腺苷预处理对心肌缺血/再灌注损伤的延迟保护作用及机制

Delayed cardioprotective effect of adenosine preconditioning on myocardium against ischemia-reperfusion injury

目的探讨ATP敏感性K+通道(KA+TP)开放和诱导型一氧化氮合酶(iNOS)表达上调在腺苷(ADO)预处理对心肌缺血/再灌注损伤延迟保护中的作用。方法48只家兔随机分成4组,每组12只。对照组:心肌缺血前24h静注生理盐水0.5ml;CC-PA组:心肌缺血前24h静注CCPA(ADOA1受体激动剂)0.1mg/kg进行药物预处理;Gli组:心肌缺血前30min静注格列苯脲(Gli)0.3mg/kg;CCPA/Gli组:在CCPA组处理的基础上,心肌缺血前30min静注Gli。采用家兔离体工作心脏模型,各组动物心肌均缺血30min,复灌30min。观察缺血/再灌注前后血流动力学和心功能的变化。测定再灌注结束后冠脉流出液中乳酸脱氢酸(LDH)、肌酸激酶(CK)、一氧化氮(NO)和心肌组织ATP含量。氯化三苯基四氮唑(TTC)染色法判断心肌梗死范围。RT-PCR测定iNOSmRNA表达水平。结果与对照组比较,CCPA组心肌缺血/再灌注后心功能明显改善,冠脉流出液LDH水平降低(30.11±3.32U/Lvs89.43±5.76U/L,P<0.01),CK含量减少(42.77±5.48U/Lvs143.76±11.20U/L,P<0.01),心肌梗死范围明显缩小(13.4%±2.3%vs32.1%±4.5%,P<0.01),心肌组织ATP浓度明显增加(1.52±0.23μmol/Lvs0.44±0.08μmol/L,P<0.01),Gli可拮抗这种保护作用。CCPA组心肌组织NO浓度较对照组升高(31.4±5.3μmol/Lvs24.6±3.3μmol/L,P<0.01),且iNOS表达上调(1.68±0.22vs0.96±0.17,P<0.01),此效应不受Gli拮抗。结论CCPA可以模拟缺血预处理的延迟保护效应;iNOS合成的NO可能激活KA+TP,并使iNOS表达上调,在ADO预处理的延迟保护中具有重要作用。

Objective To verify the hypothesis that delayed protection of adenosine against ischemia-perfusion injury is mediated by opening of ATP-sensitive K＋ channels（KATP）and up-regulation of inducible nitric oxide synthase（iNOS）.Methods Forty-eight rabbits were randomly divided into four groups（12 each）,i.e.saline control group：rabbits were given saline treatment 24h before ischemia;CCPA group：rabbits were given CCPA（an adenosine receptor A1 agonist）24h before ischemia;Gli group：rabbits were given KATP blocker glibenclamide（Gli）30min before ischemia;and CCPA/Gli group：rabbits were given CCPA 24h before ischemia and then Gli 30min before ischemia.All hearts were isolated and perfused in a working mode,and then subjected to 30min of global ischemia followed by 30min of reperfusion.Hemodynamic data were recorded before and after ischemia-reperfusion.CK,LDH,ATP and NO were determined after reperfusion.Infarct size was measured by triphenyltetrazolium chloride staining,and iNOS expression was determined by semi-quantity reverse transcriptional polymerase chain reaction（RT-PCR）.Results Compared with that in saline control group,CCPA produced improvement in postischemic CO,LVSP and LVEDP（P〈0.01）,while Gli inhibited the cardioprotective effect;CCPA caused a significant reduction in infarction size（13.4%±2.3% vs 32.1%±4.5%,P〈0.01）and concentrations of LDH（30.11±3.32U/L vs 89.43±5.76U/L,P〈0.01）and CK（42.77±5.48U/L vs 143.76±11.20U/L,P〈0.01）,while such beneficial effects were also blocked by Gli.CCPA enhanced the production of NO（31.4±5.3μmol/L vs 24.6±3.3μmol/L,P〈0.01）and up-regulated the expression of iNOS（1.68±0.22 vs 0.96±0.17,P〈0.01）,which was not inhibited by Gli.Conclusions It is suggested that delayed protective effect by ischemic preconditioning can be simulated by CCPA,and NO synthesized by iNOS may activate KATP and mildly up-regulate the iNOS expression,which plays an important role in the delayed protection effect of adenosine.