活性氧在1,4-苯醌诱导的线粒体自噬中的作用

Effect of reactive oxygen species on 1,4-benzoquinone-induced mitophagy

目的探讨苯的代谢产物1,4-苯醌(1,4-BQ)能否活化PINK1/Parkin介导的线粒体自噬,以及活性氧(ROS)在线粒体自噬发生中的作用。方法以人早幼粒白血病细胞HL60为受试细胞,将其分为对照组、1,4-BQ组、N-乙酰半胱氨酸(NAC)组和1,4-BQ+NAC组。采用透射电镜观察细胞超微结构,采用蛋白免疫印迹法检测自噬蛋白LC3、PINK1和Parkin表达,采用DCFH-DA染色法测定胞内ROS含量。结果对照组细胞线粒体呈正常杆状结构,嵴线分明;1,4-BQ组可见呈椭圆肿胀变形的线粒体和包含双层膜性结构的线粒体自噬体。与对照组比较,1,4-BQ组的LC3-Ⅱ/LC3-Ⅰ比值、PINK1、Parkin蛋白表达和ROS含量均增加(P<0.05);1,4-BQ+NAC组的LC3-Ⅱ/LC3-Ⅰ比值、PINK1、Parkin蛋白表达和ROS含量均低于1,4-BQ组(P<0.05)。结论 1,4-BQ可诱导PINK1/Parkin途径的线粒体自噬,且ROS在诱导的线粒体自噬中起重要作用。

Objective To investigate whether the metabolite of benzene,1,4-benzoquinone(1,4-BQ),can activate PINK1/Parkin-mediated mitocphagy and the role of reactive oxygen species(ROS)in 1,4-BQ induced mitophagy in vitro.Methods Human promyelocytic leukemia cells HL60 were used as the test cells,and were divided into control group,1,4-BQ group(10μM 1,4-BQ treated cells for 24 h),NAC group(5 mM antioxidant N-acetyl cysteine treated cells for 24 h)and 1,4-BQ+NAC group(5 mM NAC preincubated for 1 h prior to the treatment with 10μM 1,4-BQ for 24 h).The ultra structure of the cells were observed by transmission electron microscopy(TEM),and the expression of mitophagy related protein LC3,PINK1 and Parkin were detected by Western blot,and the intracellular ROS content was determined by DCFH-DA staining.Results The mitochondria in the control group showed a normal rod-shaped structure with clear mitochondrial cristae,while in the 1,4-BQ group,the mitochondria showed a swollen structure with less mitochondrial cristae,and typical double-membrane mitophagosomes were observed.LC3-Ⅱ/LC3-Ⅰratio,the expression of PINK1,Parkin protein and ROS content in 1,4-BQ group were increased compared with the control group(P<0.05),and this increase was markedly blocked by the co-treatment of 1,4-BQ and NAC(P<0.05).Conclusion The 1,4-BQ can induce PINK1/Parkin-mediated mitophagy and ROS plays a significant role in 1,4-BQ-induced mitophagy.