ROCK2在ARC中的表达及对晶状体上皮细胞的自噬及氧化损伤的影响

The expression of Rho kinase 2 in age-related cataracts and its effect on autophagy and oxidative damage of lens epithelial cells

目的探讨Rho激酶2(ROCK2)在年龄相关性白内障(ARC)晶状体前囊膜中的表达及其对H_(2)O_(2)诱导的人晶状体上皮细胞自噬及氧化损伤的影响。方法收集2020年6月~2021年4月在我院行超声乳化白内障吸出术的ARC患者20例,获取晶状体前囊膜,并获取眼科标本库中无眼科疾病、眼球结构完整且晶状体透明的正常晶状体前囊膜。免疫组织化学染色和Western blot检测ROCK2蛋白表达;将人晶状体上皮细胞HLE-B3随机分为对照组、H_(2)O_(2)组、sh-NC+H_(2)O_(2)组和sh-ROCK2+H_(2)O_(2)组,转染sh-NC或sh-ROCK2至对应组细胞,并用H_(2)O_(2)处理细胞,实时荧光定量PCR和Western blot检测细胞转染效率,MTT法检测各处理组细胞增殖活性,透射电镜观察细胞内自噬现象,自噬双标腺病毒mRFP-GFP-LC3转染细胞后观察自噬溶酶体和自噬体水平,Western blot检测细胞中自噬相关蛋白表达情况,DCFH-DA法检测活性氧(ROS)水平,试剂盒测定超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)的活性及丙二醛(MDA)含量。结果与正常组织比较,ARC中ROCK2表达增高(P<0.05);转染sh-ROCK2后成功下调了HLE-B3细胞中ROCK2表达水平(P<0.05);与对照组比较,H_(2)O_(2)处理细胞后,细胞增殖活性显著下降,自噬体明显增多,明显激活了自噬溶酶体和自噬体水平,Beclin-1蛋白相对表达量显著升高,LC3-II/LC3-Ⅰ比值显著上调,ROS水平显著上升,SOD和GSH-Px的活性均显著降低,MDA含量则显著升高(均P<0.05);与H_(2)O_(2)组比较,细胞转染sh-ROCK2后再用H_(2)O_(2)处理,细胞增殖活性显著提高,自噬体减少,自噬溶酶体和自噬体水平受到抑制,Beclin-1蛋白相对表达量显著降低,LC3-II/LC3-Ⅰ比值显著下调(均P<0.05),同时,细胞内ROS水平显著下降,SOD和GSH-Px的活性均显著升高,MDA含量显著降低(均P<0.05)。结论ROCK2在ARC晶状体前囊膜内高表达,下调其表达能够抑制H_(2)O_(2)诱导的人晶状体上皮细胞过度自噬,减少氧化损伤。

Objective To explore the expression of Rho-associated coiled-coil-forming protein kinase 2(ROCK2)in the anterior lens capsule of age-related cataract(ARC),and its effect on the autophagy and oxidative damage of human lens epithelial cells induced by H_(2)O_(2).Methods The anterior capsule of ARC patients and normal lens were collected,and the expression of ROCK2 protein was detected by immunohistochemical staining and Western blot;Human lens epithelial cells HLE-B3 were randomly divided into control group,H_(2)O_(2)group,sh-NC+H_(2)O_(2)group and sh-ROCK2+H_(2)O_(2)group,transfected sh-NC or sh-ROCK2 to the corresponding group of cells,and treated the cells with H_(2)O_(2),Real-time fluorescent quantitative PCR and Western blot detected the cell transfection efficiency,MTT method detected the cell proliferation activity of each treatment group,transmission electron microscopy observed the phenomenon of autophagy in cells;After autophagy double-labeled adenovirus mRFP-GFP-LC3 was transfected into cells,autophagolysosome and autophagosome levels were observed,Western blot detected the expression of autophagy-related proteins in cells,DCFH-DA method detected the level of reactive oxygen species(ROS),the kit measured the activity of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)and the content of malondialdehyde(MDA).Results Compared with normal tissues,the expression of ROCK2 in ARC was increased(P<0.05).After transfection with sh-ROCK2,the expression level of ROCK2 in HLE-B3 cells was successfully down-regulated(P<0.05).Compared with the control group,after H_(2)O_(2)treatment of cells,cell proliferation activity decreased significantly,autophagosomes increased significantly,autophagolysosome and autophagosome levels were obviously activated,and the relative expression of Beclin-1 protein increased significantly,the ratio of LC3-II/LC3-I was significantly increased,the level of ROS was significantly increased,the activities of SOD and GSH-Px were significantly reduced,and the content of MDA was significantly increased(P<0.05).Compared with the H_(2)O_(2)group,the cells were transfected with sh-ROCK2 and then treated with H_(2)O_(2),the cell proliferation activity was significantly increased,the autophagosomes were reduced,the levels of autophagolysosomes and autophagosomes were inhibited,the relative expression of Beclin-1 protein was significantly reduced,the ratio of LC3-II/LC3-I was significantly down-regulated.At the same time,the intracellular ROS level was significantly decreased,the activities of SOD and GSH-Px were both significantly increased,and the MDA content was significantly decreased(P<0.05).Conclusion ROCK2 is highly expressed in the anterior capsule of ARC lens,down-regulating its expression can inhibit H_(2)O_(2)-induced excessive autophagy in human lens epithelial cells and reduce oxidative damage.