泽泻治疗非酒精性脂肪性肝病的作用机制研究

Mechanism of Alisma orientale in treating nonalcoholic fatty liver disease

该研究将网络药理学技术、分子对接技术及实验验证相结合,初步阐明了泽泻治疗非酒精性脂肪性肝病(NAFLD)的活性成分、潜在靶点及作用机制,为其临床应用提供依据。通过中药系统药理学数据与分析平台检索,结合口服利用度(OB)和类药性(DL)筛选泽泻活性成分,通过蛋白数据库预测与活性成分及NAFLD均相关的潜在靶点。利用Cytoscape软件构建"活性成分-潜在靶点"网络,将活性成分和潜在靶点进行分子对接,通过DAVID生物信息注释数据库进行KEGG通路分析以及富集分析,采用ClueGO软件进行靶点GO注释分析。采用蛋白印迹、免疫细胞化学检测蛋白表达水平,采用荧光探针检测活性氧(ROS)的生成水平。结果显示,从TCMSP数据库与分析平台中获得7个泽泻活性成分,成分对应的靶点共140个,有效成分的靶点与疾病的靶点交集后获得59个潜在靶点。分子对接结果显示,7个泽泻活性成分能充分作用于潜在靶点3-羟基-3-甲基戊二酰辅酶A(HMGCR)、酪氨酸磷酸酯酶非受体1型(PTPN1)。另外,KEGG富集分析显示,潜在靶点主要富集在色氨酸(TRP)通道的炎性介质调节、胰岛素抵抗、神经活性配体-受体相互作用、血管平滑肌收缩、Fcγ受体(FcγR)介导的吞噬作用等相关通路。GO富集分析显示,潜在靶点主要影响G蛋白偶联受体信号传导途径、有机羟基化合物转运、脂质生物合成过程的正调控、脂质代谢过程的正调控等生物过程。蛋白印迹、免疫细胞化学与荧光探针结果确证泽泻提取物可有效降低脂肪变性肝细胞HepG2的HMGCR和PTPN1蛋白表达水平,并可降低HepG2细胞的ROS生成水平。该研究系统揭示泽泻通过多成分-多靶点-多途径共同调控NAFLD的物质基础和作用机制,为中药泽泻的临床应用提供理论基础和科学依据。

In this study,network pharmacology technology was combined with molecular docking technology and experimental verification to clarify the active ingredients,potential targets and mechanism of Alisma orientale for nonalcoholic fatty liver disease(NAFLD),providing a basis for its clinical application.The active ingredients of A.orientale were screened through traditional Chinese medicine systems pharmacology database(TCMSP),and the potential targets related to both active ingredients and NAFLD were predicted through protein databases by considering the oral bioavailability(OB)and drug-likeness(DL).The"active ingredient-potential target"network was constructed by using Cytoscape software,and the molecular docking was performed between active ingre-dients and potential targets.KEGG pathway analysis and enrichment analysis were performed through DAVID biological information annotation databases.ClueGO software was used to analyze target GO annotation.Western blot and immunocytochemistry were used to detect the protein expression levels,and fluorescent probe was used to detect the reactive oxygen species(ROS)generation level.The results revealed that 7 active ingredients of A.orientale were obtained from TCMSP database and analysis platform,140 ingredient-related targets were screened,and 59 potential targets were obtained by intersecting disease targets with ingredient-related targets.Molecular docking showed that 7 active ingredients of A.orientale could act on the potential targets including 3-hydroxy-3-methylglutaryl-coenzyme A reductase(HMGCR)and tyrosine-protein phosphatase non-receptor type 1(PTPN1).In addition,KEGG enrichment analysis showed that the potential targets were mainly enriched in inflammatory mediator regulation,insulin resistance,neuroactive ligand-receptor interaction,vascular smooth muscle contraction,FcγR-mediated phagocytosis and other related pathways of tryptophan(TRP)channel.GO enrichment analysis showed that potential targets mainly affected the biological processes of G-protein coupled receptor signaling pathway,organic hydroxyl compound transport,positive regulation of lipid biosynthesis process,positive regulation of lipid metabolic process.Western blot,immunocytochemistry and fluorescent probe confirmed that the extract of A.orientale could reduce HMGCR and PTPN1 protein expression levels effectively,and also could reduce ROS production level of HepG2 cells.This study systematically revealed the material basis and mechanism of A.orientale in regulating NAFLD through multi-component,multi-target,and multi-pathway characteristics,which provided a theoretical basis and scientific basis for the clinical application of A.orientale.