基于网络药理学预测荜茇抗肝纤维化作用及验证研究

基于网络药理学及肝纤维化(liver fibrosis, LF)体外模型方法探讨荜茇及其主要活性成分抗肝纤维化的作用机制。通过TCMSP及TCMIP数据库,结合口服生物利用度(oral bioavailability, OB)、类药性(drug-likeness, DL)、肠上皮通透性(Caco-2)和drug-likeness grading作为限定条件获取荜茇主要活性成分。利用TCMSP搜索荜茇相关靶基因,在GeneCards数据库搜索与LF相关的基因,采用Cytoscape 3.7.1软件,构建荜茇潜在活性化合物-抗肝纤维化靶点网络。使用STRING数据库进行蛋白质-蛋白质相互作用分析,构建蛋白质-蛋白质相互作用网络。使用Bioconductor数据库对荜茇抗LF作用靶点进行基因本体(GO)富集分析与KEGG通路分析。最后,通过荜茇活性成分荜茇酰胺(piperlongumine, PL)干预大鼠肝星状细胞(HSC-T6)的体外实验进行核心靶点和通路的初步验证,考察荜茇酰胺抑制大鼠肝星状细胞增殖及对肝纤维化标志物α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、Ⅰ型胶原(collagenⅠ)的影响,检测肝纤维化过程肿瘤坏死因子(tumor necrosis factor, TNF)信号通路中肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α),NF-κB p65及白细胞介素6(interleukin-6,IL-6)蛋白表达。网络药理学发现,通过对生物利用度、类药性的分析得到荜茇酰胺等12种潜在活性化合物,涉及抗肝纤维化作用靶点48个。GO功能富集分析得到GO条目1 240个,大部分与生物过程和分子功能相关。KEGG通路富集筛选得到99条信号通路,包括TNF信号通路、cGMP-PKG信号通路、钙信号通路等。CCK-8检测表明,PL可抑制转化生长因子-β1(transforming growth factor-β1, TGF-β1)诱导的HSC-T6增殖。Western blot分析表明,PL可下调HSC-T6中α-SMA和collagenⅠ表达,抑制TNF-α和p65蛋白表达。酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)结果表明,PL可降低HSC-T6培养上清中TNF-α及IL-6含量。结果显示,PL可通过调控TNF/NF-κB信号通路而发挥治疗肝纤维化作用。该研究增加了中药荜茇及其有效成分抗LF药理作用的新认识,也为进一步深入探讨其具体的调控机制及关键的靶标奠定理论基础。

Prediction of anti-liver fibrosis effect of Piperis Longi Fructus based on network pharmacology

Network pharmacology and liver fibrosis(LF) model in vitro were used to analyze the underly mechanism of anti-liver fibrosis effect that induced by Piperis Longi Fructus and its major active compounds. TCMSP and TCMIP were used to search for the chemical constituents of Piperis Longi Fructus, as well as the oral bioavailability(OB), drug-likeness(DL), intercellular permeability of intestinal epithelial cells(Caco-2) and Drug-likeness grading were set as limiting conditions. The related target genes of Piperis Longi Fructus were queried by TCMSP database, while related targets of LF were screened by GeneCards databases. Interaction network was constructed using Cytoscape 3.7.1. These above data were imported into STRING database for PPI network analysis. Enrichment of gene ontology(GO) and pathway analysis(KEGG) within Bioconductor database were utilized to note functions of related targets of Piperis Longi Fructus. Finally, the core targets and pathways were preliminarily verified by in vitro experiments. The effects of piperlongumine(PL), the major active component of Piperis Longi Fructus, on proliferation of rat liver stellate cells(HSC-T6) and expression of α smooth muscle actin(α-SMA) and collagen Ⅰ were investigated. The major factors TNF-α of tumor necrosis factor(TNF) pathway and NF-κB p65, IL-6 protein expressions of LF process were examined. A total of 12 active compounds such as PL were obtained by analyzing the bioavailability and drug-like properties, which inferred to 48 targets. The functional enrichment analysis of GO obtained 1 240 GO items, mainly involving in process of biology and molecular function. A total of 99 signaling pathways were enriched in the KEGG pathway enrichment analysis, including TNF signaling pathway, cGMP-PKG signaling pathway, calcium signaling pathways. CCK-8 assay showed that PL inhibited proliferation of HSC-T6 induced by transforming growth factor-β1(TGF-β1). Western blot analysis found that treated with PL suppressed the protein expressions of α-SMA, collagen Ⅰ, TNF-α and p65 in HSC-T6. Enzyme linked immunosorbent assay(ELISA) showed that PL inhibited the expressions of TNF-α and IL-6 in the cluture supertant of HSC-T6 cells. In conclusion, PL could play an anti-liver fibrosis role by regulating TNF/NF-κB signaling pathway. This study provided the mechanism basis of anti-LF effects induced by Piperis Longi Fructus and its major active compounds, which might help for the further study of the mechanism and key targets of Piperis Longi Fructus.