【doc】人胎盘提取液的抗炎和抗血小板聚集作用

【doc】人胎盘提取液的抗炎和抗血小板聚集作用 人胎盘提取液的抗炎和抗血小板聚集作用 ~2003,ActaPharmacologicaSinica ChinesePharmacologicalSociety ShanghaiInstituteofMateriaMedica ChineseAcademyofSciences SurTKetal,ActaPharmacolSin2003Feb;24(2):187—192 Anti—-inflammatoryandanti?-plateletaggregationactivity ofhumanplacentalextract1 SURTapasKumar,BISWASTuhinKanti.ALILiaquat,MUKHERJEEBiswapati DepartmentofPharmacology;SNPradhanCentreforNeurosciences, DrBCRoyPostgraduateInstituteofBasicMedicalSciences244B,AcharyaJCBoseRoad,Calcutta700020,India; ZResearchDivision,B1RDEM,122KaziNazrulIslamAvenue,Dhaka1000,Bangladesh KEYWORDSplacentalextracts;inflammation;carrageenin;plateletaggregation;adenosinediphosphate l87 A ?:Tofindtheanti.inflammatoryandanti.plateletaggregatoryactivityofhumanplacentalextract(HPE,Placentrex). METHODS:TheHPEwasstudiedforanti.inflammatoryeffectinWistarratsoncarrageenin,serotonin(5'HT), andprostaglandinE1(PGE1)inducededemainacutemodelandcottonpelletinducedgranulomaonsub'acute mode1.Anti.plateletaggregationwasstudiedagainstprotectionofadinosinediphosphate(ADP)-inducedaggrega- donofhumanplateletthroughinvitrostudy.RESULTS:HPEshowedpositiveresultsbothina cuteandsub'acute modelsofinfammafion.Highlysignificant(尸 <0.01)resultswereobtainedagainst5-HTinducedacuteinflamma' tionandcottonpelletinducedsub.acuteinflammationincomparisonwithstandard(diclofen acsodium)andcontrol (normalsaline)drugs.Theanti.inflammatorypropertyofHPEinanimalmodelwaswellsupp ortedwithclinical studyofplateletaggregation.Therewashighlysignificant(P<0.01)inhibitionofplateleta ggregationwithI-IPEat differentdosesagainstADP.CONCLUSION:Ourdatasuggestthathumanplacentalextract maybeusefulin suppressinginflammationandplateletaggregation. Thevarietyofbiologicalactionsofhumanpla- centalextract(HPE)isamatterofincreasinginterest. RecentresearchstudiesrevealthatHPEistherichre- sourcesofvariousbio.activesubstanceslike p0lyde0xyrib0nucle0tides(PDRN),RNA,DNA, peptides.aminoacids.enzymes,traceelements,etcIlJ. Therapeuticpropertiesinthetreatmentofpatientswitl1 woundshavebeendescribedt.Itisreportedthathu一 .SupportedbyM/s,AlbertDavidLtd,Calcutta,India CorrespondencetoDrMUKHERJEEBiswapati. E-mailimpuffer@cal2.vsn1.net.in Received2002-014)8Accepted2002—?08?-03 manplacentalextracthascorticotropinreleasingfactor (CRF).1ikeaction~".Enzyme—linkedimmunosorbant assay(ELISA)studiesrevealedthathumanplacental cVt0tf0phOblastswhichexpressedinterleukin-8,a knownmediatorofinflammation,wassuppressedby glucocorticoid【3】I Thecurrentstudywasaimedtofindtheanti?-in?- flammatoryactivityofHPEinb0thacuteandsub-acute experimentalmodels.Plateletaggregationisanimpor' tantpathogenicmarkerofinfl~/mmation.Hence,one rationalapproachintheresearchofanti-inflammatory drugsistosearchforcompoundscausinginhibitions ofplateletaggregation.Althoughtherearesomere' portsofplacentalextractfortheiranti??plateletaggrega?