Vitamin C Protected Human Retinal Pigmented Epithelium From Oxidant Injury Depending On Regulating SIRT1

Objective:oxidative stress has been considered as the most common cause of age-related macular degeneration (AMD), which the protection of retinal pigment epithelial cells (RPES) are promising treatments for. In this study, the purpose was to investigate the protective effects of vitamin C (Vit C) and the regulatory mechanism between Vit C and sirtuin1(SIRT1) in RPES during oxidative stress as vit C exerted famous effects as antioxidatants.Methods:1RPE oxidative stress model setting up and Vit C protective effects identification.Gradient concentrations of H2O2were used to ARPE-19cells to induce oxidative stress. Cell viability was measured by MTT test, ROS concentration was quantified with DCFH-DA probe. Gradient concentrations of Vit C were used to interfere with the oxidative stress in ARPE-19, cell viability, apoptosis and ROS level were also detected.2Detect relative expression of SIRT1, p53and Foxo3in ARPE-19with supplementation of100μmol Vit C during oxidative stress. Using Quantitative real-time polymerase chain reaction (qRT-PCR) to measure relative expression of SIRT1transcription factor and stress response factors (p53and Foxo3).3The protective effect of Vit C against oxidative stress was involved in the regulation of SIRT1.To analyze cell viability, apoptosis and intracellular ROS after treatment of Vit C at indicated concentrations and treatment of SIRT1activator RSV or SIRT1inhibitor NA upon12h exposure to H2O2.4Examine the expression of p53and Foxo3after silenced or over-expression of SIRT1genes in ARPE-19with supplementation of100μmol Vit C during oxidative stress. Western Blot was used to extract p53and Foxo3protein and qRT-PCR was used to detect relative expression of p53and Foxo3genes.Results:1Exogenous H2O2significantly increased intracellular ROS concentration and inhibited ARPE-19cell viability in a dose-dependent and time-dependent way, the optimal H2O2concentration was100μmol. Moderate concentration of Vit C administration decreased apoptosis and intracellular ROS concentration in oxidative stressed ARPE-19cells and increased ARPE-19cell viability under oxidative stress (p<0.05). Higher concentration (500μmol) and lower concentration (20μmol) of Vit C could not significantly influence the cell viability in the presence of H2O2.2Vit C affected the expression of the SIRT1transcription factor and stress response factors (p53and Foxo3). H2O2reduced the expression of SIRT1. The expression of SIRT1reached to significantly difference when the level of Vit C concentration was100μmol (p<0.05).Expression of p53and Foxo3were upregulated significantly when the ARPE-19were retreated with moderate concentration of Vit C.3The protective effect of Vit C against oxidative stress was involved in regulation of SIRT1. SIRT1activator RSV could promote the anti-apoptotic effects of Vit C and SIRT1inhibitor NA attenuated the anti-apoptotic effects of Vit C during oxidative stress in ARPE-19. Treatments with RSV and NA resulted in inhibition and promotion of intracellular production of ROS.4The expression of p53among indicated groups was found SIRT1knockdown and up-regulated SIRT1significantly increased and inhibited the expression of p53respectively. The expression of Foxo3was decreased slightly in SBRT1silenced ARPE-19and the upregulated SIRT1promoted the expression of Foxo3significantly.Conlusions:1Exogenous H2O2induces ARPE-19oxidative stress and cell injury. Vit C shows protective effect to oxidative stressed ARPE-19cells by reducing oxidative stress and cell death.2The protective effects of Vit C against oxidative stress are involved in regulation of SIRT1.3Combination of Vit C and RSV could exerts effective resistance to the damage of ARPE-19induced by oxidative stress, might be a promising therapeutic method for AMD.