背景:丹参(S.丹参)是一种重要的中草药(TCM),用于治疗心脑血管疾病。基于丹参的药效学物质,本研究的目的是通过系统的网络药理学方法研究丹参抗心脏纤维化(CF)的潜在机制,分子对接和动力学模拟以及体外实验研究。
方法:利用中药药理学(TCMSP)数据库进行系统的药理分析,筛选丹参的有效化学成分,然后通过瑞士目标预测和TCMSP数据库获得化合物的相应潜在目标基因。同时,GeneCards,DisGeNet,OMIM,TTD疾病数据库用于筛选CF靶标,通过检索相互作用基因/蛋白质(STING)数据库的搜索工具,在丹参/CF靶标上构建了药物-疾病靶标的蛋白质-蛋白质相互作用(PPI)网络。之后,成分-疾病-目标网络由Cytoscape3.7软件构建.对药物和疾病之间的交叉靶标进行基因本体论(GO)和京都基因和基因组百科全书(KEGG)富集分析。通过分子对接和分子动力学模拟评价了丹参有效成分与CF疾病靶点的关系。随后,体外实验研究了hub化合物对CF的潜在机制。
结果:确定了丹参有效化学成分的206个相应目标,其中,有82个目标与CF的目标重叠。Further,通过PPI分析,AKT1和GSK3β是中心目标,两者都富含PI3K/AKT信号通路,它是脂质和动脉粥样硬化途径的亚途径。随后,构建了复合-疾病-基因-通路图,根据程度值,芹菜素(APi)是顶级成分,AKT1(51)和GSK3β(22)是hub基因。分子对接和动力学模拟结果表明,APi与AKT和GSK3β具有很强的亲和力。细胞实验结果表明,APi抑制细胞活力,扩散,α-SMA和I/III型胶原的蛋白表达,TGFβ1诱导MCF中AKT1和GSK3β的磷酸化。
结论:通过系统的网络药理学方法,分子对接和动力学模拟,并通过体外细胞实验证实,这些结果表明,APi与AKT和GSK3β相互作用,破坏AKT和GSK3β的磷酸化,从而抑制TGFβ1诱导的MCF的增殖和分化,为丹参治疗CF的药理机制提供了新的见解。
BACKGROUND: Salvia miltiorrhiza Bunge (S. miltiorrhiza) is an important Traditional Chinese herbal Medicine (TCM) used to treat cardio-cerebrovascular diseases. Based on the pharmacodynamic substance of S. miltiorrhiza, the aim of present study was to investigate the underlying mechanism of S. miltiorrhiza against cardiac fibrosis (CF) through a systematic network pharmacology approach, molecular docking and dynamics simulation as well as experimental investigation in vitro.
METHODS: A systematic pharmacological analysis was conducted using the Traditional Chinese Medicine Pharmacology (TCMSP) database to screen the effective chemical components of S. miltiorrhiza, then the corresponding potential target genes of the compounds were obtained by the Swiss Target Prediction and TCMSP databases. Meanwhile, GeneCards, DisGeNET, OMIM, and TTD disease databases were used to screen CF targets, and a protein-protein interaction (PPI) network of drug-disease targets was constructed on S. miltiorrhiza/CF targets by Search Tool for the Retrieval of Interacting Genes/Proteins (STING) database. After that, the component-disease-target network was constructed by software Cytoscape 3.7. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed for the intersection targets between drug and disease. The relationship between active ingredient of S. miltiorrhiza and disease targets of CF was assessed via molecular docking and molecular dynamics simulation. Subsequently, the underlying mechanism of the hub compound on CF was experimentally investigated in vitro.
RESULTS: 206 corresponding targets to effective chemical components from S. miltiorrhiza were determined, and among them, there were 82 targets that overlapped with targets of CF. Further, through PPI analysis, AKT1 and GSK3β were the hub targets, and which were both enriched in the PI3K/AKT signaling pathway, it was the sub-pathways of the lipid and atherosclerosis pathway. Subsequently, compound-disease-genes-pathways diagram is constructed,
apigenin (APi) was a top ingredients and AKT1 (51) and GSK3β (22) were the hub genes according to the degree value. The results of molecular docking and dynamics simulation showed that APi has strong affinities with AKT and GSK3β. The results of cell experiments showed that APi inhibited cells viability, proliferation, proteins expression of α-SMA and collagen I/III, phosphorylation of AKT1 and GSK3β in MCFs induced by TGFβ1.
CONCLUSIONS: Through a systematic network pharmacology approach, molecular docking and dynamics simulation, and confirmed by in vitro cell experiments, these results indicated that APi interacts with AKT and GSK3β to disrupt the phosphorylation of AKT and GSK3β, thereby inhibiting the proliferation and differentiation of MCFs induced by TGFβ1, which providing new insights into the pharmacological mechanism of S. miltiorrhiza in the treatment of CF.