DJ-1拮抗ROS介导Beclin-1上调在缺氧复氧HL-1心肌细胞中的作用

摘要

Objective To explore the potential mechanism of DJ-1 via antagonizing ROS-induced Beclin-1 up-regulation on HL-1 cardiomyocytes during hypoxia-reoxygenation.Methods DJ-1 down-regulation was induced in mouse HL-1 cardiomyocytes by lentivirus transfection.The expression of DJ-1,LC3 and Beclin-1 protein expression change after hypoxia-reoxygenation and lentivirus transfection were detected by Western blot,autophagosomes were detected by MDC,ROS levels was detected by DCFH-DA,Cardiomyocytes death was proved by Annexin Ⅴ and PI.Results Compared to the control group cells,significant down-regulation of protein levels of DJ-1 was observed in cardiomyocytes with shDJ-1 virus,while protein level of Beclin-1 and LC3-Ⅱ in cardiomyocytes with shDJ-1 virus were up-regulated after hypoxia/reoxygenation,NAC decreased protein levels of Beclin-1 and LC3-Ⅱ in cardiomyocytes with shDJ-1 virus.ROS and autophagosome levels were significantly increased after hypoxia/reoxygenation,and apoptoses of those were significantly increased after hypoxia/reoxygenation in cardiomyocytes with shDJ-1 virus,and NAC could reverse of these effects.Conclusions The effect of DJ-1 via antaganizing ROS induces Beclin-1 upregulation and may involve in the ischemia-reperfusion injury.%目的 探讨D J-1拮抗氧化应激诱导过度自噬在缺血再灌注心肌损伤中的保护机制.方法 以慢病毒为shRNA DJ-1载体感染心肌HL-1细胞系构建缺氧复氧损伤模型.Western blot检测shRNA DJ-1沉默效率和LC3、Beclin-1和DJ-1蛋白表达,流式细胞术Annexin Ⅴ和PI染色分别检测凋亡和坏死,MDC和DCFH-DA染色分别检测细胞内自噬体和氧自由基(ROS)水平,激光共聚焦显微镜观察细胞内自噬体数量变化.结果 1)慢病毒为shRNAD J-1载体感染心肌HL-1细胞后DJ-1表达水平下调；2)缺氧复氧显著上调ROS,而shDJ-1加剧缺氧复氧ROS水平上调,NAC可以有效下调细胞内ROS水平；3)Western blot检测LC3、Beclin-1自噬标志蛋白和MDC染色细胞内自噬体结果均认为缺氧复氧促进自噬,而shDJ-1加剧缺氧复氧导致的自噬上调,NAC抑制自噬上调；4)流式细胞术检测显示shDJ-1增加缺氧复氧凋亡,NAC可以减少心肌细胞凋亡.结论 DJ-1拮抗ROS介导Beclin-1上调参与保护缺血再灌注心肌.