叔丁基过氧化氢诱导人正常肝细胞系L02自噬模型的建立及其线粒体应激机制

Involvement of mitochondria for induction of autophagy by tert-butyl hydroperoxide in human hepatic cell line L02

目的:建立叔丁基过氧化氢(t-BHP)诱导的人正常肝细胞系L02自噬模型并探讨其中的线粒体应激机制。方法:体外培养L02细胞,用不同浓度(100、200、400、800、1 000μmol/L)t-BHP及线粒体靶向抗氧化剂Mito Q或p38/MAPK特异性抑制剂SB203580进行处理,采用免疫荧光和Western blot检测自噬标志蛋白LC3-II和p62蛋白水平,以及p38/MAPK信号通路的激活情况;Mito-SOX Red染色流式法检测细胞线粒体活性氧(ROS)水平。结果:免疫荧光结果显示t-BHP剂量≥800μmol/L时可明显诱导L02细胞内LC3-II发生聚集。Western blot结果显示,与对照组比较,800和1 000μmol/L t-BHP处理可显著增加LC3-II蛋白水平,并降低p62蛋白水平(P均<0.05)。同时线粒体ROS水平在≥400μmol/L t-BHP处理后明显升高,在1 000μmol/L t-BHP处理后p38蛋白磷酸化水平显著增加(P均<0.05)。给予线粒体靶向抗氧化剂Mito Q或p38/MAPK特异性抑制剂SB203580预处理后可有效拮抗t-BHP(1 000μmol/L)处理引起的LC3-II和p62蛋白水平改变。结论:我们成功建立了t-BHP诱导体外培养人正常肝细胞系L02的自噬模型,证明线粒体ROS介导了此过程的发生,同时p38/MAPK通路在此过程中发挥了重要作用。

OBJECTIVE： To understand autophagy induction by tert-butyl hydroperoxide（t-BHP） in human hepatic cell line L02 and to explore the involvement of mitochondria. METHODS：L02 cells were treated with different concentrations of t-BHP（100,200,400,800,1 000 μmol/L） with or without specific inhibitors（mitochondria targeted antioxidant MitoQ or p38/MAPK inhibitor SB203580）. Autophagy markers LC3-II and p62 as well as the p38/MAPK pathway proteins were detected by immunofluorescence and/or Western blot. Mitochondrial reactive oxygen species（ROS） were measured by flow cytometry with Mito-SOX Red kit. RESULTS：The high concentrations of t-BHP（≥800 μmol/L） induced the accumulation of LC3 fluorescence puncta,with LC3-II protein level increased and p62 protein level decreased significantly. These changes accompanied the elevated mitochondrial ROS and the activated p38/MAPK pathway. Pretreatment with mitochondria targeted antioxidant MitoQ or specific p38/MAPK inhibitor SB203580 attenuated these changes which were induced by t-BHP（1 000 μmol/L）. CONCLUSIONS：We established the autophagy model induced by t-BHP in human hepatic cell line L02. Mitochondrial ROS and p38/MAPK pathway played critical roles in this process.