温经活络方通过调控AMPK及JAK2-STAT3信号通路影响Th17分化治疗类风湿性关节炎的机制研究

Th17 cell differentiation plays a key role in the pathogenesis of rheumatoid arthritis (RA), AMPK and JAK2-STAT3 signaling pathway is closely related to Th17 cell activation. Our previous study found that wenjinhuoluo prescription can activate synovial fibroblasts (FLS) AMPK and inhibit the activation of STAT3, thereby preventing the activation of Th17 cells, inhibiting joint swelling and bone destruction of RA mice, significantly, in additon, inhibiting the expression of IL-17, however, the mechanism has not been elucidated. Wenjinhuoluo prescription can exact the anti RA effect through inhibited differentiation and function of Th17 by the regulation of AMPK and JAK2-STAT3 pathway. Our project will verify the hypothesis: 1. Wenjinhuoluo prescription regulates the differentiation and function of Th17 through AMPK and JAK2-STAT3 pathway; 2. The effect of wenjinhuoluo prescription on AMPK, JAK2-STAT3 pathway and inflammatory factorsby; 3. T cells were co cultured with FLS system. Researching the effect of differentiation of Th17 on the cell proliferation, apoptosis of FLS, osteoclast regulation and studying the intervention effect of wenjinhuoluo prescription from Th17/RANKL approach. This project aims to study the mechanism to explore the probablity of wenjinhuoluo prescription as an new RA immune targeted therapy.

Th17细胞异常分化在类风湿性关节炎（RA）发病中起关键作用，AMPK和JAK2-STAT3信号通路与Th17细胞活化关系密切。我们前期研究发现，温经活络方可激活成纤维样滑膜细胞（FLS）AMPK抑制STAT3激活，从而阻止Th17细胞活化，显著抑制RA小鼠关节肿胀、骨质破坏，抑制IL-17表达，但作用机制尚未阐明。推测温经活络方通过调控AMPK和JAK2-STAT3通路抑制Th17分化和功能发挥抗RA作用。本项目将系统验证上述推测：1. 温经活络方通过AMPK和JAK2-STAT3通路调节Th17分化和功能；2. 温经活络方对AMPK和JAK2-STAT3通路及炎症因子的影响；3. 建立T细胞与FLS共培养体系，从Th17/RANKL途径研究Th17分化对FLS增殖、凋亡、破骨调控的影响及温经活络方的干预作用。本项目旨在通过对上述机制的研究，探索温经活络方作为RA新的免疫靶向治疗的可能。

本研究通过系统研究证明了牛II型胶原免疫DBA/1小鼠可建立稳定的类风湿性关节炎模型。首次从动物模型、体外细胞培养和代谢组学三个层次证实了温经活络方（WJHL）治疗RA的疗效。WJHL不仅能够在体外抑制滑膜细胞和脾脏细胞中胶原诱导的多种炎症因子的表达，还能在体内对胶原诱导的RA发挥治疗作用。.WJHL能使CIA模型动物的血清代谢谱向正常动物回归，代谢组学对WJHL抗CIA有效性评价与传统药效学研究结果一致。借助代谢组学方法可以分析机体内多种代谢循环通路的状态，是评价CIA小鼠体内血液代谢变化的有效手段，为探究类风湿性关节炎机制及在分子水平进行WJHL综合药效评估奠定了基础。. 本研究发现CIA小鼠脾脏中Th17细胞比例和IL-17A含量显著增加，Treg细胞比例显著下降，Th17/Treg比例失衡。CIA小鼠脾脏T细胞内JAK2/STAT3信号呈显著活化状态。本研究的意义在于发现了WJHL能显著降低CIA小鼠脾脏组织炎症损伤，主要表现在抑制Th1/Th17细胞相关促炎因子的分泌，促进Th2/Treg细胞相关抗炎因子的分泌，促使脾脏Th17细胞含量降低，Treg细胞含量升高进而显著调节了Th17/Treg细胞平衡。体外重组IL-23蛋白能显著诱导脾脏单核细胞发生Th17细胞极化，并伴随炎症因子释放，放大炎症级联反应；而WJHL治疗能显著抑制这一效应。WJHL能显著抑制外源性刺激诱导下脾脏淋巴结CD4+淋巴细胞的活化，主要表现在抑制促炎因子释放，促进抗炎因子释放，维持机体炎症平衡；且WJHL能强化脾脏中CD4+CD25+Treg细胞对CD4+CD25-效应性T细胞的抵抗力，增强CIA小鼠的免疫抑制能力.. WJHL治疗RA作用是通过阻断JAK2/STAT3信号通路，抑制Th17细胞分化和IL-17A分泌，恢复Th17/Treg比例平衡；WJHL可作为JAK2/STAT3信号通路新型抑制剂，为RA 的靶向治疗提供新的思路和线索。