FICZ/AhR通路对溃疡性结肠炎Th17/Treg细胞分化相关信号通路的影响和清热燥湿凉血方的干预作用

It is a recent immunology research focus that the aryl hydrocarbon receptor (AhR) can regulate the cell differentiation of Th17 and Treg cell in a ligand-dependent fashion. 6-formylindolo[3,2-b]carbazole (FICZ) is an endogenous ligand of AhR. It can regulate the differentiation of Th17 cell by activating AhR. We speculate that FICZ / AhR pathway may play an important role in UC intestinal mucosal immune disorder. The preliminary study found that AhR expression was significantly increased in experimental colitis mucosa and Th17 cells differentiated in vitro. FICZ activated AhR could make the expression of in vitro differentiated Th17 cell function-associated protein RORγt significantly increased. Qingrezaoshiliangxue formula can inhibit Th17 cells express RORγt. This prompted FICZ / AhR pathway may play a critical role in regulation of Th17 cell differentiation in UC mucosa. And Qingrezaoshiliangxue formula could regulate Th17 cell differentiation. On the basis of previous work, we intend to further study the effect of FICZ / AhR pathway on the the Th17/Treg differentiation and related signaling pathways in the UC mucosa, and the cross-talk between Th17 and Treg differentiation signaling pathways. Through which we intended to reveal the role of FICZ / AhR pathway in the pathogenesis of UC, and to reveal the target and mechanism of action of Qingrezaoshiliangxue formula in treatment of UC.

FICZ激活芳香烃受体（AhR）调控Th17、Treg细胞分化是新近免疫学研究的热点，而Th17/Treg失衡在UC发病中起关键作用。我们推测FICZ/AhR通路在UC肠粘膜免疫紊乱中可能发挥重要作用。前期研究发现AhR在实验性结肠炎小鼠肠粘膜和体外分化的小鼠脾脏Th17细胞中表达明显升高；FICZ激活AhR能使体外诱导分化的小鼠脾脏Th17细胞功能相关蛋白RORγt表达明显升高，清热燥湿凉血方能抑制Th17细胞表达RORγt，初步证实了我们的假设。我们拟在已有工作基础上，进一步研究FICZ/AhR通路对UC肠粘膜Th17/Treg分化和相关信号通路的影响，及Th17和Treg分化信号通路间的交叉会话，并观察清热燥湿凉血方的干预作用，拟揭示FICZ/AhR通路在UC发病机制中的作用，以及清热燥湿凉血方治疗UC的靶点和作用机制。

炎症性肠病是慢性消化道炎症性疾病，慢性炎症从本质上来说是由于Th1、Th2、Th17及Treg细胞的免疫稳态失调导致免疫应答失调极化。芳香烃受体（AHR）是残留在胞浆中的转录因子，其活化能够促进Th17细胞及Treg细胞的分化。本课题利用小鼠结肠炎模型阐述AHR在其中的作用及相关机制。（一）采用磁珠分选小鼠脾脏原代CD4+T细胞，流式细胞术检测芳香烃受体激动剂FICZ及拮抗剂萘黄酮处理后， CD4+T细胞向Th17细胞及调节性T细胞的分化的影响。结果AHR激动剂处理能够显著升高CD4+T细胞中Th17细胞（8.61% vs. 2.38%），而AHR拮抗剂萘黄酮处理能够降低CD4+T细胞中Th17细胞（1.93% vs. 2.38%）。STAT3 knockdown细胞采用FICZ处理后CD4+T细胞中Th17细胞无显著上升。（二）建立小鼠急性和慢性结肠炎模型，不同因素干预小鼠，并检测Th17与Treg相关转录及细胞因子的表达等。结果在急性结肠炎小鼠中，萘黄酮、地塞米松及中药处理均能够缓解结肠炎症状并减轻炎症反应。免疫组化结果显示，在这些处理组中出现STAT3及Treg标记FOXP3的上调，并且在FICZ及中药处理组的小鼠中出现AhR表达增加的情况。在慢性结肠炎小鼠中，不同处理对症状的缓解均无明显效应，但能够在一定程度上减轻炎症反应。（三）将造模后的小鼠分离获得脾脏细胞、LPMCs及MLNLs，给予不同因素干预，通过流式细胞术分析LPMCs 及MLNLs中Th17及Treg细胞比例变化，分析与Th17及Treg细胞因子及转录因子的变化。结果流式细胞术检测急性结肠炎小鼠来源的LPMCs的结果显示，中药处理能够调节Th17及Treg细胞比例。急性结肠炎小鼠MLNLs流式分析结果显示，而中药能够升高Treg细胞比例，综上结果提示在急性结肠炎小鼠中，中药能够通过影影响LPMCs及CD4+T细胞向Th17及Treg细胞分化及相应细胞因子水平的改变而发挥治疗结肠炎的作用。