组织蛋白酶L抑制剂经线粒体途径抑制氧化应激诱导的视网膜色素上皮细胞凋亡

Cathepsin L inhibitor suppresses oxidative stress-induced apoptosis of retinal pigment epithelial cells by targeting mitochondria

目的观察组织蛋白酶(CTS)L抑制剂对视网膜色素上皮(RPE)细胞凋亡的作用及其对线粒体氧化应激的影响。方法体外培养的人RPE细胞分为正常组、过氧化氢(H_(2)O_(2))组、H_(2)O_(2)+CTSL抑制剂组。H_(2)O_(2)组、H_(2)O_(2)+CTSL抑制剂组置于含400μmol/L H_(2)O_(2)的培养基孵育24 h;H_(2)O_(2)+CTSL抑制剂组同时加入10μmol/l CTSL抑制剂。正常组为常规培养细胞。建模后24 h进行后续实验。流式细胞仪检测细胞凋亡率;细胞免疫荧光染色、蛋白质免疫印迹法、实时定量聚合酶链反应检测细胞中CTSL表达水平;MitoSOX荧光探针检测细胞中线粒体超氧化物表达水平;MitoTracker染色观察线粒体形态,定量分析线粒体平均面积、形状因子、分支节点。两组间比较采用双尾Student t检验;多组间比较采用单因素方差分析。结果与正常组比较,H_(2)O_(2)组细胞凋亡率明显增加,差异有统计学意义(t=3.307,P=0.0297);细胞中CTSL表达水平升高(t=19.950、6.916、14.220,P<0.05)。与H_(2)O_(2)组比较,H_(2)O_(2)+CTSL抑制剂组CTSL表达水平、细胞凋亡率、细胞线粒体超氧化物水平显著降低,差异有统计学意义(t=11.940、4.718、16.680,P<0.05)。H_(2)O_(2)+CTSL抑制剂组细胞线粒体呈细长椭圆形或杆状;H_(2)O_(2)组细胞线粒体失去其连续轮廓与完整形态。4组细胞线粒体评价面积、形状因子、分支节点比较,差异有统计学意义(F=251.700、34.010、60.500,P<0.0001)。结论H_(2)O_(2)显著诱导RPE细胞的凋亡、CTSL表达升高;CTSL抑制剂可抑制H_(2)O_(2)诱导的RPE细胞凋亡,降低线粒体超氧化物水平,有效恢复线粒体形态。

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(H_(2)O_(2))group,and H_(2)O_(2)+CTSL inhibitor group.The cells of H_(2)O_(2) group and H_(2)O_(2)+CTSL inhibitor group were incubated in the medium containing 400μmol/L H_(2)O_(2) for 24 hours and 10μmol/L CTSL inhibitor was added in H_(2)O_(2)+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 H_(2)O_(2) group was significantly higher(t=3.307,P=0.0297),the expression level of CTSL was significantly increased(t=19.950,6.916,14.220;P<0.05).Compared with H_(2)O_(2) group,the expression level of CTSL,the rate of apoptosis and the mitochondrial ROS level in H_(2)O_(2)+CTSL inhibitor group were significantly lower(t=11.940,4.718,16.680;P<0.05).The mitochondria of H_(2)O_(2)+CTSL inhibitor group were elongated,oval-shaped or rod-shaped,while the mitochondria of H_(2)O_(2) 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<0.0001).Conclusions H_(2)O_(2) can significantly induce apoptosis in RPE cells and increase CTSL expression.CTSL inhibitor can inhibit the H_(2)O_(2)-induced apoptosis of RPE cells,lower the mitochondrial super oxide level,and successfully repair the mitochondrial structure.