Fan Hui-Jie, Tan Zhang-Bin, Wu Yu-Ting, Feng Xiao-Reng, Bi Yi-Ming, Xie Ling-Peng, Zhang Wen-Tong, Ming Zhi, Liu Bin, Zhou Ying-Chun
School of Traditional Chinese Medicine, Southern Medical University, Guangzhou, China.
Department of Traditional Chinese Medicine, The first hospital of Yangjiang, Yangjiang, China.
J Ginseng Res. 2020 Mar;44(2):258-266. doi: 10.1016/j.jgr.2018.12.004. Epub 2018 Dec 16.
Oxidative stress-induced cardiomyocytes apoptosis is a key pathological process in ischemic heart disease. Glutathione reductase (GR) reduces glutathione disulfide to glutathione (GSH) to alleviate oxidative stress. Ginsenoside Rb1 (GRb1) prevents the apoptosis of cardiomyocytes; however, the role of GR in this process is unclear. Therefore, the effects of GRb1 on GR were investigated in this study.
The antiapoptotic effects of GRb1 were evaluated in H9C2 cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, annexin V/propidium iodide staining, and Western blotting. The antioxidative effects were measured by a reactive oxygen species assay, and GSH levels and GR activity were examined in the presence and absence of the GR inhibitor 1,3-bis-(2-chloroethyl)-1-nitrosourea. Molecular docking and molecular dynamics simulations were used to investigate the binding of GRb1 to GR. The direct influence of GRb1 on GR was confirmed by recombinant human GR protein.
GRb1 pretreatment caused dose-dependent inhibition of tert-butyl hydroperoxide-induced cell apoptosis, at a level comparable to that of the positive control N-acetyl-L-cysteine. The binding energy between GRb1 and GR was positive (-6.426 kcal/mol), and the binding was stable. GRb1 significantly reduced reactive oxygen species production and increased GSH level and GR activity without altering GR protein expression in H9C2 cells. Moreover, GRb1 enhanced the recombinant human GR protein activity , with a half-maximal effective concentration of ≈2.317 μM. Conversely, 1,3-bis-(2-chloroethyl)-1-nitrosourea co-treatment significantly abolished the GRb1's apoptotic and antioxidative effects of GRb1 in H9C2 cells.
GRb1 is a potential natural GR agonist that protects against oxidative stress-induced apoptosis of H9C2 cells.
氧化应激诱导的心肌细胞凋亡是缺血性心脏病的关键病理过程。谷胱甘肽还原酶（GR）将谷胱甘肽二硫化物还原为谷胱甘肽（GSH）以减轻氧化应激。人参皂苷Rb1（GRb1）可防止心肌细胞凋亡；然而，GR在此过程中的作用尚不清楚。因此，本研究探讨了GRb1对GR的影响。
通过3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐、膜联蛋白V/碘化丙啶染色和蛋白质免疫印迹法评估GRb1在H9C2细胞中的抗凋亡作用。通过活性氧测定法测量抗氧化作用，并在存在和不存在GR抑制剂1,3-双(2-氯乙基)-1-亚硝基脲的情况下检测GSH水平和GR活性。使用分子对接和分子动力学模拟研究GRb1与GR的结合。通过重组人GR蛋白证实GRb1对GR的直接影响。
GRb1预处理导致剂量依赖性抑制叔丁基过氧化氢诱导的细胞凋亡，其水平与阳性对照N-乙酰-L-半胱氨酸相当。GRb1与GR之间的结合能为正值（-6.426千卡/摩尔），且结合稳定。GRb1显著降低H9C2细胞中的活性氧产生，提高GSH水平和GR活性，而不改变GR蛋白表达。此外，GRb1增强重组人GR蛋白活性，半数最大有效浓度约为2.317μM。相反，1,3-双(2-氯乙基)-1-亚硝基脲共同处理显著消除了GRb1在H9C2细胞中的凋亡和抗氧化作用。
GRb1是一种潜在的天然GR激动剂，可保护H9C2细胞免受氧化应激诱导的凋亡。
