抗氧化酶抑制剂对肿瘤坏死因子-α诱导HepG2细胞凋亡的影响

Effects of antioxidant enzyme inhibitors on apoptosis in HepG2 cells induced by TNF-α

目的探索抗氧化酶抑制剂对肿瘤坏死因子-α(TNF-α)诱导人肝癌HepG2细胞内氧化损伤以及对细胞凋亡发生的影响。方法将处于对数生长期的HepG2细胞悬液以50μg/L重组人TNF-α诱导细胞凋亡8 h,建立细胞凋亡模型。将细胞分为正常对照组、凋亡模型组和超氧化物歧化酶(SOD)抑制[2 mmol/L二乙基二硫代氨基甲酸(DETC)]组、过氧化氢酶(CAT)抑制[10 mmol/L氨基三唑(ATZ)]组、谷胱甘肽过氧化物酶(GPx)抑制[0.1 mmol/L青霉胺(PEN)]组、CAT和GPx共同抑制组,暴露2 h后,检测细胞内SOD、CAT和GPx的活力;暴露4 h后,测定细胞内过氧化氢和还原型谷胱甘肽(GSH)的含量,超氧阴离子和羟自由基的产生能力;暴露12 h后,检测细胞存活率、凋亡率和死亡率。结果凋亡模型组细胞内SOD、CAT、GPx活力及产生超氧阴离子、羟自由基能力和过氧化氢、GSH的含量与正常对照组相比,差异均无统计学意义(P>0.05)。与正常对照组和凋亡模型组相比,各抑制组细胞内相应抗氧化酶活力均下降,差异有统计学意义(P<0.05)。与正常对照组和凋亡模型组相比,SOD抑制组及CAT和GPx共同抑制组细胞产生超氧阴离子水平以及CAT和GPx共同抑制组细胞产生羟自由基水平均升高,而SOD抑制组细胞产生羟自由基水平下降,差异均有统计学意义(P<0.05);此外,与正常对照组和凋亡模型组相比,SOD抑制组细胞内过氧化氢含量和CAT抑制组细胞内GSH含量均降低,而CAT抑制组、GPx抑制组、CAT和GPx共同抑制组细胞内过氧化氢含量和GPx抑制组、CAT和GPx共同抑制组细胞内GSH含量均升高,差异均有统计学意义(P<0.05)。与正常对照组相比,其他各组细胞的存活率均降低,凋亡率和死亡率均升高,除凋亡模型组和SOD抑制组细胞死亡率外,差异均有统计学意义(P<0.05)。与凋亡模型组相比,各抑制组细胞存活率降低,SOD抑制组、CAT和GPx共同抑制组细胞凋亡率升高,CAT抑制组、GPx抑制组、CAT和GPx共同抑制组细胞死亡率也升高,差异均有统计学意义(P<0.05)。结论抗氧化酶抑制剂可通过抑制细胞内抗氧化酶活力破坏细胞内氧化还原平衡,对细胞凋亡的发生造成影响。细胞内氧化还原系统中超氧阴离子可能是促进细胞凋亡发生的关键分子,过氧化氢及其进一步产生的羟自由基可能与细胞死亡密切相关。

Objective To explore the effects of antioxidant enzyme inhibitors on the intracellular oxidative damage and the occurrence of apoptosis in human hepatoma HepG2 cells induced by tumor necrosis factor-α(TNF-α). Methods Cell apoptotic models were established using the methods that HepG2 cells in logarithmic growth phase were induced by 50 μg/L recombinant human TNF-α for 8 h. HepG2 cells were divided into six groups including normal control group, apoptotic model group,superoxide dismutase(SOD) inhibition [2 mmol/L diethyldithiocarbamic acid(DETC)] group, catalase(CAT) inhibition [10 mmol/L aminotriazole(ATZ)] group, glutathione peroxidase(GPx) inhibition [0.1 mmol/L penicillamine(PEN)] group, CAT and GPx coinhibition group. The activity of SOD, CAT and GPx intracellular were detected using the kits after the cells were treated for 2 h; The content of hydrogen peroxide and reducing glutathione(GSH), production ability of superoxide anion and hydroxyl radical were detected using the kits after the cells were treated for 4 h; The survival rate, apoptosis rate and mortality rate of cells were detected using flow cytometry after the cells were treated for 12 h. Results Compared with the normal control group,the activities of SOD,CAT and GPx, production ability of superoxide anion and hydroxyl radical, the content of hydrogen peroxide and GSH did not change significantly in apoptotic model group(P>0.05). Compared with the normal control group and apoptotic model group, the activities of antioxidant enzymes correspondingly decreased in each inhibition group(P <0.05).Compared with the normal control group and apoptotic model group, the production ability of superoxide anion increased in SOD inhibition group, the production ability of superoxide anion and hydroxyl radical increased in CAT and GPx co-inhibition group, but the production ability of hydroxyl radical decreased in SOD inhibition group(P<0.05). In addition, compared with the normal control group and apoptotic model group, the hydrogen peroxide content decreased in SOD inhibition group, the GSH content decreased in CAT inhibition group,on the contrary, the hydrogen peroxide content increased in CAT inhibition group,GPx inhibition group, CAT and GPx co-inhibition group, and the GSH content increased in GPx inhibition group, CAT and GPx co-inhibition group(P<0.05). Compared with the normal control group, the cell survival rate decreased, the cell apoptosis rate and the cell mortality rate significantly increased in other groups except for the cell mortality rate in apoptotic model group and SOD inhibition group(P<0.05). Compared with the apoptotic model group, the cell survival rate decreased in each inhibition group, the cell apoptosis rate increased in SOD inhibition group, CAT and GPx co-inhibition group, the cell mortality rate increased in CAT inhibition group, GPx inhibition group, CAT and GPx co-inhibition group(P<0.05). Conclusion Antioxidant enzyme inhibitors can destroy the intracellular redox balance by inhibiting the activity of intracellular antioxidant enzymes, then affect the occurrence of apoptosis. It is prompted that superoxide anion might be the key molecule to promote cell apoptosis,while hydrogen peroxide and its further product,hydroxyl radical, may be more relevant to cell death in an intracellular redox system.