芒果苷对心肌细胞损伤的保护作用及对NFATc4表达的影响

Protective effects of mangiferin against myocardial cell damage and its effects on the expression of NFATc4

目的研究芒果苷对过氧化氢(H_(2)O_(2))诱导的心肌细胞氧化应激损伤的保护作用,以及对活化T细胞核因子c4(NFATc4)表达的影响。方法体外培养心肌H9c2细胞,分为空白组、H_(2)O_(2)组和芒果苷50、100、150μmol/L组,芒果苷组细胞在经不同浓度芒果苷作用12 h后,再与H_(2)O_(2)组一同经H_(2)O_(2)(200μmol/L)刺激12 h,检测各组细胞的相对存活率,细胞上清液中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、丙二醛(MDA)水平,细胞中活性氧(ROS)水平,以及细胞中凋亡相关蛋白[B细胞淋巴瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)、剪切的胱天蛋白酶3(cleaved caspase-3)]、核蛋白NFATc4的表达情况。转染NFATc4干扰序列,考察NFATc4对H_(2)O_(2)诱导的心肌细胞中氧化应激指标和凋亡相关蛋白的影响。结果与空白组比较,H_(2)O_(2)组细胞的相对存活率,SOD、CAT水平,Bcl-2的相对表达量均显著降低;MDA、ROS水平,Bax、cleaved caspase-3和核蛋白NFATc4的相对表达量均显著升高(P<0.05或P<0.01)。与H_(2)O_(2)组比较,芒果苷100、150μmol/L组细胞的上述指标均得以显著逆转(P<0.05)。转染NFATc4干扰序列后,核蛋白NFATc4的表达显著下调,MDA、ROS水平和Bax、cleaved caspase-3蛋白的表达均较H_(2)O_(2)组显著降低/下调,SOD、CAT水平和Bcl-2蛋白的表达均较H_(2)O_(2)组显著升高/上调(P<0.05)。结论芒果苷能够减轻H_(2)O_(2)诱导的H9c2细胞氧化应激,减少细胞凋亡,抑制NFATc4蛋白入核,进而缓解心肌细胞损伤;降低NFATc4蛋白的核内水平与减轻H_(2)O_(2)诱导的氧化应激、细胞凋亡有关。

OBJECTIVE To study the protective effects of mangiferin against oxidative stress injury of myocardial cells induced by hydrogen peroxide(H_(2)O_(2)),and its effects on the expression of nuclear factor of activated T cell cytoplasmic 4(NFATc4).METHODS H9c2 myocardial cells were cultured in vitro and divided into blank group,H_(2)O_(2)group,and 50,100,150μmol/L mangiferin groups.Mangiferin groups were treated with different concentrations of mangiferin for 12 h,and then were subjected to H_(2)O_(2)(200μmol/L)stimulation for 12 hours together with the H_(2)O_(2)group;relative survival rate was detected in each group,and the levels of superoxide dismutase(SOD),catalase(CAT)and malondialdehyde(MDA)in cell supernatant and reactive oxygen species(ROS)in H9c2 cells were measured.Meanwhile,the expressions of apoptosis-related proteins[B cell lymphoma 2(Bcl-2),Bcl-2 associated X protein(Bax),cleaved caspase-3]and nuclear protein NFATc4 were determined.Furthermore,the NFATc4 interference sequence was transfected,and the effects of NFATc4 on oxidant stress indexes and apoptosis-related proteins in H_(2)O_(2)-induced myocardial cells were investigated.RESULTS Compared with blank group,relative cell viability,the levels of SOD and CAT,relative expression of Bcl-2 were decreased significantly,while the levels of MDA and ROS,relative expressions of Bax,cleaved caspase-3 and nuclear protein NFATc4 were increased significantly(P<0.05 or P<0.01).Compared with the H_(2)O_(2)group,the above indexes of 100 and 150μmol/L mangiferin groups were reversed significantly(P<0.05).After the transfection of the NFATc4 interference sequence,the expression of nuclear protein NFATc4 was down-regulated significantly;the levels of MDA and SOD,the protein expressions of Bax and cleaved caspase-3 were all decreased/down-regulated significantly,while the levels of SOD and CAT,and the protein expression of Bcl-2 were all increased/up-regulated significantly,compared with H_(2)O_(2)group(P<0.05).CONCLUSIONS Mangiferin can relieve H_(2)O_(2)-induced oxidative stress of H9c2 cells,reduce the apoptosis and inhibit the nuclear translocation of NFATc4,thereby alleviating myocardial cell damage;reducing the nuclear level of NFATc4 protein is related to reducing H_(2)O_(2)-induced oxidative stress and cell apoptosis.