基于外泌体调控Th17/Treg平衡探讨苗药五藤膏治疗类风湿关节炎的作用机制

The unbalance of Th17/Treg and the abnormal expression of its relevant cytokines play a key role in the development of Rheumatoid Arthritis (RA). Th17 may induce the generation of OC through highly expressed Cytokine κB receptor activation factor ligand gene which is mediated by IL-17, and they constitute Th17/IL-17 signal axis which plays a key role in RA joint inflammation and destruction. the lastest papers confirm that Exosomes(Exo) can rugulate the imbalance of Th17/Treg,and inhibit the Th17/IL-17 signal axis.our early clinical study proved the Miao medicine Wutenggao had good effect in anti-RA,based on that, we come up with a hypothesis that Miao medicine Wutenggao can alleviate joint inflammation and destruction by regulating Exo to suppress Th17/IL-17 signal axis,and it also can regulate the balance of Th17/Treg to relieve systemic inflammation. we plan to build up RA animal model, adopt molecular biological technique to probe the molecular mechanism of Miao medicine Wutenggao in inhibitting Th17/IL-17 signal axis by regulating Exo at the area of alleviating joint inflammation and destruction in joints, which called eliminating inflammation nearby,and to probe the molecular mechanism of regulating the balance of Th17/Treg, which called regulate functions of viscera.thus it can enrich the theroy of Miao medicine in treating an internal illness by external treatment, and establish the experimental foundation of the developpment of Miao medicine Wutenggao.

Thl7/Treg失衡及其相关细胞因子的异常表达是类风湿关节炎（RA）发生发展的关键。Th17通过IL-17介导滑膜细胞高表达核因子κB受体活化因子配体基因，诱导破骨细胞生成，二者共同构成Th17/IL-17信号轴在RA关节局部炎症和骨破坏中起关键作用，最新研究文献发现外泌体（Exo）能调节Thl7/Treg失衡，并抑制Th17/IL-17信号轴。我们前期临床研究证实苗药五藤膏外治具有良好的抗RA疗效。基于此，本项目提出苗药五藤膏可通过调控Exo抑制Th17/IL-17信号轴缓解关节局部炎症和骨破坏，调节Th17/Treg平衡缓解全身炎症的假说。拟通过建立RA动物模型，利用分子生物学技术研究苗药五藤膏调控Exo抑制Th17/IL-17信号轴缓解关节局部炎症和骨破坏以“就近祛邪”，调节Th17/Treg平衡“内调脏腑”的分子机制，进一步丰富苗医内病外治理论，为开发苗药五藤膏奠定实验基础。

类风湿关节炎（RA）一种慢性、进行性、侵蚀性疾病，以对称性关节炎为主要临床表现，具有发病率高、复发率高和致残率高的特点。目前RA的发病机制尚不清楚，关节慢性滑膜炎症是RA的主要病理特征，骨质侵蚀是受累关节长期慢性炎症导致局部骨破坏的结局，如何缓解关节滑膜炎症，阻断RA的骨破坏，是控制RA病情进展的关键。本研究将Wistar大鼠随机分为模型组、空白组、苗药五藤膏高、中、低剂量组、IL-17阻断组及双氯芬酸二乙胺乳胶剂组，建立CIA模型，造模第7天开始给药，28天后处死大鼠取材检测。HE染色结果：苗药五藤膏高、中剂量组可见更加清晰的关节面、关节间隙，关节腔内仅见少量炎症细胞及滑膜组织浸润，关节两端骨组织结构未见明显破坏，伴见骨质疏松。TRAP染色结果：苗药五藤膏高、中剂量组滑膜衬里细胞稍增厚，滑膜内可见少量破骨细胞及炎细胞浸润，另可见少量新生血管。ELISA检测结果：苗药五藤膏各治疗组均能降低关节组织TNF-α、IL-1、IL-6表达；苗药五藤膏各治疗组均能降低大鼠血清IL-1、IL-17、TNF-α、IL-6的表达，可提高大鼠血清IL-10、TGF-β的表达。Western Blot检测结果：苗药五藤膏各治疗组可减低关节组织中RANKL的表达；苗药五藤膏各治疗组均可提高脾脏组织Foxp3的表达，降低脾脏组织RORγt的表达。RT-PCR检测结果：苗药五藤膏高、中剂量组可降低关节组织RANKL mRNA的表达；苗药五藤膏各治疗组均可提高脾脏组织Foxp3 mRNA的表达，降低脾脏组织RORγt mRNA的表达。FCM检测结果：苗药五藤膏高、中剂量组能降低大鼠脾脏组织CD4+T淋巴细胞中Th17细胞百分比，能增加Treg细胞百分比。以上研究结果表明苗药五藤膏可“就近驱邪”从而减轻关节炎症和骨破坏，可能与调控Th17/RANKL信号轴有关；苗药五藤膏可“内调脏腑”，可能与调节Th17/Treg平衡，缓解CIA大鼠全身炎症反应有关。该研究进一步丰富了苗医内病外治理论，为开发苗药五藤膏奠定了实验基础。