Cathepsin L inhibitor suppresses oxidative stress-induced apoptosis of retinal pigment epithelial cells by targeting mitochondria
Objective To explore the effect of cathepsin L(CTSL)inhibitor on apoptosis of retinal pigment epithelial(RPE)cells and mitochondrial oxidative stress.Methods RPE cells were cultured in vitro and divided into control group,hydrogen peroxide(H2O2)group,and H2O2+CTSL inhibitor group.The cells of H2O2 group and H2O2+CTSL inhibitor group were incubated in the medium containing 400 μmol/L H2O2 for 24 hours and 10 μmol/L CTSL inhibitor was added in H2O2+CTSL inhibitor group at the same time.The cells of normal group were routinely cultured cells.The follow-up experiment was carried out 24 hours after modeling.The rate of apoptosis was detected by flow cytometry.The expression of CTSL was detected by immunofluorescence staining,Western blot and real time-polymerase chain reaction.The level of mitochondrial super oxide was detected by MitoSOX fluorescent probe,and the mitochondrial structure was observed after MitoTracker staining,the average area,form factors,and branch of mitochondria were quantitatively analyzed.The two groups were compared using two-tailed Student t test,while numerous groups were compared using one-way ANOVA.Results Compared with control group,the rate of apoptosis in H2O2 group was significantly higher(t=3.307,P=0.029 7),the expression level of CTSL was significantly increased(t=19.950,6.916,14.220;P＜0.05).Compared with H2O2 group,the expression level of CTSL,the rate of apoptosis and the mitochondrial ROS level in H2O2+CTSL inhibitor group were significantly lower(t=1 1.940,4.718,16.680;P＜0.05).The mitochondria of H2O2+CTSL inhibitor group were elongated,oval-shaped or rod-shaped,while the mitochondria of H2O2 group lost their continuous contour shape and complete structure.The differences of the average area,form factors,and brach of mitochondria among 4 groups were statistically significant(F=251.700,34.010,60.500;P＜0.000 1).Conclusions H2O2 can significantly induce apoptosis in RPE cells and increase CTSL expression.CTSL inhibitor can inhibit the H2O2-induced apoptosis of RPE cells,lower the mitochondrial super oxide level,and successfully repair the mitochondrial structure.
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