目的 基于转录组学与网络药理学探讨四妙散治疗膝骨关节炎（KOA）的作用机制。方法 将32只SD大 鼠随机分为正常对照组、模型组、阳性对照组（硫酸氨基葡萄糖 0.135 g·kg-1 ）及四妙散组（8.640 g·kg-1 ），每组 8 只。通过膝关节腔注射碘乙酸钠溶液复制KOA大鼠模型。各组大鼠灌胃给药，每日1次，持续给药4周。采 用 Lequesne MG 评分，HE 及番红-固绿染色法观察软骨及滑膜组织病理变化，ELISA 法检测血清白细胞介素 （IL）-1β、IL-18、肿瘤坏死因子（TNF）-α 水平，评估四妙散治疗 KOA 的疗效。提取大鼠膝关节软骨组织总 RNA进行转录组学测序，筛选差异表达基因并进行GO功能及KEGG通路富集分析。通过网络药理学方法预测 四妙散治疗KOA的潜在作用靶点，并通过网络拓扑分析筛选核心成分及核心靶点。对四妙散治疗KOA的潜在 作用靶点进行GO功能及KEGG通路富集分析。整合转录组学与网络药理学分析的KEGG通路富集结果，采用 Western Blot法检测大鼠软骨组织中AMPK/mTOR通路相关蛋白的表达水平。结果 （1）与模型组比较，四妙散 能显著降低KOA大鼠的Lequesne MG评分（P＜0.01），减轻关节软骨损伤和滑膜炎症，并显著降低血清中炎症因 子IL-1β、IL-18、TNF-α水平（P＜0.01）。（2）与正常对照组比较，KOA大鼠软骨组织中有876个差异表达基因， 四妙散干预后能够有效逆转其中141个差异表达基因，包括69个上调基因、72个下调基因。141个差异表达基 因主要涉及 AMPK 信号通路、钙离子信号通路和 Apelin信号通路。（3）获得四妙散的活性成分共 42个，预测得 到224个作用靶点，2 864个KOA疾病相关靶点，取交集得到128个四妙散治疗KOA的潜在作用靶点。四妙散 治疗 KOA 的核心成分包括槲皮素、山柰酚、汉黄芩素、黄芩素及 β-谷甾醇等；核心靶点为 IL-6、TNF、 AKT1、IL-1β、MMP-9等；潜在作用靶点主要参与调控PI3K-AKT信号通路、MAPK信号通路以及mTOR信号 通路等。（4）与正常对照组比较，模型组大鼠软骨组织中p-AMPK/AMPK蛋白相对表达比值显著下调（P＜0.01）， p-mTOR/mTOR 蛋白相对表达比值显著上调（P＜0.01）。与模型组比较，四妙散组大鼠软骨组织中 p-AMPK/ AMPK 蛋白相对表达比值明显上调（P＜0.05），p-mTOR/mTOR 蛋白相对表达比值显著下调（P＜0.01）。结论 四妙散可能通过槲皮素、山柰酚、汉黄芩素等活性成分，作用于 TNF、IL-1β、MMP-9 等核心靶点，调控 AMPK/mTOR等关键信号通路，改善KOA大鼠关节功能，减轻软骨损伤和滑膜炎症，从而发挥抗KOA作用。

Objective To investigate the mechanism of action of Si Miao San in the treatment of knee osteoarthritis （KOA） based on transcriptomics and network pharmacology. Methods Thirty-two SD rats were randomly divided into normal control group，model group，positive control group （glucosamine sulfate 0.135 g·kg-1 ） and Si Miao San group （8.640 g·kg-1 ），with 8 rats in each group. The KOA rat model was replicated by intra-articular injection of monosodium iodoacetate （MIA） solution. Rats in each group were given intragastric administration once a day for 4 consecutive weeks. Lequesne MG score，HE and Safranin O- Fast Green staining were used to observe the pathological changes of cartilage and synovial tissue，and ELISA was used to detect the levels of serum IL-1β，IL-18 and TNF-α，so as to evaluate the efficacy of Si Miao San in the treatment of KOA. RNA was extracted from rat knee joint cartilage tissue for transcriptomic sequencing， differentially expressed genes were screened and GO function and KEGG pathway enrichment analysis were performed. The potential targets of Si Miao San in the treatment of KOA were predicted by network pharmacology， and the core components and core targets were screened by network topology analysis. GO function and KEGG pathway enrichment analysis were performed on the potential targets of Si Miao San in the treatment of KOA. The KEGG pathway enrichment results of transcriptomic and network pharmacology analysis were integrated， and the expression levels of AMPK/mTOR pathway-related proteins in rat cartilage tissues were detected by Western Blot. Results （1） Compared with the model group，Si Miao San could significantly reduce the Lequesne MG score of KOA rats （P＜0.01）， reduce articular cartilage injury and synovitis， and significantly reduce the levels of inflammatory factors IL-1β，IL-18 and TNF-α in serum （P＜0.01）. （2） Compared with the normal control group， there were 876 differentially expressed genes in the cartilage tissue of KOA rats. After intervention with Si Miao San， 141 differentially expressed genes were effectively reversed，including 69 up-regulated genes and 72 down-regulated genes. The 141 differentially expressed genes were mainly involved in AMPK signaling pathway， calcium signaling pathway and Apelin signaling pathway. （3） A total of 42 active components of Si Miao San were obtained，224 targets and 2 864 KOA disease-related targets were predicted，and 128 potential targets of Si Miao San in the treatment of KOA were obtained by intersection. The core components of Si Miao San in the treatment of KOA include quercetin， kaempferol，wogonin，baicalein and β-sitosterol. The core targets were IL-6，TNF，AKT1，IL-1β，MMP-9 and so on. The potential targets are mainly involved in the regulation of PI3K-AKT signaling pathway， MAPK signaling pathway and mTOR signaling pathway. （4） Compared with the normal control group，the relative expression ratio of pAMPK/AMPK protein in the cartilage tissue of the model group was significantly down-regulated （P＜0.01），and the relative expression ratio of p-mTOR/mTOR protein was significantly up-regulated （P＜0.01）. Compared with the model group，the relative expression ratio of p-AMPK/AMPK protein in cartilage tissue of rats in the Si Miao San group was significantly up-regulated （P＜0.05），and the relative expression ratio of p-mTOR/mTOR protein was significantly down-regulated （P＜0.01）. Conclusion Si Miao San may act on core targets such as TNF， IL-1β and MMP-9 through active ingredients such as quercetin，kaempferol and wogonin，regulate key signaling pathways such as AMPK/ mTOR，improve joint function in KOA rats，reduce cartilage damage and synovial inflammation，and thus play an anti-KOA role.

R285.5

湖南省卫生健康委科研计划项目（202204075703）