眼科 ›› 2023, Vol. 32 ›› Issue (4): 344-350.doi: 10.13281/j.cnki.issn.1004-4469.2023.04.014

• 论著 • 上一篇    下一篇

缺氧对氧化应激诱导人晶状体上皮细胞衰老的保护作用

侯芳1  黄启明2，3  万修华1   

  1. 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
  • 收稿日期:2022-12-05 出版日期:2023-07-25 发布日期:2023-07-25
  • 通讯作者: 万修华， Email： wanxh@ccmu.edu.cn
  • 基金资助:
    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）

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

Hou Fang1, Huang Qiming2, 3, Wan Xiuhua1   

  1. 1 Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University; Beijing Key Laboratory of Ophthalmology and Visual Science, Beijing 100730, China; 2 School of Life and Science, Nanchang University, Nangchang 330031, China; 3 Jiangxi Mayobio Technology Company Ltd., Nanchang 330006, China
  • Received:2022-12-05 Online:2023-07-25 Published:2023-07-25
  • Contact: Wan Xiuhua, Email: wanxh@ccmu.edu.cn
  • Supported by:
    The Training Plan for Academic and Technological Leaders of the Main Subject, Jiangxi Province (20204BCJ22035); Key Research & Development Project of Science and Technology Plan of Department of Science and Technology, Jiangxi Province (20192ACB70004, 20201BBG71007); General Project of Science and Technology Plan of Department of Science and Technology, Jiangxi Province (20192BBH80015); National Natural Science Foundation of China (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50 μM）4-HNE处理的hLEC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50 μM 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50 μM 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ROS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。（眼科，2023，32： 344-350）

关键词: 低氧, 晶状体上皮细胞, 4-羟基壬烯酸, 衰老, 氧化应激

Abstract:  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 50 μM 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 50 μM 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 50 μM 4-HNE, P=0.015; 13.85±2.47% and 21.52±3.62% at 50 μM 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 50 μM 4-HNE, P=0.009). Aging related β-galactosidase positive rate was significantly lower than those of normoxia (35.31±4.13% and 76.4±4.21% at 50 μM 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 50 μM 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 Hypoxia can promote the proliferation of hLECs, inhibit the apoptosis. ROS and aging level of hLECs caused by oxidative stress, and protect hLECs. (Ophthalmol CHN, 2023, 32: 344-350)

Key words: hypoxia, lens epithelial cells, 4-hydroxynonenoic acid, aging, oxidative stress

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