205-OR: Docosahexaenoic Acid Suppresses Oxidative Stress-Induced Autophagy and Cell Death through the AMPK-Dependent Signaling Pathway in Immortalized Adult Rat Schwann (IFRS1) Cells

Autophagy is a catabolic process that removes damaged intracellular organelles components by lysosomal degradation, but it is also activated by oxidative stress. We previously reported that the docosahexaenoic acid (DHA) suppressed oxidative stress-induced autophagy and cell death in immortalized adult rat Schwann (IFRS1) cells. However, the detailed mechanism has not been clarified yet. Therefore, we aimed to elucidate the intracellular signal transduction pathway by which DHA suppresses oxidative stress-induced autophagy in IFRS1 cells. Treatment with tert-butyl hydroperoxide (tBHP) for 3 hours decreased cell viability measured by MTT assay in IFRS1 cells, while pretreatment with 10 μM DHA for 12 hours significantly protected tBHP-induced cytotoxicity. The signal transduction of autophagy was assessed by immunoblotting of LC3, p62, AMP-activated protein kinase (AMPK) phosphorylation and UNC51-like kinase (ULK1) phosphorylation in IFRS1 cells. 50 μM tBHP increased the LC3-II/LC3-I ratio, and AMPK phosphorylation, whereas tBHP decreased the p62 protein expression, indicating that autophagy was induced by tBHP. Pretreatment with 10 μM DHA for 12 hours significantly decreased the LC3-II/LC3-I ratio and AMPK phosphorylation that were increased by tBHP, whereas DHA increased the p62 protein expression that was decreased by tBHP. p70S6Kinase and 4E-BP phosphorylation was not changed either by tBHP or by DHA. Autophagosome and autolysosome were induced by 50 μM tBHP in IFRS1 cells, as demonstrated by DAPRed and DALGreen staining. DHA pretreatment significantly reduced the production of tBHP-induced autophagosome, but suppression of autolysosome was partial. These results suggest that DHA may prevent against diabetic neuropathy by attenuating oxidative stress-induced autophagy and cell death through the AMPK-dependent signaling pathway in Schwann cells. Disclosure Y. Tatsumi: Research Support; Self; Daiichi Sankyo, Johnson & Johnson, Kyowa Kirin Co., Ltd., Lilly Diabetes, MSD K. K., Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co., Ltd., Sanofi K. K., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co. J. Nakamura: Research Support; Self; Astellas Pharma Inc., Daiichi Sankyo Company, Limited, Eli Lilly Japan K. K., Japan Tobacco Inc., Novartis Pharma K. K., Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co., Ltd., Sanofi K. K., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Speaker’s Bureau; Self; Astellas Pharma Inc., AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Eli Lilly Japan K. K., Kowa Company, Ltd., Mitsubishi Tanabe Pharma Corporation, MSD K. K., Novartis Pharma K. K., Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. K. Sango: None. K. Kato: None. A. Kato: None. N. Niimi: None. H. Yako: None. T. Himeno: None. M. Kondo: None. S. Tsunekawa: None. Y. Kato: None. H. Kamiya: Speaker’s Bureau; Self; Eli Lilly Japan K. K., MDS Co., Ltd., Novartis Pharma K. K., Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co., Ltd., Sanofi K. K., Sumitomo Dainippon Pharma Co., Ltd. Funding Grant-in-Aid for Scientific Research (18K06763)