从Treg/Th17平衡和IL-23/IL-17轴探讨清热健脾活血法治疗UC的作用机制

Recent studies showed that the blance between Treg and Th17 maintained the intestinal normal physiologic inflammation. The balance break, especially the increase ratio of Th17 or enhancement of its function, would lead to intestinal pathological inflammation. The inflammatory axis of IL-23/IL-17can further amplify the inflammatory reaction, finally resulting in the onset of UC. Several studies showed that the main pathway of immune response is IL-23/IL-17 axis, rather than the tranditionally accepted IL-12/IFN-γ axis. Studies of our two former NSFC projects have showed that Kuijieling Decoction(KD) has obvious effects on the blance of Treg/Th17 and adjusting links of IL-23/IL-17 axis, which suggested that adjust the blance of Treg/Th17 and the IL-23/IL-17 axis are the critical therapeutical mechanism of KD in treating UC. In this study, flow cytometry, real-time quantitative RT-PCR, Western blot and other techniques will be used to observe the effect of KD on the cell ration of Th17/Treg and its relative regulatory factor, also including gene and protein expression of IL-17、IL-23 from whole level and cellular level, To investigate action targets and molecular mechanism of the method of clearning heat, strengthening spleen and activating blood in TCM, which is helpful for clinical treatment and drug R&D.

新近研究表明，Treg/Th17平衡维持着肠道的正常生理性炎症，这一平衡的打破尤其是Th17比例提高和功能加强导致了肠道病理性炎症，IL-23/IL-17炎症轴进一步放大了炎症反应，导致UC的发病。多项研究表明IL-23/IL-17轴是UC的主要免疫应答通路，而不是传统认为的IL-12/IFN-γ轴。申请者主持的2项国家自然科学基金项目表明中药复方溃结灵对Treg/Th17平衡和IL-23/IL-17炎症轴的一些调节环节具有明显作用，提示调节Treg/Th17平衡和IL-23/IL-17轴是溃结灵治疗UC的重要作用机制。本研究拟从整体和细胞水平，采用流式细胞术、实时定量PCR、Western blot等方法观察溃结灵对Th17/Treg细胞比例及相关调节因子和IL-17、IL-23的蛋白及基因表达水平的影响，探讨中医清热健脾活血法治疗UC的作用靶点及分子机理，裨益临床治疗及新药开发 。

Treg/Th17失衡导致了肠道病理性炎症，IL-23/IL-17炎症轴进一步放大了炎症反应，导致UC的发病。本研究以TNBS灌肠制作的UC大鼠模型，以及大鼠脾淋巴细胞原代培养制作的细胞模型为研究对象，采用流式细胞术检测Treg/Th17平衡，采用酶联免疫、实时定量PCR、免疫组化、Western blot、流式细胞术等方法检测调节Treg/Th17平衡的细胞因子和转录因子及IL-23/IL-17炎症轴的基因及蛋白表达，探讨溃结灵治疗UC的作用机制。整体动物实验结果表明：①溃结灵对UC大鼠模型有较好的治疗作用；②溃结灵组大鼠外周血Th17细胞比例与模型组比较显著降低, Treg细胞比例及Treg/Th17比率与模型组比较显著增高；表明溃结灵对UC大鼠Treg/TH17失衡有调节作用；③溃结灵组血浆和（或）结肠组织中TGF-β1、IL-2 和IL-10含量明显高于模型组；溃结灵组血浆和（或）结肠组织中IL-6、IL-21、IL-17和IL-23含量明显低于模型组；表明溃结灵对Treg/Th17平衡相关细胞因子及IL-23/IL-17炎症轴有明显的调节作用；④溃结灵组结肠黏膜TGF-β1、Foxp3、 IL-2、STAT-5 mRNA表达明显高于模型组；溃结灵组IL-6、RORγt、STAT-3、IL-17、IL-23、IL-21 mRNA表达明显低于模型组；表明表明溃结灵对Treg/Th17平衡相关细胞因子及IL-23/IL-17炎症轴关键分子的基因表达有明显的调节作用。⑤免疫组化法及WB检测结果均表明，溃结灵组Foxp3、STAT-5蛋白表达明显高于模型组；溃结灵组RORγt、STAT-3蛋白表达明显低于模型组。离体细胞实验结果表明：①溃结灵组Th17占CD4+细胞比例明显低于模型组；②溃结灵能逆转与Treg/TH17平衡相关的部分细胞因子和转录因子IL-2、IL-10、IL-21、TGF-β1、IL-6、Foxp3、RORγt、STAT-3和STAT-5以及IL-23/IL-17炎症轴关键因子IL-17、IL-23在模型细胞的基因和蛋白表达异常。细胞实验与整体动物实验结果基本一致。提示纠正Treg/Th17失衡及抑制IL-23/IL-17炎症轴活化是溃结灵治疗UC的部分作用机制。本研究对溃结灵在临床治疗UC的推广及高水平新药开发具有积极的意义。