基于脂代谢网络信号通路探讨艾灸对类风湿关节炎多靶向调节机制研究

Moxibustion therapy and rehabilitation therapy for Rheumatoid Arthritis (RA) could be found in both TCM classic and modern literature. Literature reports and our previous studies confirmed that the pathology and moxibustion therapy of RA are associated with the changes of lipid metabolism and signaling pathway networks. Lipids are key molecules of RA. However, the mechanism has not yet been systematically clarified. The advantage of lipidomics is the systematic study of lipids. In addition, the use of lipidomics techniques align very closely with the concepts and practices of moxibustion, which pays much attention to integrity. Thus, we hypothesized that the application of lipidomics analysis for the moxibustion treating RA could screen and identify specific target molecules, and explore the mechanism of moxibustion on RA. At present, the advanced applications of lipidomics are based on combined untargeted and targeted strategy, which can provide systematic and in-depth research. On the basis of previous studies, the present study will make use of rats with RA as models, observe the general conditions and relevant biological indicators of anti-inflammation and immunity improvement effects among normal rats, normal rats with moxibustion, RA rats, RA rats with moxibustion. And then observe changes in lipid metabolism in blood, urine and synovial tissue of rats with RA after moxibustion with the methods of the mainstream mass spectrometric technique, and integrated untargeted and targeted complementary lipidomics approaches, thus in order to explore the network channels and multiple targets which are closely related to the pathology and moxibustion therapy of RA, to identify the material foundation of moxibustion effects, and further provide the objective experimental evidence for the mechanism of moxibustion therapy and rehabilitation therapy.

艾灸对类风湿关节炎（RA）治疗康复见于古代典籍和现代文献。文献报道及本课题组研究表明RA及其艾灸治疗涉及脂质代谢网络及信号通路变化，脂质是RA关键分子，但确切机制尚未系统阐明。而系统研究脂质是脂质组学优势，脂质组学整体、系统研究特点也与艾灸整体观治疗康复特点相符。因此提出假说：通过艾灸治疗RA脂质组学分析，筛选调控物质靶点，揭示对潜在内源性标志物及效应表达影响，而发挥治疗康复效应机制。目前，联合非靶向和靶向分析是脂质组学新方向，可提供系统、深入的研究。本项目在既往研究基础上，在观察艾灸改善RA大鼠一般情况、抗炎、提高免疫力等具体效应同时，采用主流的液相色谱质谱技术，结合血、尿、滑膜组织等多样本，整合互补的非靶向和靶向脂质组学，从整体上多角度、多层次比较RA及艾灸治疗所涉及脂质代谢变化，探讨密切相关的脂代谢网络信号通路和艾灸多靶向作用靶点，明确效应物质基础，为揭示其治疗康复机制提供科学依据。

类风湿关节炎（Rheumatoid arthritis, RA）是慢性反复的临床常见病和多发病。古代典籍和现代文献表明艾灸治疗RA 历史悠久，具有独特的疗效和优势。RA及其艾灸治疗存在脂质代谢变化。借助于代谢组学及其重要的分支-脂质代谢组学（即脂质组学）技术，可以系统的比较研究脂质代谢物分子及其代谢网络和信号通路变化，这种研究思路符合中医整体观的特点。因此，本课题应用气相和液相色谱质谱技术，采用非靶向和靶向的研究策略，基于血清/血浆、尿液、关节滑膜组织开展艾灸治疗RA大鼠的差异代谢组学和脂质组学研究。重要结果发现如下：RA发生、发展及其艾灸治疗牵涉多种脂质代谢物及其代谢通路变化。（1）代谢组学研究发现：主要脂质代谢物是胆固醇和甘油。胆固醇是与RA密切相关的化合物。研究表明，RA病人及RA大鼠胆固醇水平降低，而艾灸可以使RA胆固醇水平回调。甘油磷脂代谢通路是RA及其艾灸治疗中起作用的主要代谢通路之一。（2）非靶向脂质组学研究发现：艾灸可以回调RA异常的血浆31个脂质代谢物，滑膜组织24个脂质代谢物，还可以调节正常大鼠的血浆21个脂质代谢物，滑膜组织16个脂质代谢物。通过代谢通路分析发现艾灸治疗RA的特异性病理效应机制是调控α-亚麻酸代谢、甘油磷脂代谢和鞘脂代谢通路。而艾灸的非特异性生理效应则是通过调控甘油磷脂代谢和α-亚麻酸代谢发挥作用。（3）靶向脂质组学研究发现：艾灸治疗RA的特异性病理效应机制是调控亚油酸代谢和花生四烯酸代谢。而艾灸的非特异性生理效应则是通过调控亚油酸代谢发挥作用。艾灸通过下调13-HDHA、14-HDHA、17-HDHA、8-HEPE、11-HEPE、12-HEPE、15-HEPE等花生四烯酸代谢产物发挥特异性病理效应，通过上调5-HETE、8-HETE、12-HETE、5,6-diHETE，下调FFA14:0、FFA16:1发挥非特异性生理效应。总之，艾灸对RA引起的脂质代谢改变有明显的逆转作用。艾灸的治疗效应包括特异性病理效应和非特异性生理效应。通过代谢组学和脂质组学技术，我们发现并验证了与RA病变及艾灸治疗密切相关的脂质代谢物及其代谢调节通路，为阐明艾灸的疗效机制奠定了基础。