| POLH对过氧化氢诱导的晶状体上皮细胞氧化应激和凋亡的影响 |
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| 引用本文： | 李鹏飞,王从玉,王思文,鲍思洁,孙诚浩,管怀进.POLH对过氧化氢诱导的晶状体上皮细胞氧化应激和凋亡的影响[J].眼科新进展,2022,0(6):427-431. |
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| 作者姓名： | 李鹏飞 王从玉 王思文 鲍思洁 孙诚浩 管怀进 |
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| 作者单位： | 226001 江苏省南通市，南通大学附属医院眼科 |
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| 摘 要： | 目的 探究DNA聚合酶η（POLH）对过氧化氢（H2O2）诱导的晶状体上皮细胞氧化应激和凋亡的防御机制。方法 以年龄相关性白内障患者的晶状体前囊膜组织和H2O2处理的晶状体上皮细胞系SRA01/04为研究对象，分别通过RT-PCR和免疫印迹实验验证POLH的表达并筛选H2O2最佳作用浓度用于后续实验，将培养的SRA01/04细胞分为H2O2组、H2O2+HA组和H2O2+OV-POLH组，采用RT-PCR和免疫印迹实验检测POLH mRNA和蛋白表达情况，免疫荧光法检测DNA氧化损伤标志物以及TUNEL实验检测细胞凋亡变化，免疫印迹实验检测细胞中BAX、BCL-2、Nrf2和Keap1蛋白表达。结果 RT-PCR结果显示，与透明晶状体前囊膜（1.00±0.22）相比，年龄相关性白内障患者晶状体前囊膜组织中POLH mRNA相对表达量明显下降（0.38±0.62）（P<0.000 1）。RT-PCR和免疫印迹实验均显示，200 μmol·L-1 H2O2处理的SRA01/04细胞中POLH表达量最低；TUNEL实验检测结果显示， 200 μmol·L-1 H2O2处理后细胞的凋亡明显增加。RT-PCR和免疫印迹实验结果显示，与H2O2组和H2O2+HA组相比，H2O2+OV-POLH组SRA01/04细胞中POLH表达量显著升高（P<0.000 1）。免疫荧光染色结果显示，与H2O2+HA组相比，H2O2+OV-POLH组细胞的γH2A染色明显下降。免疫印迹实验检测结果显示，与H2O2组和H2O2+HA组相比，H2O2+OV-POLH组细胞促凋亡蛋白BAX表达量明显下降，而抑凋亡蛋白BCL-2表达量明显升高（均为P<0.01）；与H2O2组和H2O2+HA组相比，H2O2+OV-POLH组细胞Keap1蛋白水平明显下降，而转录调控蛋白Nrf2表达明显升高（均为P<0.01）。结论 过表达POLH可通过增强DNA损伤修复功能抑制H2O2诱导的晶状体上皮细胞凋亡，其抑制氧化应激机制可能与Nrf2-Keap1-ARE通路有关。
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| 关 键 词： | 年龄相关性白内障 晶状体上皮细胞 氧化应激 DNA聚合酶η |
Effects of DNA polymerase η on H2O2-induced oxidative stress and apoptosis in human lens epithelial cells |
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| LI Pengfei,WANG Congyu,WANG Siwen,BAO Sijie,SUN Chenghao,GUAN Huaijin.Effects of DNA polymerase η on H2O2-induced oxidative stress and apoptosis in human lens epithelial cells[J].Recent Advances in Ophthalmology,2022,0(6):427-431. |
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| Authors: | LI Pengfei WANG Congyu WANG Siwen BAO Sijie SUN Chenghao GUAN Huaijin |
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| Affiliation: | Department of Ophthalmology, the Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu Province, China |
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| Abstract: | Objective To investigate the defense mechanism of DNA polymerase η (POLH) on H2O2-induced oxidative stress and apoptosis in human lens epithelial (HLE) cells. Methods The anterior capsule tissues and H2O2-treated HLE cells (SRA01/04) of patients with age-related cataract (ARC) were selected for the study. The expression of POLH was verified by real-time polymerase chain reaction (RT-PCR) and Western blot. The optimum concentration of H2O2 was screened for subsequent experiments. The cultured SRA01/04 cells were divided into the H2O2 group, H2O2+HA group, and H2O2+OV-POLH group. The mRNA and protein levels of POLH were detected by RT-PCR and Western blot, the DNA oxidative damage markers were detected by immunofluorescence assay, the cell apoptosis was detected by TUNEL assay, and the expression levels of Bcl-2-associated X protein (BAX), B-cell lymphoma-2 (BCL-2), nuclear factor erythroid 2-related factor 2 (Nrf2), and Kelch-like ECH-associated protein 1 (Keap1) in cells were measured by Western blot. Results RT-PCR results showed that the relative expression of POLH mRNA was significantly decreased in the ARC patients (0.38±0.62) compared with that in the normal patients (1.00±0.22) (P<0.000 1). RT-PCR and Western blot results showed that the expression of POLH in SRA01/04 cells treated with 200 μmol·L-1 H2O2 was the lowest. TUNEL results showed that apoptosis of cells treated with 200 μmol·L-1 H2O2 significantly increased. RT-PCR and Western blot results also showed that POLH expression was significantly higher in the H2O2+OV-POLH group than that in the H2O2 group and the H2O2+HA group (both P<0.000 1). Immunofluorescence results showed that compared with the H2O2+HA group, γH2A staining was significantly decreased in the H2O2+OV-POLH group. Western blot results showed that compared with the H2O2 group and the H2O2+HA group, BAX and Keap1 expression levels were significantly decreased, while BCL-2 and Nrf2 expression levels were significantly increased in the H2O2+OV-POLH group (all P<0.01). Conclusion Overexpressed POLH can enhance DNA damage repair and inhibit H2O2-induced apoptosis of HLE cells. The mechanism of inhibiting oxidative stress may be related to the Nrf2-Keap1-ARE pathway. |
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| Keywords: | age-related cataract lens epithelial cells oxidative stress DNA polymerase η |
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