Mitophagy as a potential mediator for the anti‐inflammatory effects of metformin in human retinal pigment epithelium cells

Abstract

Aims/Purpose: Mitochondrial malfunction and subsequent excessive production of reactive oxygen species (ROS), deficient autophagy and chronic inflammation are associated with many age‐related diseases, including age‐related macular degeneration. In this study, we investigated metformin and its ability to activate mitophagy and alleviate inflammation in retinal pigment epithelium (RPE) cells upon induced mitochondrial damage.Methods: Experiments were conducted using the ARPE‐19 cell line. The cells were treated with 15 mM metformin (Met) for 1 h before adding 10 μM mitochondrial complex 3 inhibitor Antimycin A (Aa) for additional 2–24 h. The levels of proinflammatory cytokines IL‐6 and IL‐8 were measured using the enzyme‐linked immunosorbent assay (ELISA). The levels of the autophagy substrate SQSTM1/p62 and the autophagy marker LC3 were studied using the Western blot method. To further analyse mitophagy, LC3 and TOM20 colocalization was detected using immunofluorescence. Mitochondrial ROS production was assessed using MitoSOX CMXRos staining.Results: The production of proinflammatory cytokines IL‐6 and IL‐8 were increased by Aa exposure. Conversely, Met treatment significantly reduced the levels of IL‐6 and IL‐8. The autophagy substrate SQSTM1/p62 was accumulated in the cells after Aa treatment implying a blockage in the autophagic flux. Met treatment induced the clearance of SQSTM1/p62 and LC3 I and II at 6‐h time point. At 24‐h time point, Met treatment increased the amount of LC3 puncta/cell and colocalization of LC3 and TOM20 suggesting enhanced mitophagy in cells suffering mitochondrial damage induced by Aa. Aa treatment led to increased production of mitochondrial ROS which was decreased by Met treatment.Conclusions: Metformin has anti‐inflammatory and antioxidant properties in human RPE cells upon antimycin A exposure. Studies on autophagy markers suggest enhanced activation of autophagy, and mitophagy, upon metformin exposure, which could explain the detected positive effects on inflammation and oxidative stress.