- tionactivityI4,51buttheobservationsnotcorrelatedwith 188SurTKPf4f,ActaPharmacolSin2003Feb;24(2):187—192 itsanti—inflammatoryactivity.Inthecurrentresearch programme,anti—inflammatoryeffectofhumanplacental extractwasobservedinexperimentalanimalmodelwhile plateletaggregationactivitywasstudiedinclinicalcases. TestdrugHumanplacentasweighingbetween 400..600gcollectedatthetimeoffu11termspontane.. OUSdeliverywereimmediatelyplacedunderice,then theamnioticmembraneandumbilicalcordwere removed.mincedintosmallpiecesandwashedwith coldnormalsaline.Aqueousextractwiththesepieces ofplacentawasprepared,sterilized,andsealedinam— pulesf2mL)underinertcondition.Theextract1mL intheampulewasderivedfrom0.1gofplacenta.This extractcontainsprotein(0.95g/L),DNA(2.8mg/L), RNA(1.6mg/L),Na*ion(27.9mmol/L),Kion(3.07 mmol/L),andCl_ion(15.1mmol/L). Acutetoxicity(LD50)Thistestwasperformed toevaluatethetherapeuticdoseaswellasforscreening oftheCNStoxicity.HPEwasadministeredforthis purposeat0.4,0.5,0.6,0.7,0.8,and0.9IIper20g ofmiceinintraperitonealroute.Thestudieswerecar- riedoutcontinuouslyfor72h. AnimalsMaleWistarratsweighing150—180g wereusedforpresentresearchprogramme.Theywere housedingroupsunder12:12hregime(1ightsonfrom 7:o0hto19:o0h)at(23?2).Cpriortotheexperiments. TheYweresuppliedpelletdietandwateradlibitum. DrugsCarrageenin(Sigma),serotoninhydrochlo— ride(Sigma),prostaglandinEl(Sigma),andadenosine diphosphate(Sigma)wereusedinthisstudy. Anti.inflammatoryactivity AcuteinflammationAcutepawedemawasin— ducedingroupsoftenrats,eachusingthreedifferent experimentalmodels.Theratsweredeprivedoffood for24hbeforetheinductionofinflammation.butwa— terwasallowedadlibitum.TheHPEwasadminis. teredatdoseof300mg/kgintramuscularlyt. Diclofenacsodium(10mg/kg,im)and0.9%NaC1(5 mL/kg,im)wereusedasreferencedrugs.Aftereach treatmentpawvolumeoftheanimalsweremeasured plethysmometrically. Carrafleenin-inducedpawedemaAcutein— flammationwasinducedbycarrageeninaccordingto themodelofWinteretal[.Forthispurpose0.1mLof 1%suspensionofcarrageenininnormalsalinewas injectedintothesub—plantertissuesofrightIlindpawin rats.Thepawvolumewasmeasuredplethysmome. tricallyat0hand3haftercarrageenininjection.The treateddrugswereadministeredintramuscularly1h priortocarrageemninjecfion. 5一HT—inducedpawedemaInthisexperiment 0.1mLof5一HTf1g/L)insterilesalinewasinjected intothesub—plantertissueofthefighthindpawofrats. Thepawvolumewasmeasuredplethysmometrically beforeandafter30minofthe5一HTinjectionis,9】.HPE. diclofenacsodium.and0.9%NaClwereadministered 1hbefore5一HTtreatment. Prostaglandin?inducedpawedemaProstag— landinEl(1mg/kg)wasadministeredintothesub—planter regionoftherighthindpawofrats.inaccordancewith themethodofriUisandCornelsen[10J.Thepawvol— umeuptotheanklejointsweremeasuredplethysmo— metricallybeforeandafter30minoftheprostaglandin E1injection. Sub—acuteinflammationSub—acuteinflamma— tionwasproducedbycottonpelletinducedgmnuloma inrats[1l】.Sterilecotton(15~1)mgwasimplantedsub— cutaneouslybilaterallyinaxillaunderetheranesthesia. Thetreateddrugswereadministeredforconsecutive6 dinthesamedoseasmentionedearlier.Theanimals