异槲皮苷调控PTP1B改善胰岛素抵抗的网络药理学研究及体外实验验证

Network pharmacological study and in vitro validation of isoquercitrin regulating PTP1B to improve insulin resistance

目的采用网络药理学法及分子对接技术探析异槲皮苷改善胰岛素抵抗的分子机制,并通过体外实验研究异槲皮苷对胰岛素抵抗的干预作用及机制。方法利用PubChem、PharmMapper、GEO、CTD、GeneCards、OMIM等多个数据库筛选异槲皮苷活性成分及胰岛素抵抗相关靶点;采用Cytoscape软件将异槲皮苷治疗胰岛素抵抗的潜在靶点构建蛋白质相互作用(PPI)网络,并根据度值筛选核心靶点。利用基因本体(GO)与京都基因与基因组百科全书(KEGG)富集分析寻找与靶点蛋白相关的生物学通路,采用AutoDock Tools软件模拟分子对接,预测异槲皮苷与关键靶点的结合度。体外实验采用异槲皮苷干预蛋白酪氨酸磷酸酶1B(PTP1B)质粒转染HepG2细胞,检测不同浓度异槲皮苷干预后PTP1B的活性;构建PTP1B质粒转染HepG2胰岛素抵抗细胞模型,给予异槲皮苷(40μmol/L)干预,葡萄糖氧化酶法、qRT-PCR、Western Blotting法检测PTP1B等相关因子的表达。结果网络药理学筛选得到异槲皮苷改善胰岛素抵抗的交集靶点21个,富集到GO条目2761个,主要涉及胰岛素受体信号通路、糖原生物合成过程的调控等,富集到KEGG通路89条,涉及包括胰岛素信号通路、胰岛素抵抗、磷脂酰肌醇-3-羟激酶(PI3K)-蛋白激酶B(Akt)信号通路、磷酸腺苷活化的蛋白激酶(AMPK)信号通路等。分子对接结果显示,异槲皮苷与靶点PTP1B、磷酸肌醇依赖性蛋白激酶1(PDPK1)、胰岛素受体(INSR)、糖原合酶激酶3β(GSK3β)、AKT2均有一定的结合活性。体外实验结果显示,异槲皮苷能有效抑制PTP1B活性,降低PTP1B过表达HepG2胰岛素抵抗细胞中PTP1B、GSK3β的表达,升高胰岛素受体底物1(IRS-1)、葡萄糖转运蛋白-1(GLUT-1)等因子的表达,改善细胞胰岛素抵抗。结论异槲皮苷可能通过抑制PTP1B调控PI3K/Akt信号通路因子活性改善胰岛素抵抗。

Objective To explore the potential mechanism of isoquercetin in treating insulin resistance based on network pharmacology and molecular docking, and verify the mechanism by cell experiment. Methods PubChem, PharmMapper, GEO, CTD,GeneCards, OMIM were used to screen the active components of isoquercetin and to predict the target. Cytoscape software was used to construct a protein interaction(PPI) network for potential targets of isoquercitrin in the treatment of insulin resistance, and the core targets were screened according to the degree value. GO and KEGG enrichment analysis were used to find biological pathways related to target proteins. AutoDock Tools software was used to simulate molecular docking and predict the binding degree of isoquercetin and key targets. HepG2 cells were transfected with PTP1B plasmid by isoquercitrin in vitro, and the activity of PTP1B after different concentrations of isoquercitrin was detected. HepG2 insulin-resistant cells were transfected with PTP1B plasmid, and treated with 40μmol/L isoquercitrin. The expression of PTP1B and other related factors was detected by glucose oxidase, qRT-PCR, and Western Blotting. Results Network pharmacological screening obtained 21 intersection targets of isoquercitrin to improve insulin resistance,enriched into 2 761 GO items, mainly involved in the insulin receptor signaling pathway and the regulation of glycogen biosynthesis process, and enriched into 89 KEGG pathways. It involves insulin signaling pathway, insulin resistance, PI3K-Akt signaling pathway,AMPK signaling pathway and so on. The results of molecular docking showed that the key active components of isoquercetin had certain binding activity to the targets PTP1B, PDPK1, INSR, GSK3β, and AKT2. The results of in vitro experiments showed that isoquercetin could effectively inhibit the activity of PTP1B, reduce the expression of PTP1B and GSK3β, increase the expression of IRS-1, GLUT-1 and other factors in HepG2 insulin resistance cells with PTP1B overexpression, and improve the insulin resistance of cells. And isoquercetin may improve insulin resistance by inhibiting the activity of PI3K/Akt signaling pathway factors regulated by PTP1B.