氯氰菊酯对非配体依赖性IL-6/JAK/STAT3/AR信号通路的效应

The anti-androgenic effect mediated by androgen receptor (AR) is the important mechanism of male reproductive toxicity caused by the pyrethroid pesticides including cypermethrin. In the preliminary studies, we found cypermethrin inhibited the ligand-independent interaction between AR and the co-activator SRC-1. Therefore, the anti-androgenic effects involve ligand-independent AR signaling pathway. In the project, the growth factor interleukin-6 (IL-6) will be used as the starting points, and the IL-6/JAK/STAT/AR signaling pathway will be used as the main clue. The methods including mammalian two-hybrid assay, Western blot, Real time RT-PCR and AR report gene assay will be applied to study the effects of cypermethrin on phosphorylation of STAT3, the interaction between AR and STAT3, the interaction between AR and SRC-1, and interaction between AR and co-repressors including NCoR and SMRT caused by IL-6. The project will elucidate the molecular targets of cypermethrin in the signaling pathway and reveal the effects of cypermethrin on ligand-independent IL-6/JAK/STAT/AR signaling pathway. The new theory will be formed that the anti-androgenic effect is related to the ligand-independent AR signaling pathway. The project will open up a new way for research of the molecular mechanism of other environmental anti-androgens.

雄激素受体（AR）介导的抗雄激素效应是氯氰菊酯生殖毒性发生的重要机制。基于前期研究发现氯氰菊酯抑制非配体依赖性SRC-1与AR相互作用，申请者提出氯氰菊酯抗雄激素效应与非配体依赖性AR信号通路相关的假说。本项目以白细胞介素6 （IL-6）为切入点，以非配体依赖性IL-6/JAK/STAT/AR信号通路为主线，应用哺乳双杂交系统、Western blot、Real time RT-PCR和AR报告基因实验等，从细胞和分子水平系统研究氯氰菊酯对IL-6作用下STAT3磷酸化、AR与STAT3相互作用、AR与共激活因子SRC-1相互作用以及AR与共抑制因子NCoR 和SMRT相互作用的影响，阐明氯氰菊酯对IL-6/JAK/STAT/AR信号通路的作用，从新的视角揭示氯氰菊酯抗雄激素效应的机制，形成抗雄激素效应与非配体依赖性AR信号通路相关的新学说，为其他环境抗雄激素的机制研究开辟新的思路和领域。

非配体依赖性雄激素受体（AR）信号通路介导的抗雄激素效应是氯氰菊酯等农药的雄性生殖毒性发生的重要机制。本项目以白细胞介素6 （IL-6）为切入点，主要研究了氯氰菊酯对非配体依赖性IL-6/JAK/STAT/AR信号通路的影响，获得较有意义的研究成果。应用Real-Time Cell Analysis (RTCA) iCELLigence 系统进行细胞增殖实验，研究氯氰菊酯对LNCaP细胞增殖的作用，结果表明IL-6能够促进LNCaP细胞的增殖，而氯氰菊酯能够抑制IL-6介导的LNCaP细胞增殖效应。应用Real time RT-PCR实验研究氯氰菊酯对雄激素效应基因前列腺特异性抗原（PSA）mRNA表达的影响，结果发现，IL-6能促进PSAmRNA的表达，而氯氰菊酯能抑制IL-6介导的PSA的mRNA表达水平。建立了哺乳动物双杂交实验用于研究蛋白质-蛋白质之间的相互作用，结果表明，IL-6能够促进AR与共激活因子SRC-1的相互作用，抑制AR与共抑制因子SMRT的相互作用，促进AR与STAT3的相互作用；而氯氰菊酯能抑制IL-6介导的AR/SRC-1的相互作用，抑制IL-6介导的AR/SMRT相互作用，抑制IL-6介导的AR/STAT3的相互作用。应用Western blot研究氯氰菊酯对STAT3磷酸化的影响，表明IL-6 能够促进STAT3磷酸化，而氯氰菊酯不影响IL-6作用下STAT3磷酸化水平。本项目阐明氯氰菊酯对IL-6/JAK/STAT/AR信号通路的作用，从新的视角揭示氯氰菊酯抗雄激素效应的机制，形成抗雄激素效应与非配体依赖性AR信号通路相关的新学说，为其他环境抗雄激素的机制研究开辟新的思路和领域。本项目为氯氰菊酯抗雄激素效应的研究积累了大量的资料，为2017年申报新的国家自然科学基金奠定基础。