weresacrificedond7.Thegranulationtissueswith cottonpelletweredriedat60.Covernightandthenthe dryweightwastaken.Theweightdifferenceswere consideredastheweightofgranulationformation. Plateletaggregationstudy SelectionofsubiectTotall5volunteersofei— thersexwereselectedfromthemedicaloutpatients' departmentofBangladeshInstituteofResearchandRe— habilitationonDiabetes.Endocrine&MetabolicDisor- ders(BIRDEM),Dhaka,Bangladesh.Acarefuldrug historywastakenfromthesubjects.Patientsnotre— ceivingforlasttwoweeksthedrugslikeaspirin, sulfinpyrazone,chlorpromazine,amitriptyline, furosemide,penicillinanditsderivatives,dextran,which interferewiththeplateletaggregationactivity,werese— lectedforthepresentresearchprogramme. CollectionofbloodSpecimensofbloodsamples werecollectedusing3.2%sodiumcitrateattheratio1: 9withthebloodinplasticcontainerwithminimum traumaorstasisatthevenipuncturesite.Testingwas performed30minaftervenipunctureattheroom temperature. PreparationofplasmaSamplesofbloodand anticoagulantsweregentlyinvertedupanddown,avoid— ingshaking.Plateletrichplasma(PRP)wasprepared bycentrifugingat100xgunder4.Cfor15min.PRP SurTKetal,ActaPharmacolSin2003Feb;24(2):187—192 thuspreparedwerecollectedbycarefulpipettingina sterilepolypropylenetubeandclosed.Plateletpoor plasma(PPP)waspreparedbycentrifugingatapproxi? mately2400xgfor20min.PPPwascollectedina polypropyleneplastictube. StudymethodsofplateletaggregationTlle aggregationwasmeasuredonadualchannelChrono— LogOpticalPlateletAggregometer(Chrono—Log Corporation,HavertownPA19083.4691)atconstant stirringof1200rpm(1000rpmin5OHzUnits)and electronicallycontrolledtemperature(37+21.C.Tlle lighttransmissionwassetat0%withPRPandat 100%withp ppt 关于艾滋病ppt课件精益管理ppt下载地图下载ppt可编辑假如ppt教学课件下载triz基础知识ppt lO1.Aggregationwasinducedwith aggregatingagentADPataconcentrationof1mmol/L. 职Ewasaddedatdifferentdosesof2.5,5,10,and2O lIIL,5minbeforeadditionofADP. StatisticalanalysisTheresultsofanimalex— perimentswereanalysedbyunpairedStudent'sttest. Pairedttestmethodwasappliedfortheanalysisofthe clinicaldata. Acutetoxicity(LD5o)Fromthisstudyitwas observedthattheHPEissafeupto45mI./kgbodyweight inintraperitonealrouteonmice. Anti-inflammatoryactivity l89 AcuteinflammationFromthestudyitwasob. servedthattherewassignificantfP<O.01)inhibitionof pawedemaintheanimalstreatedwithHPEbothon carrageenin(54.3%)andPGE1(39.7%)induced inflammation.Theseresultsarealmostsameasinthe caseofdiclofenacsodium(57.1%and44.4% respectively)treatedgroup.However,therateofinhi? bitionfP<O.01)ofedemain5一HTinducedacutein— flammationwasevenbetter(49.0%1thandiclofe:nac sodium(39.4%)treatedgroup(Tab1). Sub-acuteinfIammationIncottonpelletinduced sub—acuteinflammationmode1.,therewashighlysig— nificant(<O.01)decreaseoftheweightofgranuloma tissue(39.5%)inHPEtreatedgroup.However,the rateofinhibitionofgranulomatissueweightindiclofenac sodiumtreatedgroup(52.1%1wasfoundtobebetter (Tab21. Anti?plateletaggregationactivityResultsof