吴茱萸碱通过芳香烃受体抑制炎性巨噬细胞NF-κB活性的分子机制研究

Dysfunction of macrophages (Mφ) plays an important role in the pathogenesis of systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS) induced by severe trauma and infectious complication. Evodiamine (EVO) exerts protective effect on this pathological process. Based on our preliminary experimental finding that the inhibitory effect of EVO on nuclear factor-κB (NF-κB) action of inflammatory Mφ is mediated, at least partially, by aryl hydrocarbon receptor (AhR) and the fact that there are several AhR signal transduction pathways within nucleus, we propose the scientific hypothesis that EVO can depress NF-κB activity by facilitating the formation of AhR/RelA and AhR/p50/Stat1 complexs in inflammatory Mφ. In this study we will investigate the in vitro and in vivo effect of EVO on the formation of AhR-binding proteins complexs, their DNA binding activities and targeted gene expression in LPS stimulated murine Mφ via co-immunoprecipitation, cellular co-localization, electrophoresis mobility shift assay (EMSA) and supershift technique, detect the direct modulatory effect of EVO on the affinity between AhR and its binding proteins by dual polarisation interferometry and on DNA binding of AhR related complexs by EMSA. The results will not only help to reveal the immunomodulatory mechanism of EVO on inflammatory Mφ, but also establish the foundation for the development of this novel immune response modifier in the treatment of immune dysfunction induced by severe trauma and infectious complication.

巨噬细胞（Mφ）功能紊乱在严重创伤、感染所致SIRS及MODS病程中扮演重要角色，而吴茱萸碱（EVO）对此具有保护效应。根据"EVO对炎性Mφ核因子κB（NF-κB）的抑制作用至少部分通过芳香烃受体（AhR）而介导"这一实验现象，结合AhR在细胞核内存在多条信号通路的特点，我们提出"EVO可促进炎性Mφ核内AhR/RelA、AhR/p50/Stat1复合物形成进而抑制NF-κB活性"的科学假设。利用免疫共沉淀、细胞共定位、EMSA及supershift技术，研究离体、在体实验中EVO对LPS激活的小鼠Mφ核内AhR各复合物形成、其与DNA结合活性及靶基因表达的影响，分别利用双偏振极化干涉测量技术、EMSA检测EVO对AhR-连接蛋白亲和力及AhR相关复合物-DNA结合的直接调节作用。这不仅有助于揭示EVO对炎性Mφ的调节机制，而且可为纠正创伤、感染后免疫功能紊乱的新型调节剂的研发奠定基础。

巨噬细胞功能紊乱在严重创伤、感染所致SIRS及MODS病程中扮演重要角色，而吴茱萸碱对此具有保护效应。我们的前期研究发现EVO对炎性巨噬细胞中NF-κB的抑制作用部分依赖于芳香烃受体（AhR), 而AhR在细胞核内可与RelA、RelB、p50、Stat1等蛋白形成不同比例的复合物，因此我们推测EVO可通过改变上述复合物的形成以及其与DNA的结合活性，进而发挥抗炎活性。本项目主要研究内容包括：1）EVO对炎性巨噬细胞核内AhR各复合物形成的影响；2）EVO对炎性巨噬细胞AhR连接复合物的DNA结合活性以及靶基因表达的影响。在LPS活化的巨噬细胞中，通过免疫共沉淀技术沉淀AhR并检测与其相互作用蛋白，发现EVO可显著降低细胞总AhR的蛋白含量以及AhR与Stat1的结合, 但对其与ARNT 、RelB、RelA、p50的结合无影响；以AhR各连接复合物的DNA结合序列为探针（NCE、DRE、非共有序列的DRE、RelBAhRE以及RelB/p52连接位点），通过EMSA技术检测EVO作用下炎性巨噬细胞核蛋白与上述DNA探针结合活性的变化，发现EVO可显著减少炎性巨噬细胞核蛋白与NCE以及非共有序列DRE的结合活性，但是对DRE、RelBAhRE以及RelB/p52 序列探针的结合活性无明显影响。此外，利用微量热泳动技术（MST）检测核蛋白与NCE之间的亲和力，发现EVO可降低两者的亲和力。同时q-PCR结果显示EVO对下游靶基因IL-6、TNF- 和IL-1的mRNA表达水平具有明显的下调作用。本项目已顺利完成预期的全部研究目标，研究结果不仅有助于从全新角度阐明EVO的抗炎作用机理，而且为其后续的改构及后续成药性研究奠定基础。