[1]李鹏飞,王从玉,王思文,等.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,2022,42(6):427-431.[doi:10.13389/j.cnki.rao.2022.0087]
 LI Pengfei,WANG Congyu,WANG Siwen,et al.Effects of DNA polymerase η on H2O2-induced oxidative stress and apoptosis in human lens epithelial cells[J].Recent Advances in Ophthalmology,2022,42(6):427-431.[doi:10.13389/j.cnki.rao.2022.0087]
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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/HTML
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
42卷
期数:
2022年6期
页码:
427-431
栏目:
实验研究
出版日期:
2022-06-05

文章信息/Info

Title:
Effects of DNA polymerase η on H2O2-induced oxidative stress and apoptosis in human lens epithelial cells
作者:
李鹏飞王从玉王思文鲍思洁孙诚浩管怀进
226001 江苏省南通市,南通大学附属医院眼科
Author(s):
LI Pengfei WANG Congyu WANG Siwen BAO Sijie SUN Chenghao GUAN Huaijin
Department of Ophthalmology, the Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu Province, China
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
Keywords:
age-related cataract lens epithelial cells oxidative stress DNA polymerase η
分类号:
R776
DOI:
10.13389/j.cnki.rao.2022.0087
文献标志码:
A
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。
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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国家自然科学基金面上项目(编号:82171038,81974129)
更新日期/Last Update: 2022-06-05