维生素E对氯化镍染毒大鼠肺组织p15基因甲基化过程的影响

Effects of Vitamin E on the Methylation of p15 in Lung of Rats Exposed to Nickel Chloride

[目的]探讨维生素E(VE)对氯化镍染毒大鼠肺组织p15基因甲基化过程的影响。[方法]以0.1、0.2、0.4和0.8mg/mL氯化镍灌胃染毒大鼠,并设立对照组(生理盐水)和拮抗组(0.8mg/mL氯化镍+5mg/mL VE),持续6周。染毒结束后处死大鼠,制备肺组织匀浆,采用分光光度法,测定谷胱甘肽(GSH)和丙二醛(MDA)水平的变化;以荧光定量PCR法检测肺组织中抑癌基因p15的表达水平;以甲基化PCR法检测p15启动子甲基化状态;以Western-blot检测DNA甲基转移酶1(DNMT1)和蛋氨酸腺苷转移酶2A(MAT2A)的表达水平。[结果]随着氯化镍染毒剂量的增加,MDA含量显著升高,GSH含量显著下降(P<0.01);与相同剂量染毒组相比,拮抗组大鼠肺组织中MDA含量降低,GSH含量升高(P<0.05);对照组大鼠肺组织p15启动子为非甲基化状态,染毒组大鼠肺组织p15启动子甲基化和非甲基化条带均有表达;各染毒组p15 mRNA表达量与对照组相比均显著性降低(P<0.01),使用抗氧化剂VE后p15mRNA表达量与相同剂量染毒组相比显著性增高(P<0.05);随着染镍剂量的增高,DNMT1和MAT2A表达量显著性升高,与对照组相比,最高剂量染镍组DNMT1和MAT2A的表达量分别增高了1.52倍和1.49倍。使用拮抗剂VE后,与相同剂量染镍组相比DNMT1和MAT2A的表达量分别降低了10%和29%;双变量相关分析显示大鼠肺组织中GSH含量与DNMT1和MAT2A蛋白表达水平呈负相关关系,相关系数分别为r=-0.889(P=0.018)和r=-0.821(P=0.044)。[结论]维生素E可能通过影响GSH在氯化镍染毒大鼠肺组织中的含量而影响p15基因的甲基化过程。

[ Objective ] To investigate the effects of vitamin E on the methylation of p15 in lung of rats exposed to nickel chloride. [ Methods ] The rats were fed by gavage with the designed concentrations of NiCl2 ·6H2O （ 0.1, 0.2, 0.4 and 0.8 mg/mL ） for four different exposed groups and 0.9% NaCl solution for the solvent control group. The antagonism group was supplied with vitamin E （ 5 mg/mL ） along with 0.8 mg/mL NiCl2. After treating the rats for six weeks, we detected the levels of malondialdehyde （ MDA ）and glutathione （ GSH ）to assess oxidative stress in the lung tissues. In addition, we also evaluated the methylation status and expressions of tumor suppressor gene p15 using methylation specific PCR and quantitative real time PCR. DNA methyhransferase （ DNMT1 ）and methionine adenosyltransferase 2A（ MAT2A ）that play key role in methylation process were detected by western blot. [ Results ] Compared with the control group, the levels of MDA in lung of the rats exposed to nickel chloride increased significantly, while the levels of GSH decreased significantly （ P 〈 0.01 ）. As compared with the high-dose group （ 0.8 mg/mL NiCl2 ）, a significant increase in GSH contents was observed in the antagonism group（ NiCl2＋VE ）, while the levels of MDA decreased significantly（ P〈 0.05 ）. The tumor suppressor gene of p15 was found to be non-methylated in the control group. The methylation and non-methylation ofp15 were both observed in NiCl2 exposure groups and VE antagonism group. Compared with the control group, the levels of p15 were down-regulated significantly in the NiCl2 exposure groups（ P〈0.01 ）. As compared with the high-dose group（ 0.8 mg/mL NiCl2 ）, a significant increase in p15 levels was observed in the antagonism group （ NiCl2＋VE ）（ P 〈 0.05 ）. The levels of DNMT1 and MAT2A significantly increased as the concentrations of NiCl2 increased, and the ratios increased 1.52- and 1.49- folds in the high-dose group respectively compared with the control group. Compared with the high-dose group, the relative ratios of DNMT1 and MAT2A in the antagonism （ NiCl2＋VE ）group decreased 18% and 29% respectively. We found that the levels of GSH were correlated negatively with the relative expressions of DNMT1 （ r=-0.889, P=0.018 ）and MAT2A（ r=-0.821, P=0.044 ）. [ Conclusion ] Our findings suggest that glutathione influenced by vitamin E may play important role in DNA methylation.