异丙酚对离体大鼠心肌缺血/再灌注损伤后细胞凋亡及其机制研究

EFFECTS OF PROPOFOL ON CARDIOMYOCYTES APOPTOSIS AND ITS MECHANISM AFTER ISCHEMIA/REPERFUSION INJURY IN ISOLATED RAT HEARTS

目的:观察异丙酚对离体大鼠心肌缺血/再灌注损伤的影响并从氧化应激和线粒体介导的凋亡方面探讨其作用机制。方法:应用Langendorff离体心脏灌注系统建立心肌缺血/再灌注损伤模型。40只SD大鼠随机分为正常对照组、缺血/再灌注模型(I/R)组、异丙酚15、30、60μmol.L-1组。除正常对照组外,各组分别平衡灌注20 min后,常温全心停灌25 min,再灌注30 min。Powerlab/8s仪记录各组平衡末、缺血前及再灌30 min时的各项心功能指标并测定冠脉流出液中乳酸脱氢酶(LDH)、肌酸激酶(CK)活性;检测心肌线粒体活力、膜肿胀度、锰超氧化物岐化酶(Mn-SOD)活性和丙二醛(MDA)含量;流式细胞仪检测心肌细胞凋亡;流式细胞术检测Bcl-2和Bax的表达,免疫组化法测定天冬氨酸特异的半胱氨酸蛋白酶(caspase)-3,9,8蛋白的表达。结果:与I/R组相比,异丙酚30、60μmol.L-1能明显改善缺血/再灌注后的心功能,减弱冠脉流出液中LDH、CK的活性(P<0.05);心肌线粒体活力有所恢复,膜肿胀度减轻,Mn-SOD活性升高,MDA生成明显减少(P<0.05),心肌细胞凋亡明显减少,Bcl-2表达上调,Bax表达下调,caspase-3,9阳性表达细胞数明显减少(P<0.05)。结论:异丙酚明显减轻缺血/再灌注所致的心肌线粒体的过氧化损伤,抑制线粒体途径的凋亡,可能是其心肌保护作用机制之一。

Aim： To investigate the protective effect of propofol on iscbemia/reperfusion（I/R） injury in isolated rat hearts and clarify the possible molecular mechanism from oxidative stress and the apoptosis initiated by mitochondria pathway. Methods： The Langendorff model of ischemia/reperfusion was used. Forty isolated perfused rat hearts were randomly divided into control, I/R, propofol 15, 30, 60μmaol·L^-1 groups. Hearts were suffered globally ischemia for 25 min and 30 min with reperfusion, The cardiac function indexes such as the left ventricular developed pressure（LVDP）, the left ventricular end diastolic pressure（LVDEP）, heart rate （HR）, coronary arterial flow（CF） were recorded at the time of equilibration, before ischemia, the end of reperfusion respectively, The lactate dehydrogenase（LDH）, creatine kinase（CK） activities in the flow were measured, The swelling and activity of mitochondria, the activity of manganese superoxide dismutase（Mn-SOD） and content of malondialdehyde（MDA） in myocardium mitochondria were also determined. The incidence of cardiomyocyte of apoptosis and the expression of Bcl-2 and Bax were evaluated by Flow Cytometry （FCM）. The expression of caspase-3,8,9 was detected by immunohistochemistry, Results： Compared with I/R group, administration of propofol at the concentration of 30 and 60μmaol·L^-1 markedly ameliorated the cardiac function in CF, LVDP and LVDEP（ P 〈 0.05）, distinctly reduced the activities of the LDH and CK in the flow（ P 〈 0.05）and the mitochondrial swelling, the content of MDA in myocardium mitochondria（ P 〈 0.05）, and significantly enhanced the activity of Mn-SOD in myocardium mitochondria（ P 〈 0.05）. The apoptotic index in propofol 30, 60μmaol·L^-1 group was markedly lower than that of I/R group. The expression of Bcl-2 protein in 30, 60μmaol·L^-1 propofol was higher than that of I/R group（P〈0.05）. The expressions of Bax, easpase-9,3 protein in 30,60μmaol·L^-1 propofol were obviously lower than those of I/R group（ P〈 0.05）. There was no significant difference in expression of caspase-8 between propofol administrated groups and I/R group. Conclusion： It could be concluded that propofol administrated before iscbemia and during reperfusion has cardioprotective effects on iscbemia/reperfusion injury in the isolated rat heart. The effect is associated with diminishing oxidative stress, protecting mitochondria from peroxidative injury, thus interfering with the mitochondriadependent apoptotic pathway might be one of the major mechanisms of its cardioprotection.