缺氧后处理对缺氧复氧心肌线粒体活性氧及细胞凋亡的影响

目的 观察缺氧后处理对缺氧复氧心肌线粒体活性氧及细胞膜和线粒体Bcl-2和Bax蛋白表达的影响,探讨其调控心肌细胞凋亡的机制.方法 构建大鼠乳鼠心肌细胞缺氧复氧损伤模型,将细胞分为对照组、缺氧/复氧纽(缺氧3h后复氧6h)、缺氧后处理组(缺氧3h后行复氧5min、缺氧5 min,反复3次,再复氧6 h).应用荧光酶标仪测定线粒体活性氧量,流式细胞仪检测心肌细胞凋亡,Western blot检测细胞膜和线粒体Bcl-2和Bax蛋白的表达.结果 缺氧/复氧组和缺氧后处理组心肌细胞线粒体活性氧量较对照组显著升高(P＜0.01).缺氧后处理组心肌细胞线粒体平均荧光强度为30.74±1.88a.u./μg,显著低于缺氧/复氧组(63.17±2.75a.u./μg,P＜0.01),仍高于对照组(14.41±2.15a.u./μg).缺氧/复氧组和缺氧后处理组心肌细胞凋亡率较对照组显著升高(45.86％±3.29％和26.99％±3.35％比5.72％±1.63％,P＜0.01),缺氧后处理组低于缺氧/复氧组(P＜0.01).细胞膜和线粒体Bcl-2蛋白在缺氧后处理组显著上调,在缺氧/复氧组显著下调；Bax蛋白在缺氧后处理组显著下调,在缺氧/复氧组显著上调.结论 缺氧后处理抑制线粒体活性氧爆发,减轻缺氧/复氧诱导的心肌细胞凋亡,其抗凋亡机制可能与线粒体和细胞膜Bcl-2蛋白表达上调及Bax蛋白表达下调有关.

Effects of Hypoxia Postconditioning on Mitochondrial Reactive Oxygen Species and Cardiomyocyte Apoptosis Induced by Hypoxia/Reoxygenation

AimTo investigate the effect of hypoxia postconditioning on mitochondrial reactive oxygen species (ROS) and cardiomyocyte apoptosis induced by hypoxia/reoxygenation.MethodsThe model of cultured cardiomyocytes with hypoxia/reoxygenation (H/R) was established and the cardiomyocytes were divided into 3 groups, including control group, H/R group (hypoxia 3 h and reoxygenation 6 h), and hypoxia postconditioning (PC) group (3 intermittent cycles of 5 min H/R immediately after hypoxia 3 h, then reoxygenation for 6 h).Cardiac mitochondria and sarcolemma were isolated by differential centrifugation.ROS was detected with fluorescent probes and cardiomyocyte apoptosis was detected with flow cytometry.The expressions of Bcl-2 and Bax proteins in the cardiac mitochondria and sarcolemma were measured by Western blot.ResultsBoth mitochondrial ROS and the number of apoptotic cardiomyocytes reduced significantly in PC group compared with H/R group.Bcl-2 levels increased while Bax levels decreased in cardiac mitochondria and sarcolemma of PC group.ConclusionPC attenuated ROS and cardiomyocyte apoptosis induced by H/R, which potentially mediated by upregulating the expression of Bcl-2 and downregulating the Bax in mitochondria and sarcolemma.