Liu Junzi Soup’s Interference Protects Against Oxidative Injury In ARPE-19 Cells Associated With Activation Of P62-keap1/Nrf2 Pathway

Objective:We will take oxidative injury in ARPE-19 cells as research object,observe the protection of drug serum of Liu Junzi Soup to oxidative injury in ARPE-19 cells;study effects of Liu Junzi Soup’s interference to the p62-keap1/Nrf2 pathway,and validate the molecular mechanism by the gene knockout of ARPE-19 cells of p62、Nrf2.Methods:We used t-BHP to treat ARPE-19 cells leading to oxidative injury within cells,then detect protective effects Liu Junzi Soup against cell death in ARPE-19 cells treated with t-BHP by MTT and AnnexinV-FITC/P1 on cell proliferation and apoptosis.ARPE-19 cells are divided into four groups:normal control group,model reference group,blank serum group and herb treated group(They were divided into high,medium and low dose groups).The activities of SOD in each group were detected by colorimetry.ROS levels were detected by DCFH-DA fluorescent probe.Western blot detected the mRNA transcription and protein expression of cell HO-1 and cell NQO-1.Western blot detected the protein expression of cell HO-1、NQO-1、p62、Nrf2and LC3Ⅱ.Results:1.Compared with the normal control group,the cell viability of the model reference group significantly decreased;Compared with the model reference group,the cell in the concentration of herb treated group significantly decreased oxidative damages by t-BHP and increased viability.2.Compared with the normal control group,the cell apoptosis of the model reference group significantly increased;Compared with the model reference group,the cell apoptosis of herb treated group decreased.3.Compared with the normal control group,the activities of SOD in the model reference group decreased,ROS levels in the model reference group increased;Compared with the model reference group,the activities of SOD in herb treated group increased,ROS levels in herb treated group decreased,these showed herb treated group can reduce oxidative stress of ARPE-19 cells.4.Drug serum of Liujunzi soup could up-regulate the expression of nucleus Nrf2 protein,and reversed the reduction of cell HO-1、cell NQO-1 and up-regulate the protein expression of p62 and LC3Ⅱ;While the cell target genes were transfected by siRNA,effects of up-regulate in herb treated group decreased.Conclusion:Liu Junzi Soup had stronger protective effects against oxidative-induced damages in ARPE-19 cells which were treated by t-BHP,the mechanism is the activation of p62-keap1/Nrf2 pathway,which could up-regulate the expression of antioxidant enzymes of the downstream of the Nrf2 pathway and up-regulate the protein expression of p62 to activate the autophagy.complement control proteins and autophagy-related proteins to exert antioxidant effects.