Abstract
Purpose :
Decreased autophagic and proteasomal cleansing has been documented in the degeneration of retinal pigment epithelial (RPE) cells and the pathology of age-related macular degeneration. Recently, polyphenolic compounds has been actively studied in the prevention of age-related diseases. We examined effects of resveratrol and another stilbene compound, pinosylvin, on the regulation of autophagy and cytoprotection in ARPE-19 and mouse primary RPE cells under proteasome inhibition.
Methods :
Protein aggregation was caused with proteasome inhibitor MG-132 (1 microM). Autophagy was inhibited with bafilomycin A1 (50 nM). Resveratrol (25 microM) or pinosylvin (15 microM) were used solely and together with proteasome inhibition up to 48 hours. A stress marker Hsp70 (Heat Shock Protein 70) and autophagy regulators p62 (SQSTM1) and LC3 (microtubule-associated protein 1A/1B-light chain 3) were analyzed by Western blotting (WB). Tandem fluorescently tagged LC3 (GFP-mCherry-LC3) transfection was used to study autophagy flux in fluorescence microscopy for ARPE-19 cells.
Results :
Inhibition of proteasomes with MG-132 upregulated Hsp70, autophagy markers p62 and LC3 detected in WB. Simultaneous treatment with MG-132 and resveratrol or pinosylvin decreased amount of Hsp70, p62 and LC3. However, the levels of p62 and especially LC3-I/LC3-II fluctuated during the monitored time period revealing a dynamic autophagy process. Resveratrol and pinosylvin provided similar effects on the decreased protein aggregation under proteasome inhibition. Fluorescence microscopy showed increased autophagy flux with GFP-mCherry-LC3. Resveratrol or pinosylvin with autophagy inhibition resulted accumulation of p62 and LC3-II.
Conclusions :
Stillbenes are potent inducer of autophagy and prevent cell damage during proteasome inhibition in RPE cells.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.