摘要:
目的 探究混合砷染毒对核转录因子红细胞系-2p45（NF-E2）因子-2（nuclear erythroid 2-related factor，Nrf2）抑制的人永生化角质形成细胞（human immortalized keratinocytes，HaCaT细胞） Kelch样环氧氯丙烷相关蛋白1（Kelch-like ECH-associated protein，Keap1）和N-6-腺嘌呤DNA甲基转移酶（N-6-adenine DNA methyltransferase，N6AMT1）表达的影响。方法 细胞培养72 h，分为4组：空白对照组、Keap1抑制对照组、Nrf2抑制对照组和三个Nrf2抑制混合染砷组，混合砷染毒浓度分别为2.1 μmol/L、4.2 μmol/L、21.0 μmol/L；采用实时荧光定量PCR和蛋白质分子印记（western blot）方法测定各组细胞N6AMT1、Keap1的mRNA和蛋白水平。结果 与Nrf2抑制对照组比较Keap1和N6AMT1 mRNA表达不全相等（均有P<0.05）。与Nrf2抑制对照组比较，低、中、高剂量Keap1蛋白表达促进（均有P<0.05），N6AMT1蛋白表达呈低剂量促进（t=-3.18，P=0.034），中、高剂量抑制（均有P<0.05）。结论 Nrf2抑制状况下，N6AMT1基因可能与Keap1-Nrf2通路调控有关。低剂量砷暴露时，Keap1激活可促进N6AMT1基因表达，促进砷代谢；高剂量砷暴露N6AMT1蛋白表达下调，抑制砷代谢。Keap1-Nrf2通路可能参与N6AMT1表达调控，而影响砷毒性作用。
Abstract:
Objective To explore the effects of mixed arsenic exposure on the expression of the Kelch-like epichlorohydrin-related protein 1 (Keap1) and N-6-adenine DNA methyltransferase 1 (N6AMT1) in the cytokines cells (HaCaT cells) inhibited by the nuclear transcription factor red cell-2p45(NF-E2) correlation factor-2(Nrf2). Methods Cells were cultured for 72 hours and divided into four groups:blank control group, Keap-1 inhibitory control group, Nrf-2 inhibitory control group and three Nrf-2 inhibitory mixed arsenic groups. The concentrations of mixed arsenic were 2.1 μmol/L, 4.2 μmol/L and 21.0 μmol/L, respectively. The mRNA and protein levels of N6AMT1 and Keap1 were measured by real-time fluorescence quantitative PCR and western blot. Results Compared with Nrf2 control group, Keap1 and N6AMT1 mRNA expression was not all equal (all P<0.05). Compared with Nrf2 inhibitory control group, the expression of Keap1 protein in low, medium and high doses was increased (all P<0.05), while the expression of N6AMT1 protein was increased in low doses (t=-3.18,P=0.034), and inhibited in medium and high doses (all P<0.05). Conclusions Nrf2 inhibitory condition, the N6AMT1 gene may be related to the regulation of Keap1-Nrf2 pathway. Low dose arsenic exposure, keap1 activation can promote N6AMT1 gene expression and promote arsenic metabolism. High dose arsenic exposure, the down-regulation of N6AMT1 protein expression, inhibit the metabolism of arsenic. The Keap1-Nrf2 pathway may be involved in the regulation of N6AMT1 expression and affect arsenic toxicity.
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