Zhang Sheng-Peng, Zhang Chao, Li Li-Hua, Chen Yun-Yu, Zhu Lei, Liu Xiao-Ping, Li Ping
School of Pharmacy,Wannan Medical College Wuhu 241002,China.
Zhongguo Zhong Yao Za Zhi. 2021 Feb;46(4):845-854. doi: 10.19540/j.cnki.cjcmm.20201118.402.
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.
采用网络药理学和体外肝纤维化（LF）模型，分析荜茇及其主要活性成分抗肝纤维化作用的潜在机制。利用中药系统药理学数据库与分析平台（TCMSP）和中医综合信息平台（TCMIP）检索荜茇的化学成分，并将口服生物利用度（OB）、类药性（DL）、肠上皮细