Resveratrol as Inducer of Autophagy, Pro-Survival, and Anti-Inflammatory Stimuli in Cultured Human RPE Cells

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.

A Re-Appraisal of Pathogenic Mechanisms Bridging Wet and Dry Age-Related Macular Degeneration Leads to Reconsider a Role for Phytochemicals

“…Within RPE cells, resveratrol prevents A2E-induced mitochondrial network fragmentation [ 212 ], and it also suppresses VEGF secretion induced by inflammatory cytokines [ 213 ]. Remarkably, the administration of a resveratrol-rich formulation rescues the autophagy flux, to provide cytoprotection against either proteasome inhibition or chloroquine/hydroquinone exposure in RPE cells [ 59 , 214 , 215 ]. This is accompanied by improved mitochondrial bioenergetics, upregulated antioxidant genes, and a reduction of ROS and inflammatory molecules production [ 214 , 215 ].…”

“…Remarkably, the administration of a resveratrol-rich formulation rescues the autophagy flux, to provide cytoprotection against either proteasome inhibition or chloroquine/hydroquinone exposure in RPE cells [ 59 , 214 , 215 ]. This is accompanied by improved mitochondrial bioenergetics, upregulated antioxidant genes, and a reduction of ROS and inflammatory molecules production [ 214 , 215 ]. Similar to lutein, through induction of autophagy, resveratrol also mitigates the cytotoxic effects of vital dyes indocyanine green (ICG) and brilliant blue G in ARPE-19 cells [ 196 ].…”

Effects of Resvega on Inflammasome Activation in Conjunction with Dysfunctional Intracellular Clearance in Retinal Pigment Epithelial (RPE) Cells

“…In previous studies on ARPE-19 cells, Resvega has reduced hydroquinone-mediated ROS production and promoted autophagy during impaired protein clearance [ 29 , 30 ]. Resveratrol alone has been shown to induce autophagy and evoke anti-inflammatory responses in RPE cells as well inhibit NLRP3 expression, caspase-1 activation, and IL-1β secretion in a mouse model of acute lung injury [ 31 , 32 ].…”

New Highlights of Resveratrol: A Review of Properties against Ocular Diseases

“…Caloric restriction and RSV promotes longevity through the Sirt-1-dependent induction of autophagy [ 93 ]. Moreover, RSV induced autophagy in ARPE-19 cells, as determined by the increased presence of autophagic vacuoles, increased the LC3II/I ratio, and decreased p62 expression [ 94 ]. In this study, RSV acted similarly to proteasomal inhibition downstream of the mammalian target of rapamycin (mTOR), since upstream inhibition of autophagy by 3-methyladenine was not able to inhibit autophagy in ARPE-19 cells.…”