Study On The Protective Mechanism Of Bushen Yijing Recipe Against Oxidative Stress Injury In RPE Cells Based On Autophagy Pathway

ObjectiveTo explore the protective mechanism of Bushen Yijing Formula on RPE cells under oxidative stress through in vitro cell experiments based on autophagy pathway and to provide new ideas for the prevention and treatment of AMD and other retinal diseases with traditional Chinese medicine.Methods1.Cells were treated with different concentrations of HQ(30μmol/L,60μmol/L,90μmol/L,120μmol/L)for 12h,24h,and 36h to determine the optimal intervention concentration and time for constructing an oxidative stress model for RPE cells.2.Cells were treated with different concentrations of drug-containing serum and blank serum to select the optimal intervention concentration and time of drug-containing serum by using the CCK8 method.3.Cells were divided into six groups,including the normal group,model group(HQ group),blank serum group,low-dose group,high-dose group,and autophagy inhibitor group.The CCK8 method was used to detect the effect of different interventions on cell viability.4.The morphology of cells in each group was observed under an optical microscope.5.Real-time fluorescence quantitative PCR was used to detect the expression of autophagy-related factors and factors related to the antioxidant pathway in each group.6.Transmission electron microscopy was used to observe the formation of autophagosomes in cells of each group.7.Western blot method was used to detect the expression levels of autophagy-related factors and proteins related to the antioxidant pathway in RPE cells of each group.Results1.The experimental results showed that compared with the normal group,solutions of HQ at 90μmol/L and 120μmol/L both inhibited cell proliferation(P<0.01),but the solution of 120μmol/L HQ resulted in massive cell death.Therefore,90μmol/L HQ was selected for a 24-hour intervention to construct an oxidative stress model for RPE cells.2.After screening with the CCK8 method,the blank serum group was selected to intervene with the RPE cells that had been successfully modeled,with 10%SD rat blank serum.The low-dose group and high-dose group were intervened with drug-containing serum at concentrations of 2.5%and 10%,respectively.The CCK8 method was used to detect the effect of Bu Shen Yi Jing Formula drug-containing serum on the survival rate of RPE cells under oxidative stress.The results showed that compared with the normal group,the survival rates of all groups decreased(P<0.01),and there was no statistical difference in cell survival rate between the model group and the blank serum group(P>0.05).Compared with the model group,the survival rate of cells in the drug-containing serum group increased significantly(P<0.01),while there was no significant difference in cell survival rate between the autophagy inhibitor group and the model group(P>0.05).3.Cellular morphological observation:The cells in the normal group were spindle-shaped with regular morphology.After HQ intervention,cells in the model group showed irregular morphology;some cells changed from spindle-shaped to elongated or round shape,and a large number of suspended cells were visible under the microscope.The cell gap increased,and cell fusion was poor with sparse cell density.After intervention with drug-containing serum from Bu Shen Yi Jing Formula,compared with the model group,the cell morphology in the drug-containing serum group improved and began to restore spindle shape.There was no significant difference between the blank serum group and the model group.Compared with the drug-containing serum group,the number of spindle-shaped cells in the autophagy inhibitor group decreased,while the number of cells with abnormal morphology increased.4.Through transmission electron microscopy,we found that the normal group cells had almost no autophagosomes,while the model group had more autophagosomes.Compared with the model group,the number of autophagosomes in the drug-containing serum group decreased,and the number of autophagosomes in the autophagy inhibitor group decreased further compared with the drug-containing serum group.5.Real-time fluorescence quantitative PCR was used to detect the mRNA expression levels of antioxidant pathway-related factors and autophagy-related factors in cells.The results showed that in the HQ group,the mRNA levels of Atg5,Beclinl,and Keapl increased compared with the normal group(P<0.05),while the mRNA expression level of Nrf2 decreased(P<0.05).Compared with the HQ group,there was no statistical significance in the mRNA expression levels of various factors in the blank serum group(P>0.05).The mRNA expression levels of Atg5,Beclinl,and Keap1 in the low-dose group and high-dose group.6.Western blot was used to detect the protein expression levels of Keapl,Nrf2,Beclinl,LC3II,and p-mTOR in cells.The results showed that in the HQ group,the protein expression levels of LC3II,Beclinl,and Keapl increased compared with the normal group(P<0.05),while the protein expression levels of Nrf2 and p-mTOR decreased(P<0.05).Compared with the HQ group,there was no statistical significance in the protein expression levels of various factors in the blank serum group(P>0.05).The protein expression levels of Beclinl,LC3II,and Keapl in the low-dose group and high-dose group decreased(P<0.05),and the protein expression levels of Nrf2 and p-mTOR increased significantly(P<0.05).Compared with the low-dose group,the protein expression levels of LC3II,Beclinl,and Keap1 significantly decreased,and the protein expression levels of Nrf2 and p-mTOR increased in the high-dose group(P<0.05).Compared with the high-dose group,the protein expression levels of LC3II,Beclinl,and Keap1 decreased,while the protein expression levels of Nrf2 and p-mTOR increased in the autophagy inhibitor group(P<0.05).ConclusionHQ can successfully induce oxidative stress in ARPE-19 cells.Drug-containing serum from Bu Shen Yi Jing Formula can protect RPE cells to a certain extent.Drug-containing serum from Bu Shen Yi Jing Formula can lower the level of autophagy in RPE cells and activate the Keap1/Nrf2/ARE antioxidant pathway.This may be the reason why Bu Shen Yi Jing Formula can treat age-related macular degeneration and protect RPE cells from oxidative stress damage.The activation of the Keap1/Nrf2/ARE antioxidant pathway may be related to the regulation of autophagy levels.