The Mechanism Of Protective Effects Induced By Remote Intrathecal Morphine Preconditioning On Postischemia Mycardial Injury In Rats

Objective Brief ischemic episodes in remote non cardiac tissues or organs such as the small intestine or limb skeletal muscle can induce their own protection and remote target organ protection against ischemia and reperfusion injury, it was names as “remote ischemic preconditioning(RIPC)”. RIPC is an alternative measure to induce cardioprotection following ischemic preconditioning(IPC). Morphine is widely used as analgesic drug in clinical practice. Previous studies have found that small doses of intrathecal morphine could produce cardioprotection against ischemia and reperfusion injury in rats. The aim of this study was to explore the mechanism of cardioprotection induced by remote intrathecal morphine preconditioning(RMPC) on mycardial ischemia injury in rats. Methods After intrathecal catheter placement, the open chest anesthetized male SD rats were subject to 30 minutes of ischemia and 2 hours of reperfusion to estabish myocardial ischemia and reperfusion injury model. Then, ischemia-reperfusion was elicited by occlusion and open of left coronary artery. Before ischemia and reperfusion, RMPC was elicited by three cycles of 5-min infusions interspersed with 5-min drug free periods, to confirm whether RMPC reduced myocardial infarct size. This study consisted of three series of experiments. Series 1: The aim of this series experiments is to study the cardic signal transduction pathway of cardioprotective effects induced by RMPC. Rats were divided into three groups: SHAM, control(CON) and RMPC group, respectively. By observing the number of apoptosis cells and expression of Akt, p-Akt and eNOS in ischemia myocardial, we assessed whether the antiapoptosis mechanism involvedofpi3k-akt-enossignalpathwayplayedakeyroleinthiseffect.series2:theaimofthisseriesexperimentsistodeterminewhethertheneuralandhumoralpathwaymechanismcontributethecardioprotectionofrmpc.theexperimentswereperformedwithcgrp8-37(aselectivecgrpreceptorantagonist),hoe-140(aselectivebradykininb2receptorantagonist),8-spt(anon-selectiveadenosinereceptorantagonist)orhex(anautonomicnerveantagonist)intravenouslyadministered10minutesbeforermpcrespectivelyinrats.series3:theaimofthisseriesexperimentsistostudythecentralmechanismofsignaltransductionpathwayofthisprotectiveeffects.theexperimentswereperformedwithl-name(anon-selectivenosinhibitor),odq(aselectiveguanylatecyclaseinhibitor)orkt-5823(aselectiveproteinkinaseginhibitor)intrathecallyadministered10minbeforermpcrespectivelyinvivo.additionally,byobservingthepkgandnnosproteinexpressionandthennosmrnaexpressioninthoracicspinalcord,weassessedwhetherspinalcordno-cgmp-pkgsignalpathwayplayedakeyroleinthiseffect.indicatorstoberecordedweremadeupofmap,hrandrpp(hr×map);thevolumeofareaatrisk(aar)andinfarctsize(is);andtheareaofmyocardialinfarction,whichwasshowedbyis/aar.meanwhile,theproteinexpressionofakt,p-aktandenosintheischemiamyocardialandnnosandpkginthethoracicspinalcordweredeterminedbywesternblotting;lactatedehydrogenase(ldh),inthebloodserumwastestedbycolorimetricmethod;thecontentofcgmpinthethoracicspinalcordwasdeterminedbyradioimmunityprotocol;thecontentsofnoandactivityofnosinthethoracicspinalcordweredeterminedbynitratereductasereductionandcolorimetricmethods;