Calpain activation in retinal pigment epithelium (RPE) cells induced by oxidative stress / 眼科新进展

ABSTRACT

Objective To investigate the content of Calpain in H2O2-induced oxidative stress in cultured human retinal pigment epithelium (RPE) and the change of biologic activity of RPE cells.Methods Normal culture of human RPE cell line (ARPE19 cells) was assigned as the control group,while cells were cultured in the medium with 10 μmol · L-1 and 100 μmol · L-1 H2O2 were the experimental groups.After culture of 4 h,8 h,16 h,32 h,the proliferation rate of RPE cells induced with different concentrations of H2 O2 was measured by methyl thiazolyl tetrazolium (MTT) assay,and the Ca2 + concentration was measured with fluorescence dye Fluo-3/AM ester,laser scanning confocal microscopy (LSCM) and flow cytometry.Then irritation with 10 μmol ·L-1 H2 O2 for 8 hours,Calpain protein and mRNA in RPE cells were detected by WesternBlot and RT-PCR.Results The proliferation rates (r) of the RPE cells were increased significantly induced by 10 μmol · L-1 H2O2 for 4 h,8 h,16 h and 100 μmol ·L-1 H2 O2 for 4 h,8 h when compared with the control group,and the differences were statistically significant (all P ＜ 0.01),whereas the rates of the cells induced by 100 μ mol · L-1 H2O2 for 16 h and 32 h were less than those of the control group (all P ＜0.01).The Ca2+ concentration in RPE cells induced by 10 μmol · L-1 H2O2 for 8 h was significantly higher than that in the controls [(563.27 ± 77.10) U vs.(170.77 ± 20.11) U] (P ＜ 0.01),and the protein and mRNA levels of Calpain were significantly increased when compared with the control group (both P ＜ 0.05).However,these changes can be effectively suppressed using Calpain inhibitor SNJ-1945 (SNJ).Conclusion The Ca2+ inflow can make Calpain activation involve in oxidative stress stimulated by a certain concentration of H2 O2 in RPE cells.