Abstract
Purpose: To investigate the mechanism by which resveratrol acts upon retinal pigment epithelial (RPE) cells and to characterize its effect upon autophagy, survival, and inflammation, with consequent implications to treatment for age-related macular degeneration (AMD). Methods: Cultured ARPE-19 cells were exposed to 10 and 50 μM resveratrol. Cell survival/death was determined by annexin-FITC/propidium iodide using flow cytometry, while autophagy was studied by detecting autophagic vacuoles formation (acridine orange and transmission electron microscopy), as well as LC3II/I ratio and p62 expression by Western blot. In addition, time-lapse confocal microscopy of a pDENDRA-LC3 expression vector was performed to detect autophagy in transfected ARPE-19 cells under the different treatment conditions. Inhibition of proteasomal and autophagy-lysosomal fusion was carried out by MG-132 and chloroquine, respectively, while induction of autophagy was achieved by rapamycin treatment. Detection of secreted cytokines by ARPE-19 cells using Human XL Cytokine Array was performed under oxidative stress (H2O2) and resveratrol treatments, respectively. Results: Resveratrol induced autophagy in ARPE-19 cells as determined by augmented presence of autophagic vacuoles, increased LC3II/I ratio and decreased p62 expression, as well as time-lapse confocal microscopy using pDENDRA-LC3 expression vector. Resveratrol acted similarly to proteasomal inhibition and downstream of mammalian target of rapamycin (mTOR), since upstream inhibition of autophagy by 3-methyladenine could not inhibit autophagy in ARPE-19 cells. Co-treatmeant by rapamycin and/or proteasome inhibition showed no additive effect upon autophagy induction. ARPE-19 cells treated by resveratrol showed lower cell death rate compared to untreated controls. Resveratrol induced a specific anti-inflammatory response in ARPE-19 cells. Conclusions: Resveratrol can induce autophagy, pro-survival, and anti-inflammatory stimuli in ARPE-19 cells, properties which could be plausible to formulate future treatment modalities for AMD.
Highlights
The leading cause of blindness in the elderly in developed countries is age-related macular degeneration (AMD)
The size of the autophagic vacuoles for the untreated ARPE-19 cells was 748.4 ± 538.4 × 103 nm2, which increased under rapamycin treatment (716.0 ± 888.6 × 4 × 103 nm2), and decreased under MG-132 (174.1 ± 42.1) and resveratrol (166.4 ± 0.0) treatment
Co-treatment of rapamycin and resveratrol or MG-132 further enhanced the presence of autophagic vacuoles in the cells
Summary
The leading cause of blindness in the elderly in developed countries is age-related macular degeneration (AMD). The exact cause for this is still to be elucidated, as the underlying mechanism is not clear yet, apoptosis, autophagy, and necroptosis are believed to be involved. Autophagy is associated to apoptosis and necroptosis, as well as AMD, while the interplay of the different pathomechanisms of the disease need further clarification [2]. Autophagy as a self-digestive cellular mechanism is involved in the clearance of long-lived cytoplasmic proteins under conditions of stress, starvation and proteasomal inhibition. Proper clearance of such cellular debris is important for maintaining cell survival and vitality of cells and tissues in general. Autophagy contributes to the turnover and regulation of proteins responsible for countering oxidative stress, such as NFE2L2, that is known to interact with p62, the latter being an important autophagic protein [1,2,3]
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