Protective Effects Of SS31 On Mitochondrial Function And Apoptosis In ARPE-19 Cells

ObjectiveTo investigate the protective effects of SS31,a mitochondria-targeted peptide,on mitochondria function and apoptosis on human retinal pigment epithelial cell-19?ARPE-19?cells.MethodsThe ARPE-19 cells were treated with various concentrations of H₂O₂?0,100,150,200,250,300,500?mol/L?for 24 h and were pretreated with various concentrations of SS31?10 nM,100 nM,1?mol/L?for 2 h.The cell viability was determined by MTT assay,and the optimum concentration of H₂O₂ and SS31 was selected.Cell morphology was observed by inverted phase contrast microscope.The apoptosis of ARPE-19 cells was observed by 4?,6-diamidino-2-phenylindole?DAPI?staining under fluorescence microscope and detected by Annexin-V/PI staining under flow cytometry.The release level of reactive oxygen species?ROS?were detected using MitoSOX Red Mitochondrial Superoxide Indicator with fluorescence microscope and 2'-7'dichlorofluorescin diacetate?DCFH-DA?probe with flow cytometry.Changes in mitochondrial membrane potential were analyzed by JC-1 probe using flow cytometry.The expression levels of genes associated with apoptosis?Bax,Caspase-3,and Bcl-2?were determined by reverse transcription polymerase chain reaction?RT-PCR?,and the mRNA levels were determined by real-time PCR.One-way analysis of variance?ANOVA?test was to analyze these data statistically.ResultsThe cell viability was decreased in a concentration-dependent manner under different concentrations of H₂O₂.The cell viability was increased after pre-treatment with SS31?P<0.05?.There was no significant difference of cell viability between the SS31 group and the normal group.With the increase of H₂O₂ concentration,the number of living cells was decreased,and the cells were wizened gradually.With the increase of SS31 concentration,the number of living cells increased,and the cells tended to be normal in morphology.The result of DAPI staining showed that the cell nuclei were fluorescent spots in the H₂O₂ group,and fluorescence in the SS31+H₂O₂ group was weaker than that in the H₂O₂ group.Annexin-V/PI staining showed that the ratio of apoptotic cells was decreased in the H₂O₂+SS31 group compared with the H₂O₂ group?P<0.05?.The fluorescence intensity of ROS in the H₂O₂ group was strong,and in the H₂O₂+SS31 group,it was significantly weaker.The flow cytometry analysis showed that the level of ROS in the SS31+H₂O₂group was lower than that in the H₂O₂ group?P<0.05?.The analysis of mitochondria membrane potential showed that the ratio of low mitochondria potential cells was decreased in the SS31+H₂O₂ group compared with the H₂O₂ group.The RT-PCR result showed that the expression level of Bax gene and Caspase-3 gene was inhibited after the pretreatment with SS31 in ARPE-19 cells under oxidative damage,while anti-apoptotic gene Bcl-2 was improved.The mRNA level of each gene was significantly different between the SS31+H₂O₂ group and the H₂O₂ group?P<0.05?.ConclusionSS31 protected the mitochondrial function and cells from apoptosis on ARPE-19 cells under oxidative damage.