基于P2X7R信号通路探讨清热通络方防治痛风性关节炎的机制

Previous studies suggest that the occurrence of acute gouty arthritis (AGA) is mainly caused by the inflammatory cytokines interleukin-1beta （IL-1β） produced through monosodium urate（MSU）crystallization activating TLRs and NALP3 inflammatory signaling pathways.But the theory of gout disease is difficult to explain some clinical phenomena.For example,some patients with hyperuricemia never have got gout attacks,and serum uric acid of some patients with acute gouty arthritis is not increased.This suggests there may be other pathogenic signals in gout attacks..The latest research shows that P2X7R signaling pathway plays an extremely important role in the pathogenesis of gouty arthritis（GA）,and may be as a new target for the prevention and treatment of GA. Previous research has suggested that Qingre Tongluo Recipe has good clinical curative effect in acute gout arthritis (AGA).The mechanism of Qingre Tongluo Recipe in the treatment of gouty arthritis may be control the release of IL-1β by regulating the TLRs and NALP3 inflammatory signaling pathways.According to the research results,we put forward a scientific hypothesis on Qingre Tongluo Recipe which may prevent gout by mediating P2X7R signaling pathway.In order to reveal some molecular mechanism of Qingre Tongluo Recipe and provide the new theoretical basis for its treatments in gout,Some technologies,such as immunohistochemistry,flow cytometry（FCM）,reverse transcription-polymerase chain reaction（RT-PCR）, western-blotting ,enzyme linked immunosorbent assay（ELISA）and so on,would be adopted to study P2X7R signaling pathway in the pathogenesis of GA,and regulation function of Qingre Tongluo Recipe on rats induced monosodium urate (MSU),patients with acute gouty arthritis(AGA) and PBMCs of the patient in vitro culture.

以往研究认为，尿酸钠（MSU）结晶主要通过激活TLRs和NALP3炎性体信号通路，促进IL-1β分泌，导致急性痛风性关节炎（AGA）的发生，但这种理论难以解释部分高尿酸血症患者从不出现痛风、部分痛风急性发作者血尿酸并不增高的现象，提示痛风发病可能存在其他致病信号。.最新研究表明，P2X7R信号通路在GA发病中起着重要作用，可能作为防治GA的新靶点。前期研究表明，清热通络方有较好的疗效，能调控TLRs和NALP3炎性体信号通路，抑制IL-1β释放，而发挥治疗AGA的作用。本研究在此基础上，提出清热通络方可能介导P2X7R信号通路而发挥抗GA的科学假说。以MSU诱导的AGA大鼠、AGA患者及体外培养的AGA患者PBMCs模型为研究对象，采用免疫组化、流式细胞术、PCR等技术，探讨P2X7R信号通路在GA发病中的作用及清热通络方对其的影响，揭示该方防治GA的机制，为其治疗GA提供新的理论依据。

本项目从对P2X7R信号通路的调控作用的角度，揭示了痛风性关节炎发病及清热通络方防治该病的部分机制。并通过网络药理学研究对该方治疗急性痛风性关节炎的潜在机制和发挥疗效所依赖的物质基础进行了探讨。.1.探讨了清热通络方介导P2X7R信号通路干预急性痛风性关节炎大鼠的作用机制.研究采用改良Coderre法制备AGA大鼠模型，实验结果显示清热通络方可降低模型大鼠踝关节滑膜组织中P2X7R、IL-1β、NALP3、NF-κB(P50、65)蛋白的表达水平；并可抑制AGA模型大鼠PBMCs中P2X7R、NLRP3、ASC、Caspase-1 mRNA和蛋白的表达，及血浆中ATP、IL-1β、IL-8水平；还可降低AGA模型小鼠PBMCs的CD14+、P2X7R+细胞的表达率。.2.探讨了清热通络方介导P2X7R 信号通路干预急性痛风性关节炎患者的作用机制 .清热通络方可下调AGA患者PBMCs中P2X7R及IL-1β mRNA的表达，降低P2X7R、IL-1β、NLRP3和NF-κB（P50、P65）蛋白表达水平，及血浆中IL-1β、IL-8、ATP水平；并能降低PBMCs中CD14+、P2X7R+细胞的表达率。.3.探讨了清热通络方介导P2X7R 信号通路干预MSU诱导的急性痛风性关节炎患者PBMCs模型的体外机制. MSU或MSU+ATP干预后PBMCs 模型中 P2X7R、IL-1β、NALP3、NF-κB(P50、65)蛋白表达水平及培养上清中IL-1β、IL-8、IL-37 水平均显著升高。但清热通络方和生理盐水含药血清对各因子的调控作用并无显著差异。分析原因可能跟PBMCs包含的细胞种类较多有关。.4.分析了清热通络方的成分及治疗痛风性关节炎的靶点 .清热通络方中包含多种活性成分，可通过多条信号通路及多个生物学进程对与痛风发病密切相关的多个靶点起到潜在的调控作用。.5.探讨了清热通络方对AGA体外细胞模型（THP-1）中P2X7R信号通路的影响. MSU+ATP组的致炎作用优于MSU组和ATP组。清热通络方含药血清可抑制MSU+ATP诱导的THP-1中P2X7R、NLRP3、ASC、Caspase-1、IL-1β mRNA和蛋白的表达水平，并降低培养上清中IL-1β含量。.本项目研究结果，为清热通络方治疗AGA提供了理论依据，并可能为其进一步开发奠定了理论基础