南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (5): 673-680.doi: 10.12122/j.issn.1673-4254.2022.05.07

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金丝桃苷通过激活Keap1/Nrf2/HO-1通路保护小鼠GC-2细胞的氧化损伤

朱妍妍，王桐生，戴 宁，邓梦云，刘红娟，童小慧，李 莉   

  1. 安徽中医药大学中西医结合学院药理学教研室，中药复方安徽省重点实验室，安徽 合肥 230012；安徽中医药大学第一附属医院男科，安徽 合肥 230601
  • 出版日期:2022-05-20 发布日期:2022-06-02

Hyperoside protects mouse spermatocytes GC-2 cells from oxidative damage by activating the Keap1/Nrf2/HO-1 pathway

ZHU Yanyan, WANG Tongsheng, DAI Ning, DENG Mengyun, LIU Hongjuan, TONG Xiaohui, LI Li   

  1. Department of Pharmacology, College of Integrated Chinese and Western Medicine, Anhui University of Chinese Medicine, Hefei 230012, China; Anhui Provincial Key Laboratory of Traditional Chinese Medicine Compounds, Hefei 230012, China; Department of Andrology, First Affiliated Hospital of Anhui University of Chinese Medicine, Hefei 230601, China
  • Online:2022-05-20 Published:2022-06-02

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摘要： 目的 从Keap1/Nrf2/HO-1通路角度研究金丝桃苷（Hyp）对H2O2所诱导的小鼠精母细胞（GC-2）氧化损伤的保护作用。方法 将GC-2细胞随机分为正常组、模型组、氮乙酰半胱氨酸组（阳性组，NAC，2.5 mmol/L)以及金丝桃苷组（50、100、200 μmol/L），各组按剂量孵育48 h后，除正常组外其余各组细胞加入H2O（2 150 μmol/L）处理2 h，正常组给予等容量培养基。采用CCK-8法检测细胞的活力，流式细胞仪检测细胞凋亡水平，酶联免疫法检测超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-PX）、过氧化氢酶（CAT）活力以及丙二醛（MDA）含量，Western blot和RT-qPCR检测Nrf2、Keap1、HO-1蛋白及mRNA的相对表达水平，免疫荧光法检测Nrf2核转位水平。结果 150 μmol/L H2O2干预2 h可制备GC-2细胞氧化损伤模型。与正常组比较，模型组GC-2细胞增殖率以及SOD、GSH、CAT活力显著降低，MDA含量、细胞凋亡率，Keap1和Nrf2 mRNA表达显著升高（P<0.05）以及Keap1蛋白表达水平显著升高（P<0.01）。与模型组比较，NAC组和金丝桃苷200 μmol/L组细胞增殖率和SOD、GSH-PX、CAT活力显著升高，MDA含量、细胞凋亡率显著下降（P<0.05）；金丝桃苷200 μmol/L组Keap1 mRNA表达显著下降，HO-1 mRNA表达显著升高（P<0.01）；金丝桃苷200 μmol/L组Nrf2核蛋白（NEs-Nrf2）以及HO-1的蛋白表达显著升高（P<0.05），Keap1蛋白表达显著下降（P<0.01）；金丝桃苷组出现了明显的核转位现象（P<0.05）。结论 金丝桃苷可能通过激活Keap1/Nrf2/HO-1信号通路，对H2O2诱导的GC-2细胞氧化损伤具有保护作用。

关键词: 金丝桃苷；氧化应激；Nrf2；HO-1；Keap1；GC-2细胞

Abstract: Objective To study the protective effect of hyperoside (Hyp) against ydrogen peroxide (H2O2)- induced oxidative damage in mouse spermatocytes GC-2 cells and explore the role of the Keap1/Nrf2/HO-1 pathway in this protective mechanism. Methods GC-2 cells were treated with 2.5 mmol/L azaacetylcysteine (NAC), 50, 100, and 200 μmol/L hyperoside, or the culture medium for 48 h before exposure to H2O2 (150 μmol/L) for 2 h. CCK-8 assay was used to detect the changes in cell viability, and cell apoptosis was analyzed using flow cytometry. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), catalase (CAT) activity and malondialdehyde (MDA) in the culture medium. Western blotting and RT-qPCR were used to detect the protein and mRNA expression levels of nuclear factor erythroid 2-related factor2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap1), and heme oxygenase-1 (HO-1); the nuclear translocation of Nrf2 was detected using immunofluorescence assay. Results Exposure to H2O2 significantly lowered the proliferation rate, reduced the activities of SOD, GSH and CAT, and obviously increased MDA content, cell apoptosis rate, and the expressions of Keap1 and Nrf2 mRNA and Keap1 protein in GC-2 cells (P<0.05 or 0.01). Treatment of the cells prior to H2O2 exposure with either NAC or 200 μmol/L hyperoside significantly increased the cell proliferation rate, enhanced the activities of SOD, GSH-PX and CAT, and lowered MDA content and cell apoptosis rate (P<0.05). Treatment with 200 μmol/L hyperoside significantly decreased the mRNA and protein expressions of Keap1 and increased the expressions of HO-1 mRNA and the protein expressions of Nrf2 and HO-1 (P<0.05 or 0.01). Hyperoside also caused obvious nuclear translocation of Nrf2 in the cells (P<0.05). Conclusion Hyperoside protects GC-2 cells against H2O2-induced oxidative damage possibly by activation of the Keap1/Nrf2/HO-1 signaling pathway.

Key words: hyperoside; oxidative stress; Nrf2; HO-1; Keap1; GC-2 cells