缺氧后处理与氧自由基清除剂预处理对心肌细胞膜和线粒体Bcl-2和Bax表达的影响

目的观察缺氧后处理与氧自由基清除剂超氧化物歧化酶(SOD)联合过氧化氢酶(CAT)预处理,对心肌细胞膜和线粒体Bcl-2、Bax蛋白表达的影响,探讨两者调控心肌细胞凋亡的机制。方法建立乳鼠心肌细胞缺氧/复氧损伤模型,分对照组、缺氧/复氧组、缺氧后处理组及缺氧/复氧+SOD+CAT组。应用荧光探针测定心肌细胞线粒体活性氧水平,Hoechst染色及流式细胞仪检测心肌细胞凋亡,Wesiern blot法检测心肌细胞膜和线粒体Bcl-2、Bax蛋白表达。结果与对照组比较,缺氧/复氧组、缺氧后处理组、缺氧/复氧+SOD+CAT组细胞线粒体活性氧及细胞凋亡率明显升高(P<0.01);与缺氧/复氧组比较,缺氧后处理组、缺氧/复氧+SOD+CAT组细胞线粒体活性氧及细胞凋亡率明显降低(P<0.01);缺氧后处理组及缺氧/复氧+SOD+CAT组细胞膜和线粒体Bcl-2蛋白明显上调,Bax蛋白明显下调。结论缺氧后处理及氧自由基清除剂预处理抑制线粒体活性氧爆发,减轻缺氧/复氧再灌注心肌细胞凋亡,其抗凋亡可能机制与细胞膜和线粒体Bcl-2蛋白表达上调及Bax蛋白表达下调有关。

Effect of postconditioning and free radical scavengers on expression of Bcl-2 and Bax proteins in cardiomyocytes and mitochondria

Objective To observe the effect of postconditioning and free radical scavengers,superoxide dismutase(SOD)and catalase(CAT),on expression of Bcl-2 and Bax proteins in cardiomyocytes and mitochondria,and study the machinism of Bcl-2 and Bax underlying apoptosis of cardiomyocytes. Methods A hypoxia(H)/reoxygenation(R) model of suckling mouse cardiomyocytes was established.The suckling mouse cardiomyocytes were divided into control group,H/R group, postconditioning(PC) and H/R+SOD+CAT group.Mitochondrial reactive oxygen species(ROS) in cardiomyocytes and mitochondria were detected with fluorescence probes.Apoptosis of cardiomyocyte was assayed by flow cytometry with Hoechst 33342 staining.Expressions of Bcl-2 and Bax proteins in cardiomyocytes and mitochondria were detected by Western blot.Results The number of mitochondrial ROS and apoptotic cardiomyocytes was significantly less in PC group and H/R+SOD + CAT group than in H/R group28.59±2.93 and 28.43±1.76 vs 58.19±3.29,(26.99±3.35)%and(26.45±5.08)%vs(45.86±3.29)%,P<0.01].The Bcl-2 expression level was higher while the Bax expression level was lower in PC group and H/R + SOD + CAT group than in H/R group.Conclusion Postconditioning and free radical scavengers reduce hypoxia while reoxygenation induces apoptosis of cardiomyocytes by upregulating the Bcl-2 expression and down-regulating the Bax expression in cardiomyocytes and mitochondria.