Mitochondrial-Targeted Antioxidant Peptide SS31 Prevents RPE Cell Death under Oxidative Stress.

Abstract

This work aims at investigating the protective effects of the mitochondria-targeted peptide SS31, on mitochondria function, preventing human retinal pigment epithelial cell-19 (ARPE-19) cell apoptosis. The ARPE-19 cells were subjected to 24 h of intervention with H2O2 of various concentrations (0, 100, 150, 200, 250, 300, and 500 μmol/L). Various concentrations of SS31 (10 nM, 100 nM, and 1 μmol/L) pretreated the cells for 2 h. The MTT assay determined cell viability. ARPE-19 cell apoptosis was observed by 4′,6-diamidino-2-phenylindole (DAPI) staining under fluorescence microscope and detected by Annexin-V/PI staining under flow cytometry. The measurement of reactive oxygen species (ROS) release level used MitoSOX Red (a mitochondrial superoxide indicator) and the probe 2′-7′dichlorofluorescin diacetate (DCFH-DA). And with the use of a JC-1 probe, the mitochondrial membrane potential (MMP; ΔΨm) was analyzed. Reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR were responsible for measuring the levels of apoptosis related genes (Bcl-2, Bax, and Caspase-3). The cell viability increased significantly with SS31 pretreated (P < 0.05). In the SS31 + H2O2 group, the fluorescence of the cell nuclei with DAPI staining was weaker than H2O2 along group accordance with the decreased ratio of apoptotic cells (P < 0.05). The ROS generation decreased in SS31 pretreated group, with the increased ΔΨm. The RT-PCR result showed decreased Bax gene and Caspase-3 gene expression with SS31 pretreatment, while increased antiapoptotic gene Bcl-2 (P < 0.05). We provide evidence that SS31 promotes resilience of RPE cells to oxidative stress by stabilizing mitochondrial function.