The effect of mitochondria-targeted antioxidant peptide SS31 on oxidative damage of lens epithelial cell / 中华实验眼科杂志

ABSTRACT

Background Presbyopia is one of primary causes affecting the visual and life qualities of the agings,and its mechanism is associated with the oxidative damage of lens epithelial cells with ageing.SS31 is a mitochondria-targeted antioxidant peptide.To study the effect of SS31 on oxidative damage of lens epithelial cells has an important significance for the prevention and treatment of presbyopia.Objective This study was to investigate the effect of SS31 on in vitro oxidative damaged human lens epithelial cells.Methods Human lens epithelial cell line (HLEB-3) was cultured using DMEM with low glucose and 10％ fetal bovine serum(FBS).The cell model of oxidative damage was established by adding 200 μmol/L tea-butyl hydropeoxide (t-BHP) into DMEM for 18 hours.The cells were divided into blank control group,t-BHP model group,10 nmol/L SS31 +t-BHP group,100 nmol/L SS31 +t-BHP group,1 μmol/L SS31 +t-BHP group,10 μmol/L SS31 +t-BHP group and 100 pμmol/L t-BHP group,and then MTT assay was used to detect the survival rate of the cells and evaluate the optimal SS31 concentration for sequential study.The cells then were divided into blank control group,t-BHP model group and 1 μmol/L SS31 +t-BHP co-culture group.The change of mitochondrial membrane potential of the cells was tested by JC-1 dye and flow cytometry.Reactive oxygen species (ROS) level in the mitochondria was determined using MitoSOX staining.Results The cell survival rate in the t-BHP model group was (53.42±2.52)％,and that in the blank control group was 100％.The cell survival rate was considerably increased in various concentrations of SS31 groups,showing a significant difference among different groups (F=58.349,P＜0.01).A highest survival rate was (82.13 ±3.15) ％ in the 1 μmol/L SS31 +t-BHP co-culture group,which was statistically significant in comparison with the t-BHP model group (t =28.710,P＜0.05).JC-1 dye and flow cytometry assay showed that the ratio between red and green fluorescence intensity was 7.07 ±0.06 in the blank control group,4.46±0.14 in the t-BHP model group and 5.76±0.26 in the 1 μmol/L SS31 +tBHP co-culture group,showing significant difference among the 3 groups (F=172.332,P＜0.01).The ratios between red and green fluorescence intensity in the blank control group and 1 μmol/L SS31 +t-BHP co-culture group were higher than that in the t-BHP model (t =2.609,1.303,both at P＜0.001).ROS fluorescence cells were much more in the t-BHP model group compared with blank control group and 1 μmol/L SS31 + t-BHP co-culture group.Conclusions SS31 can protect HLEB-3 cells from oxidative stress.SS31 may serve as a potential new approach to the treatment of presbyopia and other age-related diseases of lens.