Nei核酸内切酶Ⅷ样蛋白1对过氧化氢诱导的晶状体上皮细胞凋亡与自噬的影响  

Effects of Nei endonuclease VIII-like protein 1 on H_(2)O_(2)-induced apoptosis and autophagy in lens epithelial cells

作　　者：鲍思洁 李鹏飞 康丽华 王从玉 王思文 管怀进[1] BAO Sijie;LI Pengfei;KANG Lihua;WANG Congyu;WANG Siwen;GUAN Huaijin(Eye Institute,Affiliated Hospital of Nantong University,Medical School of Nantong University,Nantong 226001,Jiangsu Province,China)

机构地区：[1]南通大学医学院南通大学附属医院眼科

出　　处：《眼科新进展》2023年第12期934-939,共6页Recent Advances in Ophthalmology

基　　金：国家自然科学基金面上项目(编号:82171038);国家自然科学青年基金项目(编号:82101101)。

摘　　要：目的探究Nei核酸内切酶Ⅷ样蛋白1(NEIL1)对过氧化氢(H_(2)O_(2))诱导的晶状体上皮细胞(LECs)凋亡与自噬的影响。方法以年龄相关性白内障(ARC)患者、接受透明晶状体摘除术的黄斑前膜患者的晶状体前囊膜样本和晶状体上皮细胞系SRA01/04细胞为研究对象,采用RT-PCR实验检测NEIL1 mRNA表达。将SRA01/04细胞随机分为对照组、OE-Vector组和OE-NEIL1组,采用RT-PCR和Western blot检测过表达效率;将细胞随机分为对照组、H_(2)O_(2)组、OE-Vector H_(2)O_(2)组、OE-NEIL1 H_(2)O_(2)组,采用CCK-8法检测细胞活力,Western blot检测自噬相关蛋白(ATG7、P62、Beclin1、LC3-I和LC3-II)和凋亡相关蛋白(Bax和Bcl-2)的表达,荧光染色检测细胞凋亡与线粒体膜电位情况。结果ARC患者晶状体前囊膜样本中的NEIL1 mRNA表达显著低于黄斑前膜患者,且H_(2)O_(2)组SRA01/04细胞中NEIL1 mRNA的表达同样低于对照组(均为P<0.05)。RT-PCR与Western blot检测结果显示,OE-NEIL1组SRA01/04细胞中NEIL1 mRNA和蛋白表达均显著高于OE-Vector组(均为P=0.000)。与对照组相比,H_(2)O_(2)组SRA01/04细胞中细胞活力显著降低;Bax蛋白表达上调,Bcl-2蛋白表达下调;Mito-Tracker标记活细胞数显著减少,Annexin V-FITC标记的凋亡细胞数显著增多,差异均有统计学意义(均为P<0.05)。与OE-Vector H_(2)O_(2)组相比,OE-NEIL1 H_(2)O_(2)组SRA01/04细胞中细胞活力显著升高;自噬相关蛋白(ATG7、P62、Beclin1、LC3-I和LC3-II)表达均显著上调;Bax蛋白表达下调,Bcl-2蛋白表达上调;Mito-Tracker标记的活细胞数增加,Annexin V-FITC标记的凋亡细胞数减少,差异均有统计学意义(均为P<0.05)。结论在H_(2)O_(2)诱导氧化应激条件下,NEIL1可通过促进LECs的自噬,维持LECs内环境稳定,增加其细胞活力从而减少LECs凋亡,参与ARC的发生发展。Objective To investigate the influence of Nei endonuclease VIII-like protein 1(NEIL1)on H_(2)O_(2)-induced apoptosis and autophagy in lens epithelial cells(LECs).Methods Reverse transcription polymerase chain reaction(RT-PCR)was used to detect the expression of NEIL1 messenger ribonucleic acid(mRNA)in the anterior lens capsule and LEC line SRA01/04 cells from age-related cataract(ARC)patients and macular epiretinal membrane patients who underwent the clear lens extraction.SRA01/04 cells were then divided into the control group,OE-Vector group and OE-NEIL1 group.RT-PCR and Western blot(WB)were used to detect the overexpression efficiency.Subsequently,the cells were divided into the control group,H_(2)O_(2)group,OE-Vector H_(2)O_(2)group and OE-NEIL1 H_(2)O_(2)group;cell viability was detected by Cell Counting Kit-8,WB was used to evaluate the expression of autophagy-related proteins(ATG7,P62,Beclin1,LC3-I and LC3-II)and apoptosis-related proteins(Bax and Bcl-2),and fluorescent staining was adopted to measure cell apoptosis and mitochondrial membrane potential.Results The expression of NEIL1 mRNA in the anterior lens capsule samples from ARC patients was significantly lower than that of macular epiretinal membrane patients,and the expression of NEIL1 mRNA in the SRA01/04 cells in the H_(2)O_(2)group was also lower than that in the control group(both P<0.05).The results of RT-PCR and WB revealed that the expressions of NEIL1 mRNA and protein in the SRA01/04 cells of the OE-NEIL1 group were significantly higher than those in the OE-Vector group(both P=0.000).Compared with the control group,the viability of SRA01/04 cells in the H_(2)O_(2)group decreased,the expression of the Bax protein was up-regulated,the Bcl-2 protein was down-regulated,viable cells labeled with Mito-Tracker decreased,and apoptotic cells labeled with Annexin V-FITC increased,with statistical significances(all P<0.05).Compared with the OE-Vector H_(2)O_(2)group,in the OE-NEIL1 H_(2)O_(2)group,the viability of SRA01/04 cells significantly improved,t

关 键 词：Nei核酸内切酶Ⅷ样蛋白1 年龄相关性白内障 晶状体上皮细胞 凋亡 自噬 

分 类 号：R776[医药卫生—眼科]