龙眼叶治疗2型糖尿病的网络药理学研究及实验验证

目的 基于网络药理学和分子对接的方法探讨龙眼叶治疗2型糖尿病（T2DM）的作用机制,并通过体外实验进行初步验证。方法 采用中药系统药理学分析平台（TCMSP）并结合化源网数据库和相关药理活性文献筛选出龙眼叶的活性成分;通过Swiss TargetPrediction网站在线分析筛选活性成分的靶点。利用OMIM、GeneCards、DrugBank三个数据库获取T2DM的相关靶点,进而利用韦恩（VENNY）2.1筛选出活性成分与T2DM的交集靶点;然后将其导入STRING数据库以进行蛋白质互作网络（PPI）网络构建;采用Cytoscape v3.8.0软件构建“中药-化学成分-关键靶基因-疾病”网络图;通过DAVID数据库对交集靶点进行GO注释和KEGG通路富集分析,GraphPad Prism 8.3软件将其可视化;运用SYBYL-X 2.0和Discovery Studio 4.5 软件对龙眼叶排名靠前的活性成分和关键靶点进行分子对接,验证其生物活性。验证实验以人肝癌细胞（HepG2）作为研究对象,高糖诱导胰岛素抵抗HepG2细胞模型,CCK8法检测细胞增殖抑制率,蛋白印迹（Western blot）检测磷脂酰肌醇3激酶/蛋白激酶B（PI3K-AKT）信号通路相关蛋白的表达情况。结果 共筛选出龙眼叶有16个活性成分,活性成分和疾病的交集靶点共96个,网络拓扑分析筛选出关键靶点16个。生物学过程包括凋亡过程的负调控、细胞质膜、酶结合等。龙眼叶在改善T2DM主要作用于HIF-1信号通路、胰岛素抵抗信号通路、PI3K-AKT信号通路等。分子对接显示山柰酚和EGFR、槲皮素和PTGS2、木犀草素和PIK3R1有比较强的结合活性。细胞实验结果表明龙眼叶提取物可以上调IR模型HepG2细胞AKT-1、p-AKT-1、p-PI3Kp85蛋白表达。结论 龙眼叶抗T2DM的作用机制可能与PI3K-AKT、HIF-1、胰岛素抵抗等信号通路有关。

Network Pharmacology and Experimental Study on the Mechanism of the Leaves of Dimocarpus longan in Treating Type 2 Diabetes

Objective To explore the mechanism of the leaves of Dimocarpus longan in the treatment of Type 2 Diabetes Mellitus (T2DM) based on network pharmacology and molecular docking technique, and to conduct the preliminary verification through in vitro experiments.Methods The Traditional Chinese Medicine System Pharmacology Analysis Platform (TCMSP) combined with the Huayuan network database and related pharmacological activity literature was used to screen the active ingredients of the leaves of Dimocarpus longan. The targets of active ingredients were screened by online analysis on Swiss TargetPrediction website. The relevant targets of T2DM were obtained by three databased of OMIM, GeneCards, and DrugBank, and then the intersection targets of active components and T2DM were screened by VENNY 2.1. Then it was imported into STRING database for PPI network construction. Cytoscape v3.8.0 software was used to construct a network diagram of "Chinese medicine-chemical components-key target genes-disease". GO annotation and KEGG pathway enrichment analysis were done through the DAVID database. GraphPad Prism 8.3 was used for visualization. SYBYL-X 2.0 and Discovery Studio 4.5 software were used to molecularly dock the top active ingredients and key targets of the leaves of Dimocarpus longan to verify their biological activity. The validation experiments used HepG2 cells as the subjects, high sugar-induced HepG2-IR cell models, CCK8 detection of cell proliferation inhibition rates, and Western blot expression of PI3K-AKt signaling pathway.Results 16 active components of the leaves of Dimocarpus longan were screened. 96 intersecting targets between active ingredients and diseases, and 16 key targets were screened out by network topology analysis. Biological processes include negative regulation of apoptosis process, plasma membrane, and enzyme binding and so on. The leaves of Dimocarpus longan mainly act on the HIF-1 signaling pathway, insulin resistance signaling pathway, prolactin signaling pathway, and PI3K-AKT signaling pathway of T2DM. Molecular docking showed that Kaempferol and EGFR, Quercetin and PTGS2, Luteolin and PIK3R1 have strong binding activity. The results of cell experiment showed that the leaves of Dimocarpus longan extract could up regulate the expression of AKT-1, p-AKT-1 and p-PI3Kp85 protein in IR model HepG2 cells.Conclusion The anti-T2DM mechanism of the leaves of Dimocarpus longan may be related to PI3K-AKT, HIF-1, insulin resistance and other signaling pathways.