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

doi:10.3969/j.issn.1004-616x.2016.02.002

癌变·畸变·突变 ›› 2016, Vol. 28 ›› Issue (2): 91-96.doi: 10.3969/j.issn.1004-616x.2016.02.002

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

师腾瑞^1,3, 李鸽^2,3, 海春旭^1,3, 秦绪军^1,2,3

1. 第四军医大学预防医学院毒理学教研室, 陕西西安 710032;
2. 第四军医大学预防医学院营养与食品卫生学教研室, 陕西西安 710032;
3. 陕西省自由基生物学与医学重点实验室, 陕西西安 710032

收稿日期:2015-12-15 修回日期:2016-01-19 出版日期:2016-03-31 发布日期:2016-03-31
通讯作者: 秦绪军,E-mail:qinxujun@hotmail.com E-mail:qinxujun@hotmail.com
作者简介:师腾瑞,E-mail:526611412@qq.com
基金资助:
国家自然科学基金(81270417,31070766,30300074);陕西省青年科技新星人才基金(2010KJXX-06)

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

SHI Tengrui^1,3, LI Ge^2,3, HAI Chunxu^1,3, QIN Xujun^1,2,3

1. Department of Toxicology, Fourth Military Medical University, Xi'an 710032;
2. Department of Nutrition and Food Hygiene, Fourth Military Medical University, Xi'an 710032;
3. The Key Laboratory of Free Radical Biology and Medicine of Shaanxi Province, School of Preventive Medicine, Fourth Military Medical University, Xi'an 710032, Shaanxi, China

Received:2015-12-15 Revised:2016-01-19 Online:2016-03-31 Published:2016-03-31

摘要/Abstract

摘要： 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、1000 μmol/L)t-BHP及线粒体靶向抗氧化剂MitoQ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≥800 μmol/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1000 μmol/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≥400 μmol/L t-BHP处理后明显升高,在1000 μmol/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MitoQ或p38/MAPK特异性抑制剂SB203580预处理后可有效拮抗t-BHP(1000 μmol/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。

关键词: 叔丁基过氧化氢, 自噬, 线粒体, 活性氧, p38/MAPK

Abstract: 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, 1000 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 (1000 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.

Key words: tert-butyl hydroperoxide, autophagy, mitochondria, reactive oxygen species, p38/MAPK

中图分类号:

R114
R992

师腾瑞, 李鸽, 海春旭, 秦绪军. 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[J]. 癌变·畸变·突变, 2016, 28(2): 91-96.

SHI Tengrui, LI Ge, HAI Chunxu, QIN Xujun. Involvement of mitochondria for induction of autophagy by tert-butyl hydroperoxide in human hepatic cell line L02[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2016, 28(2): 91-96.

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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

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