Nrf2调控水合氯醛活化树突状细胞在三氯乙烯药疹样皮炎中的作用

Occupational medicamentosa-like dermatitis due to trichloroethylene (OMDT) is a severe occupational hazard with high mortality, but whether OMDT is caused by trichloroethylene itself or its metabolites remains unknown. Earlier investigations have suggested the involvement of abnormal immune response induced by metabolic activation of trichloroethylene in the progression of OMDT. Moreover, our previous studies have shown that dendritic cells (DC) play an important role in OMDT, while chloral hydrate (CH), the major metabolite of trichloroethylene, has electrophilic property that can bind to nucleophilic functional groups in proteins and can induce the activation of THP-1 cells. Therefore, we hypothesize that CH may participate in the development of OMDT via the generation of oxidative stress signal to induce Nrf2 pathway resulting in DC activation. This project employs THP-1 cells and mouse bone marrow-derived dendritic cells (BMDC) to study the effect of CH on the activation and maturation of DC. A mouse animal model of OMDT will be constructed by adoptive immunizing naive mice with CH-activated BMDC and the effector function of T cells in the model will be detected to explore the mode of action of CH in developing OMDT. By knocking out Nrf2 gene, using ARE-luciferase reporter assay and next-generation sequencing method (including ChIP-seq and RNA-seq), the molecular mechanism, by which the CH-induced DC activation is regulated by oxidative stress signal and Nrf2 pathway, will be clarified. The proposed research is expected to provide valuable scientific clues and thoughts for risk assessment and occupational hazard prevention and treatment of trichloroethylene exposure.

职业性三氯乙烯药疹样皮炎（OMDT）是一类死亡率高且危害严重的职业病，但其致敏原与发病机制尚不明确，可能与三氯乙烯代谢活化诱导的异常免疫应答有关。前期研究显示，树突状细胞（DC）在OMDT中发挥重要作用，而三氯乙烯主要代谢产物水合氯醛（CH）具有亲电特性能与蛋白上的亲核基团结合，并且可诱导THP-1细胞活化。因此我们推测CH可能通过产生氧化应激信号激活Nrf2通路来调节DC的免疫应答，诱导OMDT的发生。本项目拟通过体外培养的DC模型，探究CH对其活化成熟功能的影响；将CH活化的DC过继免疫健康小鼠构建OMDT动物模型，检测其T细胞的效应功能，探索CH作为致敏原诱发OMDT的作用路径；通过基因敲除、荧光素酶和下一代测序（ChIP-seq、RNA-seq）的方法，阐明氧化应激和Nrf2通路对CH活化DC的分子调控机制，为三氯乙烯暴露的风险评估和职业危害防治提供科学线索和新的思路。

职业性三氯乙烯药疹样皮炎（OMDT）是一类死亡率高且危害严重的职业病，但其致敏原与发病机制尚不明确。本项目着重探讨Nrf2调控三氯乙烯的氧化代谢产物——水合氯醛（CH）介导的激活树突状细胞的免疫应答在OMDT中的免疫毒性作用路径。体外研究中使用抗氧化剂姜黄素和CRISPR/Cas9技术敲除NRF2基因，通过人细胞系活化试验建立CH诱导的树突状细胞活化模型。结果显示CH可促进THP-1细胞活化和成熟，并导致细胞处于氧化应激状态，上调Nrf2通路相关分子的基因和蛋白表达，促进Nrf2转核，而以上效应均可被姜黄素和NRF2基因敲除所抑制。这些结果说明Nrf2可能是调节CH诱导的THP-1细胞活化和氧化还原稳态的重要转录因子。进一步研究发现CH同样可促进小鼠骨髓来源的树突状细胞（BMDC）的活化和细胞因子分泌，诱导细胞氧化应激和Nrf2通路相关分子表达。在体内研究中，我们将前述CH活化的BMDC过继免疫小鼠后成功构建三氯乙烯诱发的接触性超敏反应模型。在该模型小鼠中，引流淋巴结内淋巴细胞大量增殖、T细胞数目增加、T细胞活化并分泌细胞因子。可见CH活化的BMDC可参与三氯乙烯诱发的接触性超敏反应，并且促进相应的效应T细胞增殖活化。本研究从细胞效应（激活树突状细胞）、器官效应（引流淋巴结中效应T细胞增殖活化）以及系统效应（接触性超敏反应的发生）三个层面，阐明了CH在OMDT免疫应答中的作用模式路径和关键分子机制，提示CH很可能是OMDT的致敏原，同时Nrf2可能成为调节CH免疫诱导作用的潜在靶点，为三氯乙烯职业接触危害的预防和治疗提供新的线索和思路。