Abstract
The protective effects and mechanisms of metformin against oxidative stress were evaluated both in vivo and in vitro. ARPE-19 cells comprised the normal group, the glyoxal-treated group (0.5 mM glyoxal), and the glyoxal+metformin group (0.5 mM glyoxal and 0.1 mM metformin). In the in vitro model, differences in cell viability, ROS production, NO products, cellular apoptosis, and the expressions of phospho-AMPKα, total-AMPKα, Sirt1, Nrf2, TXNIP, ZO-1, and Occludin were assessed. In the glyoxal-treated group, cell viability and NO production were decreased, while ROS production and cell apoptosis were increased (p < 0.05), compared with the control group. These changes were prevented by metformin treatment. Protein expressions of phospho-AMPKα, Sirt1, TXNIP, ZO-1, and Occludin, but not Nrf2, were decreased significantly in the glyoxal-treated group compared to normal controls. Metformin treatment significantly increased the above protein expressions and slightly increased TXNIP expression. Immunofluorescence showed that metformin prevented the glyoxal-induced, disorganized tight junctions in ARPE-19 cells. To confirm metformin's protection, Sprague-Dawley rats were injected intravenously with sodium iodate (SI) to induce oxidative stress in the retinal pigment epithelium (RPE). Metformin was then delivered intraperitoneally or intravitreally. One day and three days after SI and metformin treatments, the RPE-Bruch's membrane-choriocapillaris complex was isolated and immune-stained with ZO-1 antibodies. The morphology of the RPE showed enlarged cellular bodies and disorganized ZO-1 staining in SI-treated rats. Metformin treatment prevented these changes. The results indicated that metformin maintained the barrier functions of RPE cells both in vivo and in vitro. Metformin exerted its protection against oxidative stress possibly via activating AMPK/Sirt1 and increasing TXNIP. Metformin has been proposed as a candidate drug for age-related macular degeneration (AMD) by both preclinical and clinical studies. The cellular and animal models used in this study might be useful for the interpretation of the molecular mechanisms involved in the drug activity.