目的 基于网络药理学整合体内验证方法探索盘龙七片对膝骨关节炎疼痛大鼠的干预作用及相关机制。方法 从疾病及药物相关数据库筛选盘龙七片药物作用靶点及膝骨关节炎疼痛相关靶点，根据拓扑特征值筛选关键靶标后，进行基因本体（gene ontology，GO）功能及京都基因与基因组百科全书（Kyoto encyclopedia of genes and genomes，KEGG）通路富集分析。采用改良Hulth法建立大鼠膝骨关节炎疼痛模型，设置假手术组、模型组、美洛昔康（1.5 g/kg）组和盘龙七片低、中、高剂量（0.15、0.30、0.60 g/kg）组，给予药物干预42 d，测量给药后大鼠机械痛阈值、冷痛觉超敏反应评分及双后肢负重差，并对网络药理预测结果进行体内实验验证。结果 共筛选出175个盘龙七片治疗膝骨关节炎疼痛的潜在核心靶点，生物学功能富集在调节神经细胞对各种刺激反应、炎症反应、细胞内信号传导及代谢反应4个过表达模块，其中Wnt/Ca²⁺途径核心靶标富集较为显著，也是在调节神经细胞对各种刺激反应生物学过程富集程度最高的通路。体内实验结果显示，盘龙七片可明显升高给药3～12 d及24～30 d膝骨关节炎大鼠机械痛阈值（P＜0.05、0.01、0.001），显著降低给药3～42 d时膝骨关节炎大鼠冷痛觉超敏反应评分与双后肢负重差（P＜0.05、0.01、0.001），抑制膝骨关节炎疼痛大鼠脊髓中无翅型MMTV整合位点家族成员5A（wingless type MMTV integration site family member 5A，WNT5A）、重组磷脂酶C（phospholipase C，PLC）、蛋白激酶C（protein kinase C，PKC）蛋白表达及肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、白细胞介素-1β（interleukin-1β，IL-1b）、前列腺素E₂（prostaglandin E₂，PGE₂）水平（P＜0.05、0.01、0.001）。结论 盘龙七片对大鼠实验性膝骨关节炎急性发作期和慢性持续性的疼痛均有缓解作用，其机制可能与调节Wnt/Ca²⁺信号通路有关。

Objective To explore the interventional effect and related mechanism of Panlongqi Tablet (盘龙七片) on rats with knee osteoarthritis pain through network pharmacology analysis and in vivo validation method. Methods Drug targets of Panlongqi Tablet and targets related to knee osteoarthritis pain were screened from disease and drug related database, and key targets were screened according to topological eigenvalues. After that, modular enrichment analysis of function and pathway was conducted based on gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG). Rats with knee osteoarthritis pain model established by modified Hulth method were divided into sham group, model group, meloxicam (1.5 g/kg) group and Panlongqi Tablet low-, medium-, high-dose (0.15, 0.30, 0.60 g/kg) group, drug intervention was given for 42 d. Mechanical pain threshold, cold pain hypersensitivity score and weight bearing difference of both hind limbs were measured after treatment, and network pharmacological prediction results were verified in vivo. Results A total of 175 potential core targets of Panlongqi Tablet for treatment of knee osteoarthritis pain were selected based on topological screening parameters. Their biological functions focus on four overexpression modules including regulating nerve cell responses to various stimuli, regulating inflammatory response process, regulating intracellular signal transduction and metabolic response. Wnt/Ca²⁺ pathway was a significant pathway for core target enrichment, and also a pathway with the highest enrichment degree in the biological process of regulating nerve cell responses to endogenous, hormonal, exogenous and other stimuli. In vivo experiment results showed that Panlongqi Tablet significantly increased the mechanical pain threshold of rats with knee osteoarthritis pain from 3—12 d and 24—30 d after treatment, and decreased the cold pain hypersensitivity score and weight bearing difference on both hind limbs of rats from 3—42 d after treatment (P < 0.05, 0.01, 0.001), and inhibited wingless type MMTV integration site family member 5A (WNT5A), phospholipase C (PLC) and protein kinase C (PKC) protein expressions as well as levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and prostaglandin E₂ (PGE₂) in spinal cord of rats with knee osteoarthritis pain (P < 0.05, 0.01, 0.001). Conclusion Panlongqi Tablet can alleviate both acute onset and chronic persistent pain of experimental knee osteoarthritis in rats, and its mechanism may be related to the regulation of Wnt/Ca²⁺ signaling pathway.

R285.5

中国中医科学院科技创新工程项目(C12021A03808);国家"重大新药创制"科技重大专项(2019ZX09731-002);中国中医科学院中药研究所技术研发项目(20191306)