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