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  

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

作　　者：晏浩[1,2] 李文林[2] 徐建军[1] 朱书强[1] 龙翔[1] 车建鹏[1] 

机构地区：[1]南昌大学第二附属医院胸心外科,江西南昌330006 [2]南昌大学医学院病理生理教研室,江西南昌330006

出　　处：《基础医学与临床》2013年第6期763-768,共6页Basic and Clinical Medicine

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shRNADJ-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。Objective To explore the potential mechanism of DJ-1 via antagonizing ROS-induced Beclin-1 up-regu lation on HL-1 eardiomyocytes during hypoxia-reoxygenation. Methods DJ-1 down-regulation was induced in mouse HL-1 cardiomyoeytes by lentivirus transfection. The expression of DJ-1, LC3 and Beclin-1 protein expression change after hypoxia-reoxygenation and lentivirus transfeetion 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- 11 in cardiomyo cytes 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 up regulation and may involve in the ischemia-reperfusion injury.

关 键 词：缺血再灌注 自噬 DJ-1 凋亡 氧自由基 

分 类 号：R965[医药卫生—药理学]