theexpressionofnnosandpkginthethoracicspinalcorddorsalhornareaweredeterminedbyimmunohistochemicalmethod;theexpressionofnnosmrnawasdeterminedbyreversetranscriptionpolymerasechainreaction(rt-pcr)method.alldatawereexpressedasmean±sdanddataanalysiswasperformedwithspss11.0,astatisticalsoftware.dataofdifferentgroupswereanalyzedusinganalysisofvariancewithttestformultiplecomparisons.statisticaldifferenceswereconsideredsignificantifthepvaluewas<0.05.resultsseries1:1.hemodynamicdata:comparedtoshamgroup,hr,mapandrppsignificantlyreducedat30minutesafterischemiaand2hoursafterreperfusioninconandrmpcgroup(p<0.05).comparedtobaseline,hr,mapandrppsignificantlyreducedat30minutesafterischemiaand2hoursafterreperfusioninconandrmpcgroup(p<0.05).2.infarctsize:comparedtocongroup,rmpcsignificantlyreducedtheisandis/aar.3.tunelstainingforapoptosis:comparedtoshamgroup,thenumberofapoptosiscellsenhancedincongroup.however,comparedtocongroup,rmpcsignificantlyreducedthenumberofapoptosiscells.4.westernblottinganalysis:comparedtoshamgroup,p-aktproteinexpressionenhancedandenosreducedincongroup.then,rmpcsignificantlyincreasedtheexpressionofp-aktandenos.series2:1.hemodynamicdata:comparedtocongroup,mapwaslowerinhexgroup,andmapandrppwerelowerinhex+rmpcgroupafterpreconditioning(p<0.05).hr,mapandrppdidnotdifferamonggroupsatbaseline,30minutesafterischemiaand2hoursafterreperfusion(p>0.05).comparedtobaseline,mapandrppwerelowerinthehexgroup(p<0.05),andhr,mapandrppwerelowerinthehex+rmpcgroupafterpreconditioning(p<0.05);hr,mapandrppwerealllowerjustafter30minutesofischemiaand2hoursofreperfusion(p<0.05).2.infarctsize:cgrp8-37,hoe-140andhexabolishedthecardioprotectionofrmpconischemiaandreperfusioninjuryinrats(p<0.01),but8-sptpartiallyreservedthecardioprotectiveeffectofrmpc(p<0.05).3.ldhactivity:comparedtocongroup,ldhwaslowerinthermpcgroup.then,cgrp8-37,hoe-140,8-sptandhexallreversedtheeffectofreducingtheldhinthebloodseruminducedbyrmpc(p<0.01).series3:1.hemodynamicdata:hr,mapandrppdidnotdifferamonggroupsatbaseline,afterpreconditioning,30minutesafterischemiaand2hoursafterreperfusio(p>0.05).buthr,mapandrppatthetimepointsof30minutesafterischemiaand2hoursafterreperfusiondecreasedfrombaselineinallgroups(p<0.05).2.infarctsize:l-name,odqandkt-5823allreservedthecardioprotectiveeffectofrmpconischemiaandreperfusioninjuryratheart(p<0.01).3.westernblottinganalysis:comparedtocongroup,thennosandpkgproteinexpressioninthoracicspinalcordweresignificantlyhigherinrmpcgroup(p<0.01).4.radioimmunitydetermination:comparedtocongroup,thecontentsofcgmpinthethoracicspinalcordwashigherinthermpcgroup(p<0.01).5.nitratereductasereductionandcolorimetricdetermination:comparedtocongroup,thecontentsofnoandactivityofnosinthethoracicspinalcordwerebothhigherinthermpcgroup(p<0.05,p<0.01).6.immunohistochemicalanalysis:comparedtocongroup,theexpressionofnnosandpkginthethethoracicspinalcorddorsalhornareawashigherinrmpcgroup(p<0.05,p<0.01).7.rt-pcranalysis:comparedtocongroup,thennosmrnainthoracicspinalcordwassignificantlyhigherinrmpcgroup(p<0.01).conclusionrmpccouldproduceprotectiveeffectsagainstmyocardialischemiaandreperfusioninjuryinrats.cardiacpi3k-akt-enossignalpathwaymediatingantiapoptosismechanismplayedakeyroleinthecardioprotectionofrmpc.theneuralandhumoralpathwaymechanismcontributedtothecardioprotectionofrmpc.spinalcordno-cgmp-pkgsignalpathwaywasalsoinvolvedintheeffectofrmpc.