目的 利用网络药理学及分子对接探讨三叶青解热的作用机制，并对预测结果进行实验验证。方法 运用GeneCards、DisGeNet等数据库筛选三叶青和发热的靶点，获取两者交集靶点，构建活性成分–靶点网络。然后采用STRING数据库和Cytoscape软件构建共有靶点的蛋白相互作用（PPI）网络。使用Metascape数据库对共有靶点进行基因本体（GO）和京都基因与基因组百科全书（KEGG）通路富集分析。最后运用AutoDock软件对三叶青活性成分与核心靶蛋白进行分子对接，并将网络药理学预测结果进行体内实验验证。ip脂多糖诱导小鼠发热模型，并结合三叶青水提物（2.5、12.5、25 g/kg）及阿司匹林ig给药，随后检测各组小鼠肛温，并用ELISA试剂盒检测血清中发热相关的细胞因子[肿瘤坏死因子-α（TNF-α）、白细胞介素（IL）-6、IL-1β、前列腺素E₂（PGE₂）的水平，Western blotting检测各组小鼠下丘脑组织中对应预测靶点的相对表达。结果 网络药理学结果表明，三叶青可能作用于98个靶点，与癌症通路、糖尿病并发症中的晚期搪基化终产物及其受体（AGE-RAGE）信号通路有关。分子对接结果表明大黄素-8-O-β-D-葡糖苷、花青素B1、木犀草素与热休克蛋白90α型1（HSP90AA1）、一氧化氮合酶（NOS）2、NOS3 3个核心靶蛋白具有较强的结合活性和稳定的结合构象。动物实验显示，与模型组比较，三叶青水提物2.5、12.5、25 g/kg组体温及血清中TNF-α、IL-6、IL-1β均显著下降（P＜0.05），Western blotting检测显示，三叶青水提物2.5、12.5、25 g/kg组中下丘脑组织中HSP90AA1、NOS2、NOS3表达水平均下调（P＜0.05）。结论 三叶青在解热的同时可以降低炎症及发热相关细胞因子的表达，此外三叶青的解热作用与抑制HSP90AA1、NOS2和NOS3蛋白表达有关。

Objective To investigate the antipyretic mechanisms of Tetrastigma hemsleyanum based on network pharmacology and animal experiments. Methods GeneCards, DisGeNet, and other databases were used to screen the targets of T. hemsleyanum and fever, obtain the intersection targets of the two, and construct the active ingredient-target network. Then, STRING database and Cytoscape software were used to construct a shared target PPI network. GO and KEGG pathway enrichment analysis were performed for common targets using Metascape database. Finally, AutoDock software was used to perform molecular docking between the active ingredients of T. hemsleyanum and the core target protein, and the predicted results of network pharmacology were verified in vivo. Ip lipopolysaccharide induced fever model of mice, combined with water extract of T. hemsleyanum (2.5, 12.5, 25 g/kg) and aspirin were given ig, and then anal temperature of mice in each group was measured. The levels of fever-related cytokines (TNF-α, IL-6, IL-1β, and PGE₂) in serum were detected by ELISA kit, and the relative expression of corresponding predicted targets in hypothalamus tissues of each group was detected by Western blotting. Results Network pharmacological results showed that T. hemsleyanum may act on 98 targets related to cancer pathways, AGE-RAGE signaling pathways in diabetes complications. Molecular docking results showed that emodion-1-O-β-D-glucopyranoside, procyanidin B1, and luteolin had strong binding activity and stable binding conformation with the three core target proteins HSP90AA1, NOS2 and NOS3. The animal experiments showed that compared with the model group, the body temperature and serum TNF-α, IL-6 and IL-1β of water extract of T. hemsleyanum 2.5, 12.5, and 25 g/kg groups were significantly decreased (P < 0.05). Western blotting detection showed that, the expression levels of HSP90AA1, NOS2, and NOS3 in T. hemsleyanum 2.5, 12.5, and 25 g/kg water extract groups were down-regulated (P < 0.05). Conclusion Antipyretic effect of T. hemsleyanum can reduce the expression of inflammation and febrile related cytokines. In addition, the antipyretic effect of T. hemsleyanum is related to the inhibition of the expression of HSP90AA1, NOS2 and NOS3 proteins.

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国家自然科学基金资助项目(82304808);浙江省医药卫生科技计划项目(2024KY877);浙江省中医药科技计划项目(2023ZR076)