目的 通过网络药理学和代谢组学联合分析探讨荆防颗粒（Jingfang Granules，JFG）对类风湿关节炎（rheumatoid arthritis，RA）的保护作用及机制。方法 采用完全弗氏佐剂诱导建立关节炎大鼠模型，检测足趾肿胀度、脏器指数和踝关节组织病理学变化，酶联免疫吸附法（ELISA）检测白细胞介素-6（interleukin-6，IL-6）、IL-1β、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）水平，Western blotting检测Toll样受体4（Toll-like receptor 4，TLR4）、磷酸化核因子-κB（Phosphorylated nuclear factor-κB，p-NF-κB）、髓样分化因子88（myeloid differentiation factor 88，MyD88）蛋白表达水平，评价JFG对RA的保护作用。利用UPLC-MS技术结合代谢组学寻找JFG调节的血清代谢产物和代谢途径。基于JFG入血成分，进行网络药理学分析，预测JFG入血成分的抗RA作用靶点和信号通路，并利用Western blotting技术验证JFG改善RA的关键信号通路。结果 JFG显著改善关节炎大鼠足趾肿胀程度（P＜0.05、0.01）及病理损伤，降低脾脏和胸腺指数，降低血清炎症细胞因子TNF-α、IL-1β和IL-6含量（P＜0.05、0.01），下调滑膜组织中TLR4、p-NF-κB、MyD88蛋白表达水平（P＜0.05、0.01）。血清代谢组学发现13种内源性代谢物是JFG治疗RA的潜在生物标志物，这些代谢物主要参与了不饱和脂肪酸合成、脂肪酸代谢等代谢途径。网络药理学共鉴定出JFG与RA的交集靶点364个，其中关键靶点54个。京都基因与基因组百科全书（kyoto encyclopedia of genes and genomes，KEGG）富集分析表明其潜在机制可能与磷脂酰肌醇3激酶（phosphatidylinositide 3-kinases，PI3K）/蛋白激酶B（protein kinase B，Akt）信号通路有关。此外，Western blotting结果显示，与模型组比较，JFG组大鼠滑膜组织中PI3K、Akt、哺乳动物雷帕霉素靶蛋白（mechanistic target of rapamycin kinase，mTOR）磷酸化水平明显下调（P＜0.05、0.01）。结论 JFG可能是通过抑制PI3K/Akt/mTOR通路，调节脂肪酸代谢，抑制关节滑膜炎症，改善RA大鼠关节损伤。

ObjectiveTo investigate the protective effect and mechanism of Jingfang Granules (荆防颗粒, JFG) on rheumatoid arthritis (RA) through a combined analysis of network pharmacology and metabolomics. Methods Rat model of arthritis was established using complete Freund’s adjuvant induction. Degree of toe swelling, organ index, and pathological changes in ankle joint tissue were detected. Enzyme linked immunosorbent assay (ELISA) was used to detect levels of interleukin-6 (IL-6), IL-1β and tumor necrosis factor-α (TNF-α). Western blotting was used to detect Toll like receptor 4 (TLR4), phosphorylated nuclear factor-kappa B (p-NF-κB), and myeloid differentiation factor 88 (MyD88) to evaluate the protective effect of JFG on RA. Using UPLC-MS technology combined with metabolomics to identify serum metabolites and metabolic pathways regulated by JFG. Based on the blood components of JFG, network pharmacology analysis was conducted to predict the anti RA targets and signaling pathways, and Western blotting technology was used to verify the key signaling pathways of JFG improving RA. Results JFG significantly improved the degree of toe swelling (P< 0.05, 0.01) and pathological damage in arthritis rats, decreased the spleen and thymus index (P< 0.05, 0.01), decreased the contents of serum inflammatory cytokines TNF-α, IL-1β and IL-6 (P< 0.05, 0.01), and down-regulated the expression levels of TLR4, p-NF-κB and MyD88 protein in synovial tissue (P< 0.05, 0.01). Serum metabolomics had identified 13 endogenous metabolites as potential biomarkers for JFG treatment of RA, which mainly participated in metabolic pathways such as unsaturated fatty acid synthesis and fatty acid metabolism. Network pharmacology identified 364 intersecting targets between JFG and RA, including 54 key targets. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis indicated that the underlying mechanism may be related to phosphatidylinositide 3-kinases (PI3K)/protein kinase B (Akt) signaling pathway. In addition, Western blotting results showed that compared with the model group, the phosphorylation levels of PI3K, Akt and mechanistic target of rapamycin kinase (mTOR) in synovial tissue of JFG group were significantly down-regulated (P< 0.05, 0.01). Conclusion JFG may regulate fatty acid metabolism, inhibit joint synovial inflammation, and improve joint injury in RA rats by inhibiting PI3K/Akt/mTOR pathway.

R285.5

山东省自然基金创新发展联合基金项目（ZR2022LZY029）