基于网络药理学和动物实验探讨柠檬苦素抗肝纤维化的作用机制

Explore the Mechanism of Limonin Against Hepatic Fibrosis Based on Network Pharmacology and Animal Experiments

关键词:  网络药理学  分子对接  柠檬苦素  肝纤维化  血管新生

Key words:  network pharmacology  molecular docking  limonin  hepatic fibrosis  angiogenesis

摘要:

目的 通过网络药理学方法探讨柠檬苦素治疗肝纤维化的作用机制，并运用分子对接和动物实验进行验证。方法 首先，利用SwissTargetPrediction、GeneCards和DisGeNet等数据库筛选柠檬苦素和肝纤维化的靶点，并运用微生信网站获得柠檬苦素与肝纤维化的共同靶点。然后运用STRING数据库和Cytoscape软件构建共同靶点的蛋白质相互作用网络，并利用CytoNCA插件筛选核心靶点；使用Metascape数据库对共同靶点进行GO功能注释和KEGG通路富集分析，以预测其可能的作用机制。最后运用AutoDock Vina软件对柠檬苦素与核心靶蛋白进行分子对接验证，并将网络药理学预测结果进行动物实验验证。结果 预测结果表明柠檬苦素可能作用于AKT1、VEGFA、HIF1A等86个靶点，参与激素应答、蛋白磷酸化、血管生成等生物过程和PI3K/AKT通路、HIF-1通路、VEGF通路等与肝纤维化相关的信号通路。蛋白质相互作用分析结果显示核心靶点包括AKT1、VEGFA、HIF1A、PIK3CA等11个靶点。分子对接结果表明柠檬苦素与AKT1、VEGFA、HIF1A 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，柠檬苦素高剂量(high-dose group of limonin，LH)组、柠檬苦素低剂量(low-dose group of limonin，LL)组中血清透明质酸(hyaluronidase，HA)、层黏连蛋白(laminin，LN)含量(LL组中LN除外)均下降(P<0.01或P<0.05)，肝组织炎症和纤维化程度均减轻；Western blotting和实时荧光定量PCR(qPCR)检测显示LH、LL组肝组织中AKT、HIF-1α、VEGF蛋白和mRNA表达水平(LL组中VEGF除外)均下调(P<0.01或P<0.05)。结论 柠檬苦素主要作用于AKT1、VEGFA、HIF1A等核心靶点来干预肝纤维化血管新生，其机制可能与其抑制AKT/HIF-1α/VEGF信号通路有关。

Abstract:

OBJECTIVE To explore the mechanism of limonin treating in hepatic fibrosis through network pharmacology, and validate its mechanism by molecular docking and animal experiments. METHODS Firstly, the targets of limonin and hepatic fibrosis were screened from the SwissTargetPrediction, GeneCards and DisGeNet database, etc. Meanwhile, the common targets of limonin and hepatic fibrosis were obtained from the bioinformatics website. The protein protein interaction network of common target was constructed by using STRING database and Cytoscape software, and the CytoNCA plug-in was used to screen core targets. And then the enrichment analysis of GO and KEGG on the common target was performed by Metascape database. Thereby, the possible mechanism of limonin against hepatic fibrosis were predicted. Finally, the AutoDock Vina was used for molecular docking verification, and the prediction results of network pharmacology were verified by animal experiments. RESULTS The prediction results indicated that limonin might acted on 86 targets including AKT1, VEGFA and HIF1A, and participated in biological processes including hormone response, protein phosphorylation, angiogenesis, and PI3K-Akt pathway, HIF-1 pathway, VEGF pathway and other signaling pathways related to hepatic fibrosis. The results of protein protein interaction network topology analysis showed that the 11 core targets including AKT1, VEGFA, HIF1A and PIK3CA, etc. Molecular docking results showed that limonin had strong affinity and relatively stable binding conformation with the core targets. In the animal experiments, compared with the model group, hyaluronidase(HA) and laminin(LN) in rat serume in high-dose group of limonin(LH) and low-dose group of limonin(LL)(except for LN in LL group) were declined(P<0.01 or P<0.05), and the degree of inflammation and hepatic fibrosis were relieved to different degrees in liver tissue of the LH group and LL group; Western blotting and qPCR detection showed that protein and mRNA expression levels of AKT, HIF-1α and VEGF(except for VEGF in LL group) was down-regulated in the LH group and LL group(P<0.01 or P<0.05). CONCLUSION Limonin may acts on AKT1, VEGFA, HIF1A and other core targets to treat hepatic fibrosis angiogenesis, which may be related to the inhibition of AKT/HIF-1α/VEGF signaling pathway.