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

基础医学与临床 ›› 2013, Vol. 33 ›› Issue (6): 763-768.

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

晏浩¹,李文林²,徐建军¹,朱书强¹,龙翔¹,车建鹏¹

1. 南昌大学第二附属医院心胸外科
2. 南昌大学医学院

收稿日期:2012-04-01 修回日期:2012-09-19 出版日期:2013-06-05 发布日期:2013-05-29
通讯作者: 徐建军 E-mail:xujianjun3526@163.com

Protective effects of DJ-1 via antagonizing ROS-induced Beclin-1 up-regulation in hypoxia-reoxygenated HL-1 cardiomyocytes

Received:2012-04-01 Revised:2012-09-19 Online:2013-06-05 Published:2013-05-29

摘要/Abstract

摘要： 目的探讨DJ-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AnnexinⅤ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shRNA DJ-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。

关键词: 缺血再灌注, 自噬, DJ-1, 凋亡, 氧自由基

Abstract: 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 blotting, autophagosomes were detected by MDC, ROS levels were detected by DCFH-DA, Cardiomyocytes death was detected by AnnexinⅤ 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 levels 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. Conclusion The effect of DJ-1 via antaganizing ROS induced Beclin-1 up-regulation might involve in the ischemia-reperfusion injury.

Key words: Ischemia-Reperfusion, Autophagy, Apoptosis, DJ-1, ROS

晏浩李文林徐建军朱书强龙翔车建鹏. 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[J]. 基础医学与临床, 2013, 33(6): 763-768.

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