕部致昏,迅速剪开颈动脉放血,取出心脏经Langendorff灌 流,用胶原酶(CollegnaseP,BoehringerMannheim;0.3g/L,德 国)分离出单个心室肌细胞,保存于高钾KB液备用.KB液 组成为(mmol/L):KOH85,L—slut~icacid50,KC130,taurine 2.0,KH2PO430,MsSO41.0,HEPES10,slu~o~10,EGTA0.5,用 KOH将pH调节为7.4. 2全细胞膜片钳技术 使用Axopatch200A膜片钳放大器和PCI^MP5.51程序 (AxonInstruments,USA),应用全细胞钳制方法记录膜电流. 记录电极用两步拉制法(Narrishage,Japan)拉制,充灌电极内 (下转第181页)