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BOWEN WANG, Jing Zhong, Jin Yuan; Oxidative Stress Induces Autophagy through Inhibition of mTOR in Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3856.
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© ARVO (1962-2015); The Authors (2016-present)
Autophagy is an important cellular self-digestion process. In this study, we tended to investigate the role of autophagy in corneal epithelial cells (CECs) under oxidative stress and its potential mechanism.
CECs were exposed to tert-butyl hydroperoxide (TBH) to create an oxidative stress. Reactive oxygen species (ROS) and 3-Nitrotyrosine fluorescent assay was applied to measure ROS levels. Selected studies were undertaken in the presence of the autophagy activator rapamycin or the autophagy inhibitor 3-MA. Western blot was used to measure autophagy-related protein such as LC3-II, ATG-5, Beclin1, SQSTM1 and the phosphorylation of mTOR. Immunostaining was used to measure the formation of autophagosomes and autophagy flux. MTT assay was used to illustrate the change of cell viability. Annexin V-PI flow cytometry analysis was used to measure the apoptosis of CECs.
TBH induced autophagy in CECs in a dose-dependent manner, with autophagy-related proteins LC3-II, BECN1 and ATG5 increased significantly and SQSTM1 reduced significantly. Immunofluorescent staining also indicated TBH caused an increase of autophagy flux. Furthermore, in CECs treated with TBH, we found P-mTOR decreased in correspond with a decrease of downstream factors S6. Moreover, 3-MA or by knockdown of ATG5 attenuated TBH-induced CECs autophagy, increased ROS generation and reduced cell viability. However, we had not observed a protective role of rapamycin against TBH-induced toxicity by increasing autophagy.
In this study, oxidative stress was proved to induce autophagy through blocking mTOR pathway in CECs. Inhibition of autophagy exacerbated oxidative stress-induced reduction of cell viability and increased apoptosis in CECs. These results suggest that autophagy plays a protective role in CECs under oxidative stress. This would shine us a light to prevent and cure relevant corneal epithelial diseases by regulating autophagy.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
TBH induces autophagy in CECs in a dose-dependent manner
Autophagy inhibitor 3-MA exacerbates CECs apoptosis under oxidative stress
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