基于Th17/Treg细胞失衡的乌头汤治疗类风湿关节炎风寒湿痹证方证对应研究

To elucidate the modern scientific connotation of the chinese medicine theory of prescription corresponding to Zheng is the key scientific problem of prescription discipline. Bi Zheng is one of the common disease categories in traditional chinese medicine(TCM) which is characterized by pain, numbness, feeling of sourness and heaviness,and swelling of the muscles, tendons, and joints, difficulty in flexion and extension or even ankylosis and deformity of joints. It is recorded in the chapter of "Su Wen •Bi Lun" that Bi Zheng is caused by the synergistic invasion of exogenous evils wind, cold, and damp into the muscles, tendons, and joints. Rheumatoid arthritis (RA) is a common autoimmune disease which falls into the category of Bi Zheng based on its clinical signs and symptoms. Although the etiology of RA is unknown, environmental factors have also been shown to contribute to disease pathogenesis. .Based on the chinese medicine theory and the contemporary cognition, we established the model of rheumatoid arthritis with Wind-Cold-Damp Bi Zheng. We gavage the rats of Wind-Cold-Damp Bi Zheng to Wutou Decoction(WTD) and Simiao Pills(SMP), shows that WTD significantly improved the syndrome of model disease, and the effect of SMP was not obvious. AIA is a T cell mediated model, we found that CD4+ Th17 and IL-17 in the peripheral blood increased significantly in the model of Wind-Cold-Damp Bi Zheng. WTD was inhibited the expression of IL-17A and IL-17F.JAK/STAT pathway regulates the differentiation of CD4+T cells into Th17 and Treg, and Th17/Treg cellss imbalance mediate the development of RA. Therefore, we hypothesize that the molecular mechanism of Wutou Decoction in treatment of RA with Wind-Cold-Damp Bi Zheng is that WTD by regulating JAK/STAT pathway, inhibiting the differentiation of Th17 and promote the differentiation of Treg to regulate the imbalance of Th17/Treg cells..To prove the hypothesis, we will utilize the AIA model with Wind-Cold-Damp Bi Zheng as the useful tool, and focus on the JAK/STAT pathway. The data and results obtained from our research will shed a light on the molecular mechanisms underlying of WTD in RA characterized with a Wind-Cold-Damp Bi Zheng that based on the chinese medicine of prescription corresponding to Zheng.

阐明方证对应现代科学内涵是方剂现代研究亟待解决的关键科学问题。基于“风寒湿三气杂至合而为痹”，利用佐剂性关节炎（AIA），构建了类风湿关节炎（RA）风寒湿痹证模型。乌头汤佐证及四妙丸反证模型显示，乌头汤显著改善模型病证而四妙丸效果不明显。AIA是T细胞介导模型，检测发现模型外周血CD4+Th17及IL-17显著升高，而乌头汤抑制IL-17A和IL-17F表达。JAK/STAT通路调控CD4+T细胞分化为Th17和Treg，Th17/Treg细胞失衡介导RA发生发展。由此，我们提出假说：乌头汤通过调控JAK/STAT通路，抑制Th17分化并促进Treg分化从而调节Th17/Treg细胞失衡，是其治疗RA风寒湿痹证的分子机制。本项目以AIA风寒湿痹证模型为平台，针对调控Th17/Treg分化的JAK/STAT通路，采用分子生物学技术检测分析，阐明乌头汤方证对应治疗RA风寒湿痹证的分子机制。

阐明方证对应现代科学内涵是方剂现代研究亟待解决的关键科学问题。基于“风寒湿三气杂至合而为痹”，利用佐剂性关节炎（AIA），构建了类风湿关节炎（RA）风寒湿痹证模型。乌头汤佐证及四妙丸反证模型显示，乌头汤显著改善模型病证而四妙丸效果不明显。AIA是T细胞介导模型，检测发现模型外周血CD4+ Th17及IL-17显著升高，而乌头汤抑制IL-17A和IL-17F表达。JAK/STAT通路调控CD4+T细胞分化为Th17和Treg，Th17/Treg细胞失衡介导RA发生发展。由此，我们提出假说：乌头汤通过调控JAK/STAT通路，抑制Th17分化并促进Treg分化从而调节Th17/Treg细胞失衡，是其治疗RA风寒湿痹证的分子机制。. 本项目组基于AIA风寒湿痹证模型对成模条件进行了优化与改良，以乌头汤和四妙丸为佐证和反证，进一步确证了模型的证候属性。我们研究发现，乌头汤显著延迟AIA风寒湿痹证的发病时间，改善其体质量，改善其机械性痛敏阈值、爬杆停留时间及足肿胀体积。乌头汤显著抑制血清IL-17A、VEGF、IL-23表达，促进血清IL-35和IL-10表达。乌头汤显著抑制AIA风寒湿痹证CD4+Th17细胞比例，促进CD4+CD25+Foxp3+ Treg细胞比例表达。上述研究提示，乌头汤对AIA风寒湿痹证大鼠Th17/Treg细胞失衡的调节作用。我们研究发现，乌头汤可显著抑制AIA风寒湿痹证大鼠脾脏及滑膜JAK1、p-STAT3、RORγt蛋白表达，促进AIA风寒湿痹证大鼠脾脏及滑膜JAK3及Foxp3蛋白表达。乌头汤可显著抑制AIA风寒湿痹证大鼠脾脏RORγt mRNA表达，促进Foxp3 mRNA表达。提示乌头汤可调控JAK/STAT通路而抑制Th17分化并促进Treg分化。上述研究结果证实乌头汤通过调控JAK/STAT通路，抑制Th17分化并促进Treg分化从而调节Th17/Treg细胞失衡，是其治疗RA风寒湿痹证的分子机制。. 因此，本研究不仅阐明了乌头汤方证对应治疗RA风寒湿痹证的分子机制，也为中医学的方证对应现代科学内涵研究提供了范式。