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为探索大豆异黄酮对过氧化氢(H_2O_2)致肝细胞损伤的保护作用,以H_2O_2损伤Chang Liver细胞建立肝细胞氧化应激损伤模型,并以10,20和40 mg·L~(-1)大豆异黄酮进行干预。采用四甲基偶氮唑盐比色(MTT)法检测肝细胞存活率;以微板法检测细胞培养液中乳酸脱氢酶(LDH)、谷丙转氨酶(ALT)、谷草转氨酶(AST)活性和肝细胞超氧化物歧化酶(SOD)活性,以及肝细胞还原型谷胱甘肽(GSH)和丙二醛(MDA)含量;采用蛋白印迹技术检测肝细胞核因子-E2相关因子2(Nrf2)蛋白含量。在10~40 mg·L~(-1)范围内,大豆异黄酮对Chang Liver细胞不显示细胞毒作用。300μmol·L~(-1)H_2O_2刺激后,模型组肝细胞存活率与正常组比较下降,细胞外液中ALT、AST、LDH水平上升,说明Chang Liver细胞损伤显著;而模型组肝细胞中SOD和GSH水平与正常组比较下降,丙二醛水平上升,说明模型组Chang Liver细胞氧化应激增强。大豆异黄酮可剂量依赖性地提高Chang Liver细胞存活率,降低ALT、AST、LDH向细胞外液的释放;降低细胞MDA水平,升高细胞SOD和GSH水平,增高核中Nrf2蛋白水平。研究结果显示大豆异黄酮对H_2O_2致肝细胞氧化应激损伤具有保护作用。
In order to explore the protective effect of soy isoflavones on hepatocytes induced by hydrogen peroxide (H 2 O 2), a model of hepatocellular oxidative stress injury was established by H 2 O 2 -treated Chang-liver cells and treated with 10, 20 and 40 mg · L -1 of H 2 O 2, Soy isoflavones for intervention. The survival rate of hepatocytes was detected by MTT assay. The activities of lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in cell culture medium were detected by microplate method. (SOD) and hepatic reduced glutathione (GSH) and malondialdehyde (MDA) were detected by Western blotting. Western blotting was used to detect the expression of nuclear factor-E2-related factor 2 (Nrf2) Protein content. In the range of 10-40 mg · L -1, soy isoflavones showed no cytotoxic effect on Chang Liver cells. After stimulated with 300μmol·L -1 H 2 O 2, the survival rate of hepatocytes in model group decreased compared with that in normal group, and the levels of ALT, AST and LDH in extracellular fluid increased, indicating that Chang liver cell injury was significant; while in model group, And GSH levels decreased compared with the normal group, MDA levels increased, indicating that the model group Chang Liver cells increased oxidative stress. Soy isoflavones can increase the survival rate of Chang liver cells in a dose-dependent manner, reduce the release of ALT, AST and LDH to the extracellular fluid, decrease the level of MDA, increase the level of SOD and GSH, and increase the level of Nrf2 in the nucleus. The results showed that soy isoflavones have a protective effect on H 2 O 2 induced oxidative stress injury in hepatocytes.