蠲痹汤调节淋巴管内皮细胞TNFα/iNOS/NO信号通路促进淋巴回流防治类风湿性关节炎的研究

Rheumatoid arthritis (RA) can lead to irreversible joint deformity, causing serious pain to patients. Previously, we found that lymphatic system plays an important role in RA, inflammatory cytokines stimulate lymphatic endothelial cells (LECs) express iNOS and produce nitric oxide (NO) to damage the structure and function of smooth muscle cells (LSMCs) at collect lymphatic vessels, lead to dysfunction of lymphatic drainage, and aggravate arthritis. But the molecular mechanism of iNOS expression of LECs induced by TNFα is not clear. Juanbi Tang of <YiXueXinWu> is commonly used as traditional Chinese medicine for the treatment of RA. Our previous studies suggested that Juanbi Tang could attenuate joint inflammation and promote lymphatic drainage in a mouse model of RA, and reduce iNOS expression and NO production of LECs induced by TNFα. But the mechanisms need further study. The aim of current project is to apply TNF-Tg RA mouse models and in vitro culture of LECs to research the molecular mechanisms of TNFα induced iNOS expression of LECs and Juanbi Tang block TNFα/iNOS/NO signaling pathway of LECs to reduce the impairment of LSMCs, improve the lymphatic drainage function, and alleviate the inflammation severity of RA.

类风湿性关节炎（RA）可导致不可逆的关节畸形，给患者带来严重的痛苦。本课题组前期研究发现淋巴系统与RA发病紧密相关，RA慢性炎症造成淋巴管内皮细胞（LECs）表达iNOS，产生一氧化氮（NO），损伤集合淋巴管平滑肌细胞（LSMCs）的结构和功能，导致淋巴回流障碍，加重关节炎症。但炎症因子诱导LECs表达iNOS的信号通路分子机制并不清楚。《医学心悟》之蠲痹汤是中医治疗RA的常用方剂，前期研究发现其改善RA小鼠关节炎症，促进淋巴回流，减轻炎症诱导LECs表达iNOS和NO, 但药理机制仍需深入研究。本项目以TNF-Tg RA小鼠模型和体外培养LECs为研究对象，研究炎症因子TNFα诱导LECs表达iNOS的分子机制及蠲痹汤调控LECs的TNFα/iNOS/NO信号通路，减少NO损伤LSMCs，改善淋巴回流功能，减轻RA的药理机制。

目的：研究蠲痹汤通过保护淋巴管回流功能免于炎症损害，以减少关节内炎性产物的累积，从而改善炎症性关节炎的机制；初步探索蠲痹汤中具有以上效应的有效成分；探讨保护淋巴回流功能是否为蠲痹汤治疗炎症性关节炎的作用靶点，为指导中医临床用药提供新的思路。.主要研究内容：研究蠲痹汤对TNF-Tg小鼠淋巴管回流功能及关节炎的作用，蠲痹汤冻干粉对淋巴管内皮细胞的作用，蠲痹汤有效单体成分的筛选及作用机制及紫花前胡苷对TNF-Tg小鼠淋巴管回流功能及关节炎的作用机制。.结果：ICG-NIR检测显示，蠲痹汤治疗12周后，可促进淋巴管搏动次数及清除率的恢复；踝关节形态学染色显示，蠲痹汤可抑制炎症及破骨细胞，改善骨及软骨侵蚀。HPLC显示，蠲痹汤冻干粉质量稳定；NO测定显示，蠲痹汤冻干粉可抑制炎症刺激下LEC产生过量的NO，Q-PCR及Western blot 显示，蠲痹汤冻干粉可抑制iNOS的表达。在共培养条件下，蠲痹汤冻干粉可促进LSMC功能标志物的恢复，并无明显细胞毒性。通过NO测定，筛选出紫花前胡苷对iNOS具有抑制作用，并可通过改效应对LSMC起到保护作用。紫花前胡苷治疗12周后，可促进淋巴管搏动次数及清除率的恢复并抑制炎症及破骨细胞，在一定程度上改善骨及软骨侵蚀。.结论：蠲痹汤可治疗TNF-α高表达下踝关节炎症及骨侵蚀，其作用机制可能通过抑制LEC炎症下产生的NO对LSMC收缩功能的损伤，从而保证淋巴管对炎性物质的回流功能。蠲痹汤的以上效应可能是通过羌活中的紫花前胡苷起作用。