HMGB1在过氧化氢诱导的晶状体上皮细胞DNA损伤和衰老中的作用  

The impact of high mobility group box 1 on hydrogen peroxide-induced DNA damage and senescence in lens epithelial cells

作　　者：吴苗苗 李鹏飞 徐林慧 康丽华[1,2] 季敏 管怀进[1,2] WU Miaomiao;LI Pengfei;XU Linhui;KANG Lihua;JI Min;GUAN Huaijin(Department of Ophthalmology,the Affiliated Hospital of Nantong University,Nantong 226000,Jiangsu Province,China;Nantong University,Nantong 226000,Jiangsu Province,China)

机构地区：[1]南通大学附属医院眼科,江苏省南通市226000 [2]南通大学,江苏省南通市226000

出　　处：《眼科新进展》2025年第4期257-262,共6页Recent Advances in Ophthalmology

基　　金：国家自然科学基金项目(编号:81974129、82171038)。

摘　　要：目的探究高迁移率族蛋白B1(HMGB1)对过氧化氢(H_(2)O_(2))诱导的晶状体上皮细胞(LECs)氧化损伤环境下DNA损伤和衰老的影响。方法采用实时荧光定量聚合酶链式反应(RT-PCR)技术检测年龄相关性白内障(ARC)患者(ARC组)和黄斑前膜疾病患者(对照组)前囊膜组织中HMGB1的mRNA相对表达水平。Western blot检测LECs细胞株SRA01/04细胞中不同浓度(0、100、200、400μmol·L^(-1))H_(2)O_(2)处理后HMGB1蛋白表达变化,并筛选出最佳作用浓度用于后续构建细胞氧化损伤模型。将培养的SRA01/04细胞分为Control组(未经任何处理)、HA组(转染对照质粒HA)和OE-HMGB1组(转染质粒HMGB1),并通过RT-PCR和Western blot检测HMGB1的mRNA和蛋白相对表达水平。将培养的SRA01/04细胞分为H_(2)O_(2)组(400μmol·L^(-1)H_(2)O_(2)处理)、H_(2)O_(2)+HA组(转染对照质粒HA,同时400μmol·L^(-1)H_(2)O_(2)处理)和H_(2)O_(2)+OE-HMGB1组(转染质粒HMGB1,同时400μmol·L^(-1)H_(2)O_(2)处理),运用免疫荧光法检测各组细胞DNA氧化损伤,Western blot检测各组细胞中磷酸化的组蛋白H2A(γH2A)、肿瘤蛋白53(P53)、细胞周期依赖性激酶抑制因子1A(P21)、细胞周期依赖性激酶抑制因子2A(P16)蛋白相对表达水平,β-半乳糖苷酶(SA-β-gal)染色检测各组细胞衰老变化。结果RT-PCR检测结果显示,与对照组相比,ARC组患者前囊膜组织中HMGB1的mRNA相对表达水平明显下降,差异有统计学意义(P<0.001)。此外,在H_(2)O_(2)诱导的氧化损伤模型中,HMGB1蛋白相对表达水平随H_(2)O_(2)浓度上升呈下降趋势。RT-PCR与Western blot检测结果显示,与HA组相比,OE-HMGB1组细胞中HMGB1 mRNA和蛋白相对表达水平均显著升高,差异均有统计学意义(均为P<0.001)。免疫荧光染色结果显示,与H_(2)O_(2)组和H_(2)O_(2)+HA组相比,H_(2)O_(2)+OE-HMGB1组细胞中γH2A蛋白相对表达水平和荧光强度均显著下降,差异均有统计学意义(均为P<0.001)。与H_(2)O_(2)组及H_(2)O_(2)+HA�Objective To investigate the impact of high mobility group box 1(HMGB1)on hydrogen peroxide(H_(2)O_(2))-induced DNA damage and senescence in lens epithelial cells(LECs)under oxidative stress conditions.Methods Fluorescent quantitative real-time polymerase chain reaction(RT-PCR)technology was used to detect the mRNA expression of HMGB1 in the anterior capsule tissue of patients with age-related cataract(ARC group)and epiretinal membrane(control group).Western blot analysis was employed to examine the changes in the protein expression of HMGB1 in the LEC line SRA01/04 after treatment with varying concentrations of H_(2)O_(2)(0,100,200,and 400μmol·L^(-1)).The optimal concentration was selected for subsequent establishment of a cellular oxidative damage model.The cultured SRA01/04 cells were divided into three groups:Control(untreated),HA(transfected with the control plasmid HA),and OE-HMGB1 groups(transfected with the HMGB1 plasmid).The mRNA and protein expression levels of HMGB1 were detected by RT-PCR and Western blot.The cultured SRA01/04 cells were divided into three groups:H_(2)O_(2)(treated with 400μmol·L^(-1)H_(2)O_(2)),H_(2)O_(2)+HA(transfected with the control plasmid HA and simultaneously treated with 400μmol·L^(-1)H_(2)O_(2)),and H_(2)O_(2)+OE-HMGB1 groups(transfected with the HMGB1 plasmid and simultaneously treated with 400μmol·L^(-1)H_(2)O_(2)).Immunofluorescence was used to detect DNA oxidative damage in cells from each group.Western blot analysis was performed to assess the protein expression levels of phosphorylated histone H2A(γH2A),tumor protein p53(P53),cyclin-dependent kinase inhibitor 1A(P21),and cyclin-dependent kinase inhibitor 2A(P16)in cells from each group.Additionally,senescence-associated-β-galactosidase(SA-β-gal)staining was conducted to detect senescent changes in cells from each group.Results RT-PCR results indicated that the relative mRNA expression level of HMGB1 in the anterior capsule tissue of the ARC group was significantly decreased,compared with that in the control

关 键 词：年龄相关性白内障 晶状体上皮细胞 DNA损伤 高迁移率族蛋白B1 

分 类 号：R776.1[医药卫生—眼科]