加味牛蒡子汤调控巨噬细胞TNF/TNFR1/VEGF-C信号通路促进淋巴管生成减轻类风湿性关节炎的研究

Rheumatoid arthritis (RA) can lead to synovial inflammation and irreversible joint deformity. Previously, we found that lymphatic system plays an important role in RA, promoting lymphangiogenesis attenuates, while blocking lymphangiogenesis accelerates inflammatory tissue damage of RA. It was reported that macrophage plays an important role in inflammation induced lymphangiogenesis, but at RA joints, the deep mechanism how inflammatory cytokine induced VEGF-C expression of macrophage deserves further research. JiaWeiNiuBangZi Tang is formed by professor Shi Qi, a famous traditional Chinese medicine doctor of Shi’s Traumatology, which is commonly used for the treatment of RA in phlegm and blood stasis type. Our previous studies suggested that JiaWeiNiuBangZi Tang could attenuate joint inflammation and promote lymphatic drainage in a mouse model of RA, and its component Panax Notoginseng Saponins promotes lymphangiogenesis by stimulating VEGF-C expression, but the mechanisms of JiaWeiNiuBangZi Tang on joint inflammation induced lymphangiogenesis need further study. The aim of current project is to apply TNF-Tg RA mouse models and in vitro culture of macrophage and lymphatic endothelial cells to research the molecular mechanisms of TNFα induce macrophage to produce VEGF-C and evoke lymphangiogenesis and JiaWeiNiuBangZi Tang modulates TNF/TNFR1/VEGF-C signaling pathway of macrophage to promote VEGF-C production and lymphangiogenesis, 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和功能受限。本课题组前期发现淋巴系统与RA发病紧密相关，促进淋巴管生成可减轻、而抑制淋巴管生成则加重关节炎症；巨噬细胞在慢性炎症状态下促进淋巴管生成，但炎症因子诱导巨噬细胞表达VEGF-C的信号通路分子机制并不清楚。加味牛蒡子汤是上海石氏伤科施杞教授创立治疗痰瘀互结型RA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；其有效组分三七总皂苷通过刺激VEGF-C表达，促进淋巴管生成。但加味牛蒡子汤通过何种机制促进炎症下巨噬细胞表达VEGF-C及刺激淋巴管生成仍有待深入研究。本项目以TNF-Tg慢性RA小鼠模型和体外培养巨噬细胞和淋巴管内皮细胞为研究对象，研究炎症因子TNFα诱导巨噬细胞表达VEGF-C的分子机制以及加味牛蒡子汤调控巨噬细胞TNF/TNFR1/VEGF-C信号通路促进VEGF-C表达以及促进淋巴管生成、减轻RA的机制。

目的：研究加味牛蒡子汤及有效成分毛蕊异黄酮抑制巨噬细胞炎症诱导的LECs炎症、调控淋巴系统结构与功能，防治类风湿关节炎的抗炎免疫效用及其药理机制。.方法：体内建立TNF-α过表达的动物模型（TNF-Tg小鼠），给予加味牛蒡子汤或毛蕊异黄酮灌胃给药治疗，观察治疗后小鼠体重、RA症状评分、热痛阈行为学等改变，并从腘窝淋巴结三维成像体积大小、淋巴系统功能以及踝关节病理形态变化等方面评价药物通过调控淋巴系统对RA炎症的影响；体外分离、以20ng/mL的M-CSF诱导培养原代巨噬细胞，并培养淋巴管内皮细胞株，通过流式分析鉴定细胞、RT-qPCR、Western blot、细胞免疫荧光等方法研究毛蕊异黄酮对5ng/mL TNF-α刺激巨噬细胞炎症的药理效应及其诱导淋巴管内皮细胞炎症反应的影响，探索其防治RA的药理作用机制。.结果：加味牛蒡子促进TNF-Tg小鼠淋巴管新生，减轻关节炎症部位M1型巨噬细胞聚集的炎症免疫反应，促进踝关节处软骨与骨修复；毛蕊异黄酮能够改善TNF-Tg小鼠下肢淋巴回流功能，促进淋巴管的新生，改善炎症局部微循环，清除更多的炎症性M1型巨噬细胞，减轻RA。毛蕊异黄酮在一定浓度时可有效抑制TNF-α（5ng/mL）刺激的巨噬细胞炎症，抑制巨噬细胞NF-κB信号通路p65蛋白表达和磷酸化入核、上游信号分子IKKα/β蛋白表达，抑制MAPK信号通路的JNK蛋白表达。TNF-α（5ng/mL）刺激的巨噬细胞炎症会诱导淋巴管内皮细胞炎症，形成炎症循环，但50μM毛蕊异黄酮干预后，巨噬细胞炎症对淋巴管内皮细胞的炎症刺激作用减弱。.结论：加味牛蒡子汤可修复RA状态下淋巴管结构，其有效成分毛蕊异黄酮通过抑制巨噬细胞NF-κB和JNK信号通路，减轻巨噬细胞炎症，降低其对淋巴管内皮细胞促炎作用，改善淋巴回流功能，减轻TNF-Tg小鼠的关节炎症。该结果进一步阐明了加味牛蒡子汤及其有效成分调控淋巴管功能治疗类风湿关节炎的药理机制，为其临床应用提供了临床前证据。