异丙酚预处理通过上调Caveolin-3增加缺血心肌细胞线粒体膜稳定性的机制研究

Propofol increases mitochondrial membrane stability,decreases apoptosis and cytochrome C release in rats with myocardial ischemia by up-regulating Caveolin-3

目的探讨异丙酚通过上调小窝蛋白-3(Caveolin-3)增加缺血心肌线粒体膜稳定性的机制。方法筛选健康SD大鼠36只,体质量范围为250~300 g,采用随机数字表法分为3组,每组12只。①正常心肌细胞组:仅开胸不结扎;②心肌缺血再灌注组:单纯给予以5%葡萄糖稀释的脂肪乳,持续静脉滴注;③缺血心肌细胞+异丙酚预处理组:缺血前10 min开始持续静脉滴注异丙酚12 mg·kg^-1·h^-1,异丙酚用5%葡萄糖稀释。通过蛋白质免疫印迹检测Caveolin-3和细胞色素C;通过荧光探针DCFH-DA测定ROS产生;通过阳离子染料JC-1评估线粒体膜电位;caspase-3比色测定试剂盒测定心肌细胞caspase-3活性。结果心肌缺血再灌注组Caveolin-3蛋白活性(0.57±0.09)低于正常心肌细胞组(2.24±0.25)和缺血心肌细胞+异丙酚预处理组(2.02±0.14),异丙酚改善心肌缺血诱导的Caveolin-3下调(P<0.05);心肌缺血再灌注组DCF荧光强度(2.79±0.21)高于正常心肌细胞组(1.00±0.12),缺血心肌细胞+异丙酚预处理组心肌细胞DCF荧光强度(1.23±0.14)低于心肌缺血再灌注组(P<0.05);与正常心肌细胞组(1.00±0.06)相比,经受心肌缺血的心肌细胞显示出线粒体去极化降低(0.13±0.02),缺血心肌细胞+异丙酚预处理组(0.83±0.11)稳定了线粒体膜电位(P<0.05)。正常心肌细胞组线粒体细胞色素C为(1.00±0.08),心肌缺血再灌注组为(2.76±0.24),心肌缺血增加线粒体细胞色素C释放到胞质溶胶中,缺血心肌细胞+异丙酚预处理组(1.37±0.18)降低细胞色素C释放(P<0.05);心肌缺血再灌注组显示出caspase-3和caspase-9活性增加。与心肌缺血再灌注组相比,异丙酚降低了caspase-3和caspase-9活性(P<0.05)。结论异丙酚在心肌缺血下的保护作用取决于Caveolin-3的上调,后者降低活性氧产生、增加线粒体膜电位、减少细胞凋亡和细胞色素C释放。

Objective This study was designed to investigate whether propofol reduces mitochondrial membrane stability in patients with myocardial ischemia by up-regulating Caveolin-3.Methods 36 healthy SD rats,weighing 250 to 300 g,were screened by the laboratory animal center of the academy of military medical sciences.36 rats were randomly divided into 3 groups with 12 rats in each group.(1)Normal myocardial cell group:only thoracotomy without ligation;(2)Ischemic myocardial cell group:in the experiment,fat milk diluted with 5%glucose was given,and continuous intravenous drip was given.(3)Ischemic myocardial cells+propofol treatment group:During the experiment,continuous intravenous infusion of propofol 12 mg·kg^-1·h^-1 was started 10 min before ischemia,and propofol was diluted with 5%glucose.Waveolin-3 and cytochrome c were detected by Western blotting;fluorescent probe DCFH-DA was used.ROS production was measured;mitochondrial membrane potential was evaluated by cationic dye JC-1;caspase-3 activity was measured by caspase-3 colorimetric assay kit.Results Caveolin-3 protein was decreased in myocardial ischemia group,propofol improved Caveolin-3 induced by myocardial ischemia[(2.24±0.25)vs.(0.57±0.09),(2.02±0.14),P<0.05],DCF fluorescence increased in myocardial ischemia,and DCF fluorescence decreased in propofol group[(2.79±0.21)vs.(1.00±0.12),(1.23±0.14),P<0.05];Compared with the control,cardiomyocytes subjected to myocardial ischemia showed a decrease in mitochondrial depolarization,and the propofol group stabilized mitochondrial membrane potential[(1.00±0.06)vs.(0.13±0.02),(0.83±0.11),P<0.05].Myocardial ischemia increased mitochondrial cytochrome C release into the cytosol,propofol group decreased cytochrome C release[(1.00±0.08)vs.(2.76±0.24),(1.37±0.18),P<0.05];myocardial ischemia group showed increased caspase-3 and caspase-9 activity.Propofol reduced caspase-3 and caspase-9 activity compared with myocardial ischemia(P<0.05).Conclusion The protective effect of propofol under myocardial ischemia depends on the upregulation of Caveolin-3,which reduces the production of reactive oxygen species,increases mitochondrial membrane potential,and reduces cytochrome c and apoptosis.