The Effect And Mechanism Of Rg1 Protects Against Oxidative Damage In Retinal Pigment Epithelium(RPE)cell

Background: Ischemia retinopathy is characterized by anoxic changes of retina tissue and cells.RPE cell function loss is the key to progress in the I/R.Thus,it is important to inhibit or delay the damage of RPE cell in controlling the disease.Ginsenoside Rg1 is a medicinal ingredient extracted from ginseng which shows antioxidative and anti-apoptotic properties.However,the effects of Rg1 on ischemia retinopathy have not been reported.This study aimed to investigate the protective effect of Rg1 on the RPE cells which damaged by hypixia.Experiment is divided into three parts:Part 1Objective: Investigate effects of Rg1 on the damage of RPE cells induced by hypoxia.Methods: we adopted ARPE-19 cells for vitro study.After treated with Rg1 for 2 hours,the RPE cells were cultured with Co Cl2 to induce cytotoxicity.Cell viability was assessed with MTT assays,Annexin V/propidium iodide staining,terminal deoxynucleotidyl transferase d UTP nick-end labeling and caspase3 activity were used to detect RPE cells apoptosis.Cells were further cultured in an anaerobic chamber,cell viability was assessed by MTT assays and ELASA was used to detect apoptosis of RPE cells.Result: Rg1 can significantly inhibit the toxic effects on ARPE-19 cells caused by Co Cl2 and physical hypoxia.It can also maintain normal cell morphology and activity,inhibition of apoptosis induced by Co Cl2 and physical hypoxia,reducing the expression caspase 3.Conclusion: This study demonstrates the ability of Rg1 to inhibit the apoptosis and damage of ARPE-19 cells induced by hypoxia and maintain RPE cells activity.It plays an important role to preserve the normal environment of retina.Part 2Objective: Investigate the effect of Rg1 on ROS levels and protein expression of RPE cells induced by hypoxia.Methods: we adopted ARPE-19 cells for vitro study.we used the flow cytometry to evaluate the ROS level,Western blots to detect the expression of associated proteins in P38,JNK-MAPK,m TOR(4EBP-1,S6)and AMPK(AMPKα,ACC).Result: When RPE cells were pretreated with Rg1 at for 2 hours,the ROS level of ARPE-19 cells induced by Co Cl2,phosphorylted JNK /P38-MAPK and AMPK decreased.S6 and 4E-BP1(m TORC1 downstream target proteins)expression was elevated.Conclusion: These results indicate that Rg1 may inhibit ROS generation and regulation the activity of downstream signaling pathways(JNK/ P38-MAPK,m TORC1 /AMPK)associated with ROS.It plays an important role to protect ARPE-19 cells against hypoxia.These results suggest that Rg1 may exert a potential therapeutic value of Rg1 in ischemia retinopathy.Part 3Objective: Observe the role of Nrf2/HO-1 and the upstream pathways in the couse of inhibition of Rg1.Methods: ARPE-19 cells and mouse primary RPE cells were cultured in vitro,after treatment of Rg1 and Co Cl2,the role of Nrf2/HO-1 and the effect of Akt/m TORC1 on Nrf2/HO-1 were detected by Western Blot,PCR,immunoprecipitation(IP)and RNA interference.Result: Rg1 increased the protein and m RNA expression of Nrf2 and HO-1 in a dose-dependent and time-dependent manner.Rg1’s effect on Nrf/HO-1 may achieved by the upstream pathway Akt/m TORC1.Conclusion: Rg1 protects RPE cells against oxidative stress by activation of Nrf2-HO-1 axis.Akt/m TORC1 activation is found to be responsible for Rg1-induced Nrf2 activation and HO-1 expression,as well as its cytoprotective effect.