Experimental Study On Protective Effect Of Astragalus Polysaccharide Onhuman Retinal Pigment Epithelium Cells Against H₂O₂-induced Oxidative Damage

Objective:To observe the protective effect of astragalus polysaccharide(APS) on human retinal pigment epithelium cells(ARPE-19) against H2O2-induced oxidative damage, and investigateapoptosisand the relevantmolecularmechanisminhumanretinal pigmentepitheliumoxidativestress procedureand APS protection.Methods:1. ARPE-19 cells were cultured in DMEM/F-12 medium,then were treated with different concentrations of APS, further the cell viability was detected by MTT assay. Then the ideal concentrations of APS were detected. 2.Gradient concentrationof hydrogen peroxide was used in ARPE-19 cells to induceoxidativestressmodel.Cellviabilitywasmeasuredby MTT method to determine the appropriate concentration of hydrogen peroxide. 3.ARPE-19 cellswere treated with different concentrations of APS and then treated with 200μM H2O2. The cellular morphology was observed by inverted fluorescence microscopy; the cell viability was measured using MTT assay and apoptosis was evaluated using DAPI staining; the cell proliferation was dynamically monitored using the real-time cell electronic sensing system(RT-CES); the caspase-3 activity was evaluated using the caspase-3 cellular activity assay kit.Results:1.The ideal concentrations of APS were 250μg/ml、500μg/ml and 1000μg/ml. 2.Exogenous H2O2 significantly inhibits ARPE-19 cell viability in a dose-dependent way.The optimal H2O2 concentration is 200μM. 3.Cells disposed in H2O2 were sparse and shrinkage, but it could be improved by the adding of APS.MTT assay determination indicated that the cell viability treated with APS was not significantly different with the normal control group. However, the cell viability treated with APS was higher than H2O2 group, and the difference was both significant(P<0.05). DAPI staining revealed that characteristic apoptotic changes,such as convoluted nuclei with cavitationsand strong fluorescent spots appeared in ARPE-19 cells treated with H2O2, these apoptotic changes were alleviated in APS treated group. RT-CES result demonstrated that cell growth in H2O2 group was suppressed, while cells in other groups grew well. Meanwhile, the caspase-3 activity was inhibited by APS compared with that in H2O2 group.Conclusions: Exogenous H2O2 induces ARPE-19 oxidative stress and cell injury. APS shows protection effect to oxidativestressed ARPE-19 cellsbyreducingoxidativestressand celldeath.Oxidative stressed ARPE-19 cell injury may be related to increasedapoptosis. APS decreases ARPE-19 cell apoptosis,possibly by reducing the expression of caspase-3.