Protective Effect Of Mitochondrial Targeting Peptide SS31 On 661W Cell Necroptosis Induced By H₂O₂

ObjectiveThe present study aimed to investigate the ability of SS31,a novel mitochondria-targeted peptide to protect against H₂O₂-induced mitochondrial dysfunction and necroptosis in 661W cell lines.MethodsThe cell model of oxidative damage was established by adding 400?mol·L^-11 hydrogen peroxide?H₂O₂?,According to the MTT results,100 nmol·L^-11 SS31 and 50?mol·L^-1 Nec-1evaluate the optimal concentration for sequential study.The cells were divided into control group,H₂O₂ group,SS31+Nec-1 group,SS31+H₂O₂ group,SS31+Nec-1+H₂O₂ group,and Nec-1+H₂O₂ group.MTT and assay were used to determine cell viability and observe the morphological and structural changes of treated cells in each group.The generation of intracellular reactive oxygen species?ROS?was detected using MitoSOXTM and dichlorodihydrofluorescin diacetate?DCFH-DA?with photofluorography and flow cytometry.Malondialdehyde?MDA?lipid peroxidation assay was performed to determine the degree of oxidative stress.Mitochondrial membrane potential was measured using the JC-1 assay.The PI positive rate were by flow cytometry after Annexin V/Propidium-dide?PI?staining.Membrane changes were observed by DIPA nuclear staining and PI staining followed by photofluorography.In addition,the protein levels of RIP3?an indicator of necroptosis?were detemined by Western blot.ResultsThe rate of cell viability was lower in the H₂O₂ group than in the control group?all P<0.05?,The viability of the cells improved following treatment with SS31and Nec-1compared with H₂O₂ group,and the differences were statistically significant?all P<0.05?.MitoSOXTM fluorescence staining showed that red fluorescence cells were much more in the H₂O₂ group compared with SS31+H₂O₂ group,SS31+Nec-1+H₂O₂ group and Nec-1+H₂O₂ group?all P<0.05?,Flow cytometry analysis further confirmed that treatment with SS31,Nec-1and combined treatment of SS31+Nec-1 could significantly reduced ROS production?all P<0.05?.The level of MDA in the in the SS31,Nec-1 and combined treatment of SS31+Nec-1 group decreased significantly compared with the H₂O₂ group?all P<0.05?.Flow cytometry analysis confirmed that the proportion of cells with decreased mitochondrial membrane potential in the H₂O₂ group was higher than that those in the SS31+H₂O₂ group and Nec-1+H₂O₂ group,SS31+Nec-1+H₂O₂ group,and the difference was statistically significant?all P<0.05?.AnnexinV/PI double staining results showed that the PI-positive rate of 661W cells in H₂O₂ group was significantly higher than those in the control group,SS31+H₂O₂ group,SS31+Nec-1+H₂O₂ group and Nec-1+H₂O₂ group?all P<0.05?.Western blot analysis showed that the expression of RIP3 began to increase after9h treatment of 661W cells with H₂O₂,and the maximum level was found at 24 h?all P<0.05?.Compared with the control group,the relative expression levels of RIP3 in H₂O₂group was significantly increased?P<0.05?,but the relative expression levels of RIP3 in SS31 and Nec-1 treated H₂O₂ group were down-regulated when compared with H₂O₂ group?all P<0.05?.ConclusionsSS31 partially protects 661W Cells against H₂O₂-induced injury by inhibiting necroptosis.