A-Lipoic Acid Protects The Oxidative Stressed Human Retinal Epithelial Cells By Regulating Apoptosis And Autophagy

Objective:To observe the protective effect of alpha lipoic acid(ALA) to the oxidative stressed human retinal pigment epithelium cell line ARPE-19, and investigate apoptosis and autophagy alterations and their relevant molecular mechanisms in human retinal pigment epithelium oxidative stress procedure and ALA protection.Methods:1. RPE oxidative stress model and ALA protection effect identification. Gradient concentrations of H2O2 were used to ARPE-19 cells to induce oxidative stress model. Cell viability was measured by MTT test, ROS concentration was quantified with DCFH-DA probe. ALA was used to interfere with the oxidative stress in ARPE-19, ROS and cell viability were also detected.2. Apoptosis and Akt-bcl-2/Bax-Caspase-3 pathway changes of Oxidative stressed RPE and ALA treatment. Oxidative stressed ARPE-19 cells (treated by 12.5mM H2O2) were detected for cell apoptosis by Hoechst 33258 staining, quantitative measurement of apoptosis by Annexin V FITC-PI double staining and flow cytometry; Caspase-3 activity detection by cpp32 colorimetric assay; Bcl-2 and Bax expression were measured by Western Blot.37.5μM ALA was administered to oxidative stressed ARPE-19 and apoptosis detections was carried out as above.3. Lysosomal stability and Autophagic factor atg-5, Beclin 1, LC3Ⅰ/Ⅱchanges of Oxidative stressed RPE and ALA treatment. Oxidative stressed ARPE-19 cells (treated by 12.5mM H2O2) were detected for autophagy:lysosomal membrane stability was detected by acridine orange(AO) staining. Autophagy was semiquantified by real-time PCR of Beclin-1 and Atg-5 mRNA, Western Blot of Atg-5 and LC3Ⅲexpression, treated by 0,37.5μM ALA,37.5μM ALA was administered to oxidative stressed ARPE-19 and autophagy detections was carried out as above.Results:1. Exogenous H2O2 significantly increases intracellular ROS concentration and inhibits ARPE-19 cell viability in a dose-dependent way, the optimal H2O2 concentration is 12.5mM; ALA administration decreases intracellular ROS concentration in oxidative stressed ARPE-19 cells and increases ARPE-19 cell viability under oxidative stress.2. Oxidative stressed ARPE-19 cells show early apoptosis increase (p<0.05) with significant Akt phosphorylation activation Bax upregulation, and Caspase-3 activity increase; ALA administration to oxidative stressed ARPE-19 shows early apoptosis decrease in2,4,6,8,10 hrs (p<0.05), with upregulation of Akt phosphorylation in 4,6 hrs and Bax downregulation in 4,6,8 hrs. Meanwhile, Caspase-3 activity and Bcl-2 expression shows no significant changes. 3. Oxidative stressed ARPE-19 cells show significantly increased acidic vesicular organelles (AVO) formation and more lysosomal rupture. Autophagy related genes Atg-5, Beclin 1, LC3Ⅱexpression increased; ALA administration to oxidative stressed ARPE-19 shows increased lysosomal stability and downregulated Atg-5 and LC3Ⅱexpression. Beclin 1 and LC3Ⅰshows no significant expression changes.Conclusion:1. Exogenous H2O2 induces ARPE-19 oxidative stress and cell injury; Alpha lipoic acid(ALA) shows protection effect to oxidative stressed ARPE-19 cells by reducing oxidative stress and cell death.2. Oxidative stressed ARPE-19 cell injury may be related to increased apoptosis. Alpha lipoic acid decreases ARPE-19 cell apoptosis, possibly by p-Akt activation and Bax expression downregulation.3. Oxidative stressed ARPE-19 cell injury may be related to increased autophagy. Alpha lipoic acid attenuates ARPE-19 autophagic cell death, possibly by Atg-5 and LC3Ⅱexpression downregulation.