缺氧对氧化应激诱导人晶状体上皮细胞衰老的保护作用  

Hypoxia protects human lens epithelial cells from senescence induced by oxidative stress

作　　者：侯芳[1] 黄启明 万修华[1] Hou Fang;Huang Qiming;Wan Xiuhua(Beijing Tongren Eye Center,Beijing Tongren Hospital,Capital Medical University,Beijing Key Laboratory of Ophthalmology and Visual Science,Beijing 100730,China;School of Life and Science,Nanchang University,Nangchang 330031,China;Jiangxi Mayobio Technology Company Ltd.,Nanchang 330006,China)

机构地区：[1]首都医科大学附属北京同仁医院、北京同仁眼科中心、眼科学与视觉科学北京市重点实验室,100730 [2]南昌大学生命科学学院,330031 [3]江西美奥生物技术有限公司,南昌330006

出　　处：《眼科》2023年第4期344-350,共7页Ophthalmology in China

基　　金：江西省主要学科学术与技术带头人培养计划(20204BCJ22035);江西省科技厅科技计划重点研发项目(20192ACB70004、20201BBG71007);江西省科技厅科技计划一般项目(20192BBH80015);国家自然科学基金(82171037)。

摘　　要：目的观察缺氧对氧化应激分子4-羟基壬烯酸(4-HNE)造成的人晶状体上皮细胞(hLEC)衰老的保护作用。设计实验研究。研究对象人晶状体上皮细胞。方法采用不同浓度(0、1、2.5、10和50μM)4-HNE处理的LEC在3个不同O2浓度(1%、5%和21%)下经过24、48、72和96小时的培养,通过CCK-8检测出其增生活性水平;使用Annexin-V/PI染色、荧光探针(DCFH-DA)、β-半乳糖苷酶染色和BrdU结合测定方法,分别对缺氧和常氧培养hLEC中的细胞凋亡水平、活性氧(ROS)和β-半乳糖苷酶进行测量;采用蛋白印迹法对衰老相关蛋白P53和P16进行检测。主要指标hLEC的增生活性、细胞凋亡、衰老和相关蛋白表达水平。结果与正常氧浓度下相比较,1%低氧的hLEC活力在48、72和96小时培养时显著升高,尤其是在50μM的4-HNE浓度下,96小时时低氧和常氧hLEC活力为对照组细胞的(4.75±0.45)%和(2.17±0.52)%(P=0.008)。流式细胞术检测发现50μM 4-HNE处理后缺氧条件下hLEC的存活率(39.92±5.31)%,明显高于常氧的(10.67±3.22)%(P=0.003);早期和晚期细胞凋亡率均明显低于常氧(33.83±4.32)%和(48.81±4.99)%(P=0.015)、(13.85±2.47)%和(21.52±3.62)%(P=0.007);缺氧条件下的RROS水平显著低于常氧(28.85±5.77)%和(63.44±8.73)%(P=0.009);衰老相关β-半乳糖苷酶阳性率(35.31±4.13)%显著低于常氧的(76.4±4.21)%(P=0.004),而BrdU掺入阳性率(6.55±1.74)%显著高于常氧的(3.20±0.93)%(P=0.021);蛋白印迹法显示,4-HNE处理可激活衰老主要调控因子P53和P16表达。但与常氧相比,低氧组中促衰老基因P16和P53蛋白相对表达量上升程度显著低于常氧组(P=0.031,P=0.005)。结论缺氧可以抑制4-HNE诱导氧化应激引起的hLEC凋亡、ROS和衰老水平,对hLEC产生保护作用。Objective To analyze the protection of hypoxia on the aging of human lens epithelial cells(hLECs)induced by 4-hydroxynonaenoic acid(4-HNE).Design Experimental research.Participants Hypoxia and hLECs.Methods hLECs were treated with 4-HNE at different O2 concentrations and their proliferative activity were detected by CCK-8.The apoptosis levels,reactive oxygen species(ROS)and senescence of hLECs in hypoxic and normoxic condition were analyzed by Annexin-V/PI staining,fluorescent probe(DCFH-DA),β-galactosidase staining and BrdU combined assay respectively.Main Outcome Measures Proliferation activity,apoptosis,senescence and expression level of related proteins of hLECs.Results By compared with normal oxygen concentration,the cell viability of 1%hypoxia was significantly higher than that of 21%normoxia at 48 h,72 h and 96 h,especially at 50μM 4-HNE concentration at 96 h(4.75±0.45%and 2.17±0.52%,P=0.008).The survival rate of hLECs under hypoxia was significantly higher than that under normoxia(39.92±5.31%and 10.67±3.22%at 50μM 4-HNE,P=0.003)detected by flow cytometry.The early and late apoptosis rates were significantly lower than those of normoxia(33.83±4.32%and 48.81±4.99%at 50μM 4-HNE,P=0.015;13.85±2.47%and 21.52±3.62%at 50μM 4-HNE,P=0.007).The level of reactive oxygen species decreased significantly under hypoxia,which was significantly lower than that of normal oxygen(63.44±8.73%and 28.85±5.77%at 50μM 4-HNE,P=0.009).Aging relatedβ-galactosidase positive rate was significantly lower than those of normoxia(35.31±4.13%and 76.4±4.21%at 50μM 4-HNE,P=0.004)and the positive of BrdU incorporation significantly higher than those of normoxia(6.55±1.74%and 3.20±0.93%at 50μM 4-HNE,P=0.021).Western blot results showed that 4-HNE treatment could activate the expression of P53 and P16,the main regulators of aging.However,compared with the hypoxia group,the relative expression of P16 and P53 protein in the normoxia group was significantly higher than that in the hypoxia group(P=0.031,0.005).Conclusion Hypo

关 键 词：低氧 晶状体上皮细胞 4-羟基壬烯酸 衰老 氧化应激 

分 类 号：R776.1[医药卫生—眼科]