过氧化氢对NIH/3T3细胞蛋白磷酸酶2A催化亚基C甲基化的影响  

Effect of hydrogen peroxide on protein phosphatase 2A catalytic subunit C methylation in NIH/3T3 cells

作　　者：刘银品 唐深[2] 陆彩玲[1] 秦富[1] 孙斌[1] 许扬[1] 李习艺[1] 

机构地区：[1]广西医科大学公共卫生学院营养与食品卫生教研室,广西南宁530021 [2]广西医科大学基础医学院,广西南宁530021

出　　处：《中国药理学与毒理学杂志》2015年第2期272-276,共5页Chinese Journal of Pharmacology and Toxicology

基　　金：国家自然科学基金(81360428);广西壮族自治区自然科学基金(2014GXNSFAA118188)~~

摘　　要：目的探讨过氧化氢(H2O2)对NIH/3T3细胞中蛋白磷酸酶2A催化亚基C(PP2Ac)甲基化的影响。方法 1 H2O20,0.1,1,5,10和25 mmol·L-1刺激NIH/3T3细胞1 h,刃天青还原率检测细胞活性;2 Western蛋白印迹法检测H2O20,0.1,1和5 mmol·L-1作用1 h后细胞内去甲基PP2Ac的表达;3分别用羧基端甲基酯酶(PME-1)抑制剂AMZ30 0,0.1,0.5,1.0,5.0和10.0μmol·L-1和抗氧化剂N-乙酰半胱氨酸(NAC)0,0.5,1和5 mmol·L-1预处理细胞30 min,再用H2O21 mmol·L-1作用细胞1 h,Western蛋白印迹法检测细胞内去甲基PP2Ac的表达;4免疫荧光实验观察NIH/3T3细胞H2O21 mmol·L-1处理1 h、AMZ30 10.0μmol·L-1或NAC 1 mmol·L-1分别预处理30 min后再加H2O21 mmol·L-1处理1 h细胞内去甲基PP2Ac的表达。结果 1刃天青检测实验结果显示,H2O2对细胞增殖具有显著的抑制作用,浓度≥0.1 mmol·L-1细胞活性显著降低(P<0.01);2 Western蛋白印迹检测结果显示,H2O2可致NIH/3T3细胞内去甲基PP2Ac蛋白表达升高,H2O20.1,1和5 mmol·L-1组与空白对照组相比差异有统计学意义(P<0.01);3 Western蛋白印迹结果显示,AMZ30与NAC二者均可拮抗H2O2对细胞内PP2Ac去甲基的作用,与H2O21 mmol·L-1组相比,加入AMZ30 5.0和10.0μmol·L-1或NAC 1和5 mmol·L-1细胞内去甲基PP2Ac含量明显降低(P<0.01);4免疫荧光实验结果显示,加入H2O21 mmol·L-1刺激1 h后,细胞内去甲基PP2Ac的表达要高于正常细胞组,而用AMZ30 10.0μmol·L-1与NAC 1 mmol·L-1干预后,去甲基PP2Ac表达降低。结论 H2O2可诱导NIH/3T3细胞内PP2Ac去甲基,而PME-1抑制剂AMZ30和抗氧化剂NAC均能拮抗H2O2诱导的PP2Ac去甲基作用。OBJECTIVE To investigate the effect of hydrogen peroxide（H2 O2 ） on protein phospha-tase 2A catalytic subunit C（PP2Ac） methylation. METHODS After H2O2 0, 0.1, 1, 5, 10 and 25 mmol.L-1 treatment of NlH/ 3T3 cells for 1 h, the rate of resazurin reduction was observed to determine the cell proliferation while Western blotting was used to detect the expression of un-methylated PP2Ac. PP2A methylesterase （PME-1） inhibitor AMZ30 0, 0.1, 0.5, 1, 5 and 10 μmol.L-1 and antioxidant NAC 0, 0.5, 1 and 5 mmol.L-1 were used to intervene in the effect of H2 O2 1 mmol.L-1 on NlH/ 3T3 cells. After various treatments, the expression of un-methylated PP2Ac was detected by Western blotting and indirect immunofluorescence. RESULTS Compared with normal control group, H2 O2 0.1, 1, 5 and 25 mmol.L-1 significantly decreased the cell proliferation （ P 〈 0.01）. The expression of un-methylated PP2Ac in NlH/ 3T3 cells and in H2 O2 0.1, 1 and 5 mmol.L-1 groups was significantly increased（P〈0.01） compared with normal control group. Western blotting results revealed that both NAC （1 or 5 mmol.L-1 ） and AMZ30 （5 or 10 μmol.L-1 ） significantly antagonized the effect of H2 O2 on PP2Ac（P〈0.01）. lndirect immunofluorescence results showed that the un-methylated PP2Ac expression of H2 O2 1 mmol.L-1 group was higher than that of normal control group, while the expression of PP2Ac methylation was reduced after the use of AMZ30 10 μmol.L-1 or NAC 1 mmol.L-1. CONCLUSION H2 O2 can induce demethylation of PP2Ac in NlH/ 3T3 cells, while the PME-1 inhibitor AMZ30 and antioxidant NAC can antagonize the methylation of PP2Ac induced by H2 O2 .

关 键 词：过氧化氢 磷蛋白磷酸酶 甲基化 

分 类 号：R363[医药卫生—病理学]