异烟肼对人肝细胞CYP1A1启动子区甲基化的影响

摘要

Objective To observe the effect of isoniazid on methylation of CpG island in cytochrome P4501A1 (CYP1A1) promoter region of human hepatocytes and to explore the mechanism of isoniazid-induced hepatocyte injury.Methods Liver cells were divided into the experimental group and control group.Cells in the experimental group were treated with 200, 400, and 800 μg/mL isoniazid, respectively.Cells in the control group were added with the same volume of DMSO culture medium.The LDH level in the supernatant was measured by lactate dehydrogenase (LDH) assay kit.The relative expression of CYP1A1, DNA methyltransferase 1 (DNMT1), DNMT3a, and DNMT3b mRNA was detected by real-time fluorescent quantitative PCR.The protein levels of DNMT1, DNMT3a, and DNMT3b were detected by enzyme-linked immunosorbent assay.The methylation of CYP1A1 promoter was detected by bisulfite sequencing PCR.Results The level of LDH in the supernatant of the experimental group was higher than that in the control group, and the LDH level was the highest in the 800 μg/mL isoniazid group (all P<0.05).Compared with the control group, the expression of CYP1A1 mRNA in the experimental group first increased and then decreased with the increasing concentrations of isoniazid (all P<0.05).DNMT1, DNMT3a, and DNMT3b mRNA and protein expression increased with the increase of isoniazid concentration, and the 800 μg/mL isoniazid group was the highest (P<0.05).The methylation rate of CYP1A1 gene promoter region was 82.9%, which was higher than that of control group (79.6%) (P<0.05).Conclusion Isoniazid can cause human hepatocyte injury, and the mechanism may be related to CYP1A1 gene promoter hypermethylation.%目的 观察异烟肼对人肝细胞细胞色素P4501A1(CYP1A1)启动子区CpG岛甲基化的影响,探讨异烟肼致肝细胞损伤的机制.方法 将肝细胞分为观察组和对照组,观察组分别加入200、400、800 μg/mL异烟肼,对照组加入同体积含DMSO的培养液.采用乳酸脱氢酶(LDH)检测试剂盒检测细胞培养上清液中的LDH水平来观察细胞损伤情况,实时荧光定量PCR方法检测细胞CYP1A1、DNA甲基转移酶1(DNMT1)、DNMT3a、DNMT3b mRNA的相对表达量,酶联免疫吸附法检测细胞DNMT1、DNMT3a、DNMT3b蛋白水平,采用亚硫酸盐修饰后测序法检测CYP1A1启动子区甲基化状态.结果 观察组细胞培养上清液中的LDH水平均高于对照组,800 μg/mL异烟肼组最高(P均<0.05).与对照组比较,观察组随异烟肼浓度升高,CYP1A1 mRNA表达呈先升高后降低趋势,差异均有统计学意义(P均<0.05);DNMT1、DNMT3a、DNMT3b mRNA和蛋白表达均随异烟肼浓度升高而升高,800 μg/mL异烟肼组高于其他组(P均<0.05).细胞CYP1A1基因启动子区甲基化率为82.9%,高于对照组的79.6%(P<0.05).结论 异烟肼可导致肝细胞CYP1A1基因启动子区高甲基化,进而引起肝细胞损伤.