Effect Of Lycium Barbarum Polysaccharides On Autophagy Induced By H₂O₂ In Human Retinal Pigment Epithelial Cells

Objective:To study the effect of Lyciumbarbarum polysaccharides on the expression of Beclin-1 and LC3B in human retinal pigment epithelial cells injured by hydrogen peroxide.Methods:1.Observe the effect of hydrogen peroxide and Lycium barbarum polysaccharide on the survival rate of ARPE-19 cells:The CCK8 method was used to detect the effects of different concentrations of hydrogen peroxide on the survival rate of ARPE-19 cells,and the effect of Lycium barbarum polysaccharide on hydrogen peroxide-induced retinal pigment epithelial cell oxidative damage was examined.2.Hydrogen peroxide-induced ARPE-19 cell autophagosomes observed by transmission electron microscope:After induction of ARPE-19 cells with different concentrations of hydrogen peroxide for 12 hours,the cells were harvested and harvested.The cells were harvested and fixed in 3%glutaraldehyde,washed with PBS,fixed with 1%citric acid,dehydrated with gradient ethanol and acetone,embedded,and polymerized.After ultra-thin sections and lead staining,cell autophagosomes were observed under a projection electron microscope.3.The effect of hydrogen peroxide on the expression of Beclin-1,LC3BI,and LC3BII in ARPE-19 cells was detected by Western blotting,and the effect of Lycium barbarum polysaccharides on expression was detected:A group of ARPE-19 cells were treated with different concentrations of hydrogen peroxide for 12 hours and washed twice with 4℃pre-cooled PBS.RIPA lysate was added to extract the total protein,and the expression of LC3B,Beclinl,and β-actin were measured by Western blot.The other group added four concentrations of Lycium barbarum polysaccharide to the cell culture medium,and after culturing the cells to a certain density,400umol/L hydrogen peroxide was added for 12 hours,the treatment method was as same as the first group,two sets of data were collected,and the data was set using ImageLab 5.0 analysis.Results:1.After treatment of ARPE-19 cells with different concentrations of hydrogen peroxide,hydrogen peroxide inhibited cell proliferation and this inhibition was gradient dependent.At the lower concentration(1μg/mL,10μg/mL,100μg/mL),LBP had no significant effect on the growth of ARPE-19 cells.When the concentration of Lycium barbarum polysaccharide is 1000μg/ml,it can promote cell proliferation and increase cell viability.Addition of Lycium barbarum polysaccharides cultured ARPE-19 cells can significantly increase cell viability in a concentration-dependent manner.The results showed that the survival rate of cells in the medium supplemented with LBP group was significantly higher than that of the untreated group after 12 hours of hydrogen peroxide induction.Increased with the increase of polysaccharide concentration in a certain range2.After treatment of ARPE-19 cells with different concentrations of hydrogen peroxide for 12 hours,After 12 hours of ARPE-19 cells were induced by different concentrations of hydrogen peroxide,there was no significant increase in autophagosome expression in the 200μmol/L hydrogen peroxide group,and 400μmol/L hydrogen peroxide group had more autophagosome expression than the control group.Increased,while 600μmol/L hydrogen peroxide group significantly increased autophagosome expression.3.After induction of ARPE-19 cells with different concentrations of hydrogen peroxide,the expression of Beclin-1 and LC3BI and LC3BII were increased.With the increase of hydrogen peroxide concentration,the expression of Beclin-1,LC3BI and LC3BII was increased,showing a concentration-dependent manner.Another group of cell culture medium was added with different concentrations of Lycium barbarum polysaccharides,and as the concentration increased,the expression of Beclin-1 and LC3B decreased.Conclusion:1.A certain concentration of hydrogen peroxide reduced the survival rate of ARPE-19 cells,inhibited cell proliferation,and the inhibitory effect was gradient dependent,and the expression of autophagosome in ARPE-19 cells increased with the increase of hydrogen peroxide concentration after 12 hours of hydrogen peroxide treatment,and the expression of Beclin-1,LC3BⅠ and LC3BⅡ also increased.2.LBP can reduce the inhibition of cell proliferation caused by hydrogen peroxide on ARPE-19 in a concentration-dependent manner.LPS could decrease the expression of Beclin-1,LC3BⅠ and LC3BⅡ,and inhibit the autophagy abnormality of ARPE-19 cells induced by hydrogen peroxide.The inhibitory effect was most significant at the concentration of 1000μg/mL.