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

• Published in: International journal of molecular sciences 2020-01, Vol.21 (3), p.813
• Main Authors: Josifovska, Natasha, Albert, Réka, Nagymihály, Richárd, Lytvynchuk, Lyubomyr, Moe, Morten C, Kaarniranta, Kai, Veréb, Zoltán J, Petrovski, Goran
• Format: Article
• Language: English
• Subjects: Acridine orange; age-related macular degeneration; Apoptosis; Autophagy; Cell culture; Cell death; Chloroquine; Cluster analysis; Confocal microscopy; Cytokines; Disease; Flow cytometry; Growth factors; Hydrogen peroxide; Inflammation; Inflammatory response; Kinases; Macular degeneration; Microscopy; Oxidative stress; Phagocytosis; Propidium iodide; proteasomal inhibition; Proteasomes; protein array; Proteins; Rapamycin; Resveratrol; retinal pigment epithelium; Survival; TOR protein; Transmission electron microscopy; Vacuoles
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms21030813

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). 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 (H O ) and resveratrol treatments, respectively. 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. 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.