痛风清消方调控痛风性关节炎自行消散及基于TGF-β1信号通路的中性粒细胞外捕作用研究

Gout is a crystal associated arthropathy caused by the deposition of monosodium urate and refers to especially characteristic acute arthritis and chronic tophaceous disease. Neutrophil extracellular traps participate in the acute flare of gouty arthritis and the formation of chronic gouty tophus. Induction of transforming growth factor beta1 not only drives the process of inflammatory spontaneous resolution, but also improves the inflammatory function of granulocytes. Gouty arthritis has a typical self-limiting feature, but there is still a lack of reasonable explanation for its recurrent attacks. The applicant believes that self remission after acute episodes is a transient immune balance which stands for inflammatory spontaneous resolution overcoming chronic inflammatory state triggered by the sedimentary crystal. The nature, intensity and duration of the inflammatory reaction are closely regulated by the body, and the single-target drug cannot find the fit point. The Chinese herbal compound embodies the holistic view of Traditional Chinese Medicine and has the systematic pharmacological effect with the characteristics of various channels at multiple levels through multiple targets. It is expected to mobilize innate immune system to regulate a series of key molecules in continuous inflammation, such as interleukin-1 beta and transforming growth factor β1. Tongfengqingxiao prescription comes from Simiao Powder, and has a reliable curative effect on gouty arthritis. The applicant intends to prepare a rat model for the combination of disease and syndrome，subsequently to investigate the effect of monosodium urate microcrystal stimulation on NETosis（neutrophil extracellular traps formation）by the method of seropharmacology，and then to explore the effect of Tongfengqingxiao prescription network modulating on the transforming growth factor β1 signaling pathway in the spontaneous resolution phase of gouty arthritis.

痛风是由单钠尿酸盐沉积所致的晶体相关性关节病，特指急性特征性关节炎和慢性痛风石疾病。中性粒细胞胞外诱捕网参与痛风急性发作及慢性痛风石形成。它诱导的TGF-β1既促进炎症自行消散，又改善粒细胞炎性功能。痛风性关节炎（GA）有典型的自限性特征，但对其反复发作目前尚缺乏合理解释。申请者认为GA发作后自发缓解是炎症自行消散暂时战胜由晶体触发慢性炎症的免疫平衡。炎症反应的性质、强度和持续时间受机体严密调控，单靶点药物无法找到契合点。中药复方有多途径、多层次、多靶点的系统药理学作用，也体现中医整体观，有望通过调动固有免疫系统调控持续炎症中一系列关键分子（如IL-1β、TGF-β1）。痛风清消方乃自四妙散化裁而来，临床治疗GA疗效确切。申请者拟制备病证结合大鼠模型，再用血清药理学方法研究单钠尿酸盐微晶体刺激中性粒细胞后胞外诱捕网的形成，并探讨痛风清消方在GA自行消散中网络调控TGF-β1信号通路的作用。

痛风是由单钠尿酸盐沉积所致的晶体相关性关节病，特指急性特征性关节炎和慢性痛风石疾病。中性粒细胞胞外诱捕网参与痛风急性发作及慢性痛风石形成。它诱导的TGF-β1既促进炎症自行消散，又改善粒细胞炎性功能。课题组考虑GA发病与TFQXF治疗GA的作用机制可能与中性粒细胞的活性密切相关，并提出假说：TFQXF可能通过TGF-β1信号通路介导的中性粒细胞外捕网，调节机体炎症因子水平，促进炎症自行消散，从而达到治疗GA的目的。申请者通过制备病证结合大鼠模型并进行药物干预，再用血清药理学方法研究单钠尿酸盐微晶体刺激中性粒细胞后胞外诱捕网的形成，并探讨痛风清消方在GA自行消散中网络调控TGF-β1信号通路的作用。结果表明①血脂代谢指标及水通道蛋白可作为湿热蕴结型GA“湿”证表现的客观评价指标，ET、HSP70及CGRP可作为湿热蕴结型GA“热”证表现的客观评价指标，免疫炎症指标可作为湿热蕴结型GA炎症程度的客观评价指标。②湿热蕴结型GA发病与机体中的免疫细胞浸润密切相关，受MSU晶体刺激的单核细胞可极化为M1巨噬细胞，然后通过吞噬MSU晶体并将其递送至NLRP3炎性小体，从而产生TNF-α、IL-1β等促炎因子，并促进继发性中性粒细胞生理性流入炎症部位。中性粒细胞活化后进一步导致大量趋化因子、促炎性细胞因子、ROS、PGE2及溶酶体酶等物质的释放，从而放大GA局部炎症反应。③TFQXF可能通过槲皮素、山柰酚、柚皮素、木犀草素、汉黄芩素等113个活性成分作用于IL-6、MAPK3、IL-1β、PTGS2、CCL2等43个作用靶点，从而发挥正向调控TGF-β1水平并激活相应的信号通路，有效抵御JAK2依赖的促炎细胞因子形成，改善MSU晶体诱导的无菌性炎症，最终使机体达到免疫自稳。④TFQXF可通过TGF-β1信号通路活化，抑制中性粒细胞募集及NETs的释放，从而调节中性粒细胞外捕作用，促进抗炎因子的释放并抑制促炎因子的分泌，减轻MSU晶体诱发的炎症反应，促使炎症自行消散，避免关节软骨及软骨下骨的破坏。旨在前期TFQXF治疗GA的相关机制研究基础上进一步探索和延伸，期望继续深入探寻TGF-β1信号通路介导的中性粒细胞外捕网在TFQXF调节GA自行消散中的作用机制，为全面认识GA的发病机制及临床治疗提供更充分的理论和实验依据。