目的 利用網(wǎng)絡藥理學及分子對接探討三葉青解熱的作用機制，并對預測結(jié)果進行實驗驗證。方法 運用GeneCards、DisGeNet等數(shù)據(jù)庫篩選三葉青和發(fā)熱的靶點，獲取兩者交集靶點，構(gòu)建活性成分–靶點網(wǎng)絡。然后采用STRING數(shù)據(jù)庫和Cytoscape軟件構(gòu)建共有靶點的蛋白相互作用（PPI）網(wǎng)絡。使用Metascape數(shù)據(jù)庫對共有靶點進行基因本體（GO）和京都基因與基因組百科全書（KEGG）通路富集分析。最后運用AutoDock軟件對三葉青活性成分與核心靶蛋白進行分子對接，并將網(wǎng)絡藥理學預測結(jié)果進行體內(nèi)實驗驗證。ip脂多糖誘導小鼠發(fā)熱模型，并結(jié)合三葉青水提物（2.5、12.5、25 g/kg）及阿司匹林ig給藥，隨后檢測各組小鼠肛溫，并用ELISA試劑盒檢測血清中發(fā)熱相關(guān)的細胞因子[腫瘤壞死因子-α（TNF-α）、白細胞介素（IL）-6、IL-1β、前列腺素E₂（PGE₂）的水平，Western blotting檢測各組小鼠下丘腦組織中對應預測靶點的相對表達。結(jié)果 網(wǎng)絡藥理學結(jié)果表明，三葉青可能作用于98個靶點，與癌癥通路、糖尿病并發(fā)癥中的晚期搪基化終產(chǎn)物及其受體（AGE-RAGE）信號通路有關(guān)。分子對接結(jié)果表明大黃素-8-O-β-D-葡糖苷、花青素B1、木犀草素與熱休克蛋白90α型1（HSP90AA1）、一氧化氮合酶（NOS）2、NOS3 3個核心靶蛋白具有較強的結(jié)合活性和穩(wěn)定的結(jié)合構(gòu)象。動物實驗顯示，與模型組比較，三葉青水提物2.5、12.5、25 g/kg組體溫及血清中TNF-α、IL-6、IL-1β均顯著下降（P＜0.05），Western blotting檢測顯示，三葉青水提物2.5、12.5、25 g/kg組中下丘腦組織中HSP90AA1、NOS2、NOS3表達水平均下調(diào)（P＜0.05）。結(jié)論 三葉青在解熱的同時可以降低炎癥及發(fā)熱相關(guān)細胞因子的表達，此外三葉青的解熱作用與抑制HSP90AA1、NOS2和NOS3蛋白表達有關(guān)。

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.

R285

國家自然科學基金資助項目(82304808);浙江省醫(yī)藥衛(wèi)生科技計劃項目(2024KY877);浙江省中醫(yī)藥科技計劃項目(2023ZR076)