plateletaggregationwereexpressedasapercentofag— gregationatagiventimeinterval(5min)fromreagent addition.Centpercentaggregationwasdefinedasthe differencesbetweenthe0%baseline(PRP1and10o% baseline(PPP).HigMysignificantresponses(P<O.01) ofanti—plateletaggregationwereobservedwithallthe dosesofHPE.Therewere83.9%,76.6%, 59.2%.and60.2%aggregationofplateletwithHPEat thedosesof2.5,5,10,and20Lofplasma(PRP) Tab1.Effectofhumanplacentalextract(HPE)anddiclofenacsodiumoncarrageenininduced ratpawedema.Mean~SD. n=lO.cp<0.Ol坩contro1. Carrageenininduced 5.HTinduced PGElinduced 1.O?0.5 1.04~0.16 0.63~0.09 0.5~0.4 0.53~0.25 0.38~0.22 54.3 49.0 39.7 0.45~0.25 0.63~0.09 0.35~0.19 57.1 39-4 44-4 Tab2.Effectofhumanplacentalextract(I-WE)anddiclofenac$odiulnonsub-acuteinflamm atorymodelinrat.Mean~SD. n=lO.cp<0.Ol坩contro1. 192 SurTKn,/ActaPharmacolSin2003Feb;24(2):187— Tab3.Effectsofhumanplacentalextract(HPE)against ADP-inducedplateletaggregationinhumanPRP. Mean+SD.n=10.P<0.O1vscontro1. TreatmentgroupsanddosePlateletaggregation againstADP/% Control HPE 2.51L(n=6) 5.0mL(n=6) 10.0mL(n=12) 20.0I/mI(n=6) 84~13 77?16 59~33 6O?14 PPPbaseline Fig1.EffectofdifferentdosesofHPEforanti-plateletag- gregationactivity. respectivelywithrespectto100%PPPcontrol(Tab3, Fig1). DlSCUSSloN Inflammationcoversaseriesofreparativeand protectiveresponsesintissueinjury,whethercaused byinfection.auto—immunestimuliormechanicaliniury. Severalclassesofcompoundssuchasplasmaproteins, vasoactiveamines,tissuedigestiveenzymes,biologi— callyderivedoxidantandeicosanoidsareallassociated withinflammatoryresponse[0,钔.Mostofallinvestiga— torshavereportedthatinhibitionofcarrageenin—induced inflammationinratsisoneofthemostsuitabletestpro— ceduretoscreenanti—inflammatoryagents【.Thede— velopmentofcarrageenin—inducededemaisbi—phasic, thefirstphaseisattributedtothereleaseofhistamine, 5一HT.andkinins.while.thesecondphaseisrelatedto thereleaseofprostaglandins[7,15】.Theinhibitoryaction observedafterHPEtreatment(Tabl1oncarrageenin— inducedpawedemainratsmaybemediatedthrough eitheranyofthesemediatorsaloneorincombination. Hence,HPEwasfurtherstudiedagainstpawedema inducedbyindividualagentlike5一HTorPGE1. 5一HTispresentinmastcellsandisconsiderablymore potentthanhistamineinincreasingvascularpermeabil- itvinrats【制.Astherewasconsiderablereduction (49.0%1ofinflammationin5一HTpre—treatedratsby applicationofHPE,itmightbeconjecturedthatthein— hibitoryactionwas5一HTmediated.Prostaglandins (PGs)biosyntheticpathwayorthecyclooxygenase (COX1activityoftheenzymeisthesiteofactionof non—steroidalanti—inflammatorydrugsorNSAIDS【l7.1. DiclofenacsodiumisawidelyusedpotentNSAIDS withpronouncedanalgesicandanti—pyreticactivity.It isusedmainlyforlongtermsymptomatictreatmentof rheumatoidarthritis.osteoarthritis,andankylosing spondylitis[,们.Therefore.diclofenacsodiumwasse— lectedinthisstudyaspositivecontrol(Tabl.2).Hence itmaybeassumedthatHPEexhibitstheiranti..inflam.. matoryresponseseitherthroughinhibition/inactivation ofchemicalmediatorsorbydirectlymodulatingPG productionbysuppressionofCOX.Ithasbeenrecog— nizedrecentlythatmammaliancellsexplaintwoforms ofCOXactivity.COX—listllemaiorformpresentin platelets.whileCOX.2isonlyfoundinelevatedlevels ininflammatoryexudates[0,".TheactionofHPEon PGE,inducededema(Tab1)explainsthatitmaymodu— latePGsproductionbyinhibitionofCOX. Thesub—acuteanti—inflammatoryactivityofHPE wasstudiedbyinvestigationofitsinhibitoryeffecton thegranulomaformation(Tab21.Cottonpelletgranu— lomaisamodelofnon.immunologicaltypeofinflam.. mationmediatedbytheactivationofthechemicalme— diatorsofinflammation.mostlykinins【引.Theaction ofkinininvolvestheactivationoftwomembrane receptors,B1andB2.B1一receptorplaysanimportant roleininflammatoryprocesses[,.Intllispresentre— searchprogrammehighlysignificant(p<0.01)result wasobtainedwithHPEincottonpelletinducedsub— acuteinflammationmodelindicatingthatitmayactby thewayofinhibitingtheB1-receptor. Plateletsareessentialfornormalhaemostasis. Activationoftheclottingcascadebytraumaresultsin plateletactivation,whichisfollowedbyaggregation. ThemajorCOXmetaboliteinplateletsisthromboxane A2(TXA2),whichiscapableofinitiatingaggregation. NSAIDSinhibitT》(A.productionandthusinflamma一 0m加?加卯? o/0,u0Is?一暑?lI_||-;眦一厂I SurTKetal,ActaPharmacolSin2003Feb;24(2):187—192 tion【26'.Theaggregationofhumanplateletsinduced byADPwasusedt12]tostudytheanti.plateleteffectOf HPE(Tab31.Therewas84%士13%aggregationof plateletagainstADPwithadoseof2.5uL/mLHPE whichgraduallydecreased(77%士16%for5II,) withtheincreaseindose,reachingminimum(59%士 33%)withadoseof10I,,mITherewasnoappre? ciablechangeonfurtherincreaseofthedose(60%士 14%for20UL/mL).ADPiscontainedwithinthe plateletinstorageorganellesandreleasedfromtheplatelet duringformationoftheprimaryhaemostaticplugand therebyinducefurtherplateletaggregation【28..There aresomanyactivationpathwaysleadingtoplatelet aggregation.PGsand5?HTareconsideredasthema? jorchemicalmediatorsofplateletaggregation".The clinicalstudyofplateletaggregationreflectsthatHPE caneitherinhibitPGssynthesispathwayor5?HTrelease. Thesedataindicatethattheanti?inflammatoryef- fectofHPEmightbemediatedthroughthesuppression ofchemicalmediators.Furtherexperimentsareneeded toconfirmthemodeofactionofanti?inflammatoryand anti.plateleteffectofHPE. ACKNo,vLEDGE=M】TSAuthorsarethankfultoM/ s,Albe~DavidLimited,Calcuttaforthefinancialsup. portoftheresearchprogramme.Sincerethanksare alsoduetotheHeadoftheDepartmentofPharmacology, UniversityCollegeofMedicine,Calcuttaandtothetech— nicalstaffsofthedepartmentofpathologyandresearch divisionBIRDEM,Dhaka,BangladeshandDirector, CentralDrugLaboratory.Calcuttaforvarioushelp. 2 4 5 6 ShibasakiT,OdagiriE,ShizumeK,LingN.Corticotropin— releasingfactorlikeactivityinhumanplacentalextracts.J CfinEndocrinolMetab1982;55:384.6. GoldfarbG,DoanBaTriR,DuranA.Humanplacental extractforchroniclegulcer.Lancet1980;2:40. RosenkunG,MaW_柚gEY,LockwoodCJ,Guller S.Chronicantagonismofnuclearfactor-kappaBactivityin cytoblastsbydexamethasone:apotentialmechanismforanti. inflamma