目的 探讨p66Shc在亚砷酸钠介导的心肌细胞氧化应激中的作用. 方法 利用亚砷酸钠诱导的H9C2心肌细胞氧化应激模型,分别用CCK-8比色法检测细胞活力;双氯荧光素探针检测细胞内活性氧(ROS)水平;Western Blot法检测p66Shc、磷酸化p66Shc的表达变化;免疫荧光共聚焦检测p66Shc的亚细胞定位. 结果 与对照组相比,5 μM亚砷酸钠处理组细胞活力显著降低(P<0.05),细胞内ROS水平显著升高(P<0.001);砷引起了p66Shc表达量增加,而且磷酸化p66Shc与p66Shc蛋白总量的比值也显著升高(P<0.05),p66Shc在砷刺激下从细胞浆向线粒体转位.过表达野生型p66Shc质粒增加砷介导的心肌细胞内ROS水平,而过表达突变型p66Shc质粒和敲低p66Shc的表达都降低砷处理组细胞内ROS水平. 结论 研究显示亚砷酸钠可能通过上调p66Shc的表达和磷酸化水平诱导心肌细胞ROS水平的升高.%Objective: To explore the role of p66Shc in sodium arsenite-mediated oxidative stress in cardiomyocytes. Methods: The oxidative stress model of H9C2 cells was induced by sodium arsenite. The cell viability was measured by CCK-8 colorimetry. The intracellular reactive oxygen species (ROS) level was measured by dichlorofluorescein probe. Western blot was used to detect the expression of p66Shc and phosphorylated p66Shc. Sub-cellular localization of p66Shc was detected by immunofluorescence confocal detection. Results: Compared to the control,sodium arsenite treatment significantly decreased cell viability,but increased intracellular ROS level (P<0.05). Western blotting showed that the ratio of phosphorylated-p66Shc/p66Shc was significantly increased,as well as the total expression of p66Shc (P<0.05). Immunohistochemistry showed that p66Shc was translocated from cytoplasm to mitochondria. Over-expression of wild-type p66Shc increased arsenic-mediated ROS levels in H9C2 cells,whereas over-expression of mutant p66Shc and knockdown p66Shc decreased the levels of ROS in arsenic-exposed H9C2 cells. Conclusions: The study results suggested that sodium arsenite induces elevation of ROS level in H9C2 cells by upregulating p66Shc expression and phosphorylation levels.
展开▼
