NRF2持续激活与砷甲基化代谢差异的关系及在砷致皮肤角化紊乱中作用

Skin is the arsenic target organ in an early period of arsenic poisoning. Sustained activation of NRF2 could induced by interaction between metabolism of arsenic methylation and inhibiting or competition factor of NRF2 and Estrogen or Estrogen receptors,which play an impor- tant role in skin lesion during the occurrence and development of arsenic poisoning.The project intends to discuss the following contents by the experiment of rabbits in skin and HaCaT cells:(1) To discuss the effect of the arsenic methylation metabolism disturbing Keap1 or Bach1 on sustained activation of NRF2,and the mechanism of inflammatory skin condition and prolifera tion and the abnormal expression of cytokeratin regulated by them.(2) The influence of sustained activation of NRF2 caused by inhibited Keap1 on arsenic methylation meta- bolism and gene expression of N6AMT1 and As3MT，and the mechanism of inflammatory skin condition and proliferation and the abnormal expression of cytokeratin caused by them.(3)The impact of Estrogen or Estrogen receptors on arsenic methylation metabolism which re- gulated by intermediary metabolism，and regulation effect of NRF2 phosphorylation level influenced by Estrogen or Estrogen receptors on sustained activation of NRF2.(4)the abnormality of arsenic methylation metabolism and Keap1 or Bach1 and Estrogen or Estrogen recep- tors could result in sustained activation of NRF2,which is critical factor and the relationship between the three in skin.The project would provide the scientific basis for studying the toxicity effects of arsenic and further lay an important foundation for clarifing the mechanism of arsenic toxicity in skin.

皮肤是砷中毒早期靶器官，机体砷甲基化代谢与NRF2抑制/竞争因子和雌激素及受体相互作用诱导NRF2持续激活在砷致皮肤病损中发挥重要作用。本项目拟通过家兔皮肤和HaCaT细胞实验，探讨以下内容：(1)砷甲基化代谢在干扰Keap1和Bach1引起Nrf2持续激活中的作用，及对皮肤炎症、增殖和角化蛋白异常的调控机制。（2）抑制Keap1诱发Nrf2持续激活，对砷甲基化代谢及N6AMT1和As3MT基因表达的影响，及导致下游炎症、增殖和角化蛋白异常表达的机制。（3）雌激素及受体通过中间代谢对砷甲基化代谢的影响，及雌激素及受体通过影响Nrf2磷酸化水平，对Nrf2持续激活的调控作用。（4）砷甲基化代谢异常与Keap1、Bach1和雌激素及受体哪一个是导致Nrf2持续激活的关键因素及他们之间的关联性。这将为研究砷致皮损效应及毒性机制提供科学依据并奠定重要基础。

项目背景：砷中毒的皮肤毒性机制还不清楚；探究砷暴露后机体砷代谢与NRF2通路和雌激素及受体相互作用，诱导NRF2蛋白过表达在砷皮肤毒性中的作用，具有现实意义。主要研究内容：通过Keap1和Nrf2抑制细胞和新西兰大耳兔（简称家兔）实验，研究砷暴露及砷代谢对Keap1和Bach1的影响，探讨其和雌激素及受体的关系；通过分别抑制Keap1和Nrf2基因表达，探讨其对砷甲基化代谢的影响，观察Nrf2过表达对Bach1水平、MAPK及NF-кB通路指标的影响，及他们在Nrf2激活及皮肤角化紊乱中作用。重要结果：本项目发现（1）Nrf2通路参与砷甲基化代谢调控。SFP和5-Aza-DC干预可影响砷甲基化代谢。（2）介导调控N6AMT1表达，Nrf2通路与砷甲基化代谢存在交互作用。（3）砷暴露介导ERK和NF-кB调控Nrf2基因表达。（4）砷暴露发挥雌激素干扰作用，参与砷致Nrf2激活，促进砷甲基化代谢。（5）Nrf2通路影响皮肤角化整体进程；中、高剂量砷暴露促进皮肤角化进程。关键数据及其科学意义：（1）砷暴露皮肤细胞通过改变keap1和Bach1基因表达，调控Nrf2通路，参与细胞砷甲基化代谢；砷暴露诱导家兔Keap1基因CpG位点甲基化率升高，促进Nrf2蛋白表达，激活Nrf2通路；低砷暴露促进、高砷暴露抑制磷酸化Nrf2蛋白表达，调控Nrf2通路，参与砷甲基化代谢。（2）家兔砷暴露增强N6AMT1基因表达，提高砷甲基化代谢能力，促进砷甲基化代谢，参与Nrf2通路激活。（3）低砷暴露诱导细胞ERK和NF-кB表达上调，介导CBP表达增强，激活细胞Nrf2通路；高砷暴露正好相反。高砷暴露诱导家兔皮肤中NF-кB表达上调，促进皮肤Nrf2基因表达。（4）砷暴露抑制家兔E2水平，诱导ERα表达升高，抑制ERβ mRNA表达，发挥雌激素干扰效应，参与Nrf2通路和砷代谢调控。（5）Keap1抑制会促进细胞的皮肤角化进程；中剂量或高砷暴露促进皮肤细胞和家兔皮肤角化进程。高砷暴露通过上调家兔皮肤K1 mRNA表达，促进皮肤角化进程。Nrf2蛋白、As3MT mRNA和N6AMT1蛋白及ERα mRNA高表达，Keap1蛋白和ERβ mRNA及E2低表达，会促进家兔皮肤角化进程。本项目为阐明砷中毒机制奠定基础，在砷中毒早期预防中具有理论价值。