Abstract
Purpose:
Age-related macular degeneration (AMD) is the leading cause of central vision loss in the elderly. A2E, a major component in lipofuscin, is deposited in the RPE with age. We aim to explore the role of autophagy in the inflammatory response induced by A2E in RPE.
Methods:
(1) ARPE-19 cells were stimulated with different concentrations of A2E at different time points. Cell morphology changeswere observed. Cell viability was assessed by CCK-8 assays.<br /> (2) Transmission electron microscopy was performed to examine the autophagosomes formation treated by A2E. The p62 and LC3 protein levels and localization were analyzed by immunofluorescence staining. The expression of mTOR, p-mTOR, P62, LC3, TMEM166 were detected by Western blot. 14 different human inflammatory chemokines and cytokines in the RPE cell culture supernatants were detected simultaneously by ProcartaPlex Human 14-plex kit.<br /> (3) Autophagy specific inhibitor 3-methyladenine (3-MA) or the mTOR inhibitor rapamycin were used to observe the changes induced by A2E in RPE cells. The expression of mTOR, p-mTOR, P62, LC3, TMEM166 were detected by Western blot. The changes of inflammatory chemokines and cytokines were determined by Procarta.
Results:
(1) The cell proliferation was inhibited by A2E with dose-dependent manner.<br /> (2) Autophagosomes and LC3 were found in RPE treated with 25μmol/L A2E from 30min.<br /> (3) The expression of LC3 II, p62 and TMEM166 in RPE cells were increased, while p-mTOR was down-regulated by A2E treatment. ICAM, IL1β, IL2, IL6, IL8, IL17A, IL22, MCP-1, SDF-1 and VEGFA in the RPE cell culture supernatants were increased by A2E treatment.<br /> (4) After the treatment with 3-MA, LC3 puncta and the transformation of LC3 I to LC3II significantly decreased, and blocked the activation and degradation of p62. The expression of autophagy-associated protein also had varying degrees of decline. ICAM, IL1β, IL2, IL6, IL8, IL17A, IL22, SDF-1 and VEGFA in the RPE cell culture supernatants were increased.<br /> (5) mTOR inhibitor rapamycin atenuated RPE death by CCK-8 assay. Both the number of autophagosomes and the LC3 puncta increased. The expression of inflammatory chemokines and cytokines in the RPE cell culture supernatants had different degree of inhibition.
Conclusions:
A2E could induce autophagy in RPE cells and increase the expression of autophagy-associated protein through the mTOR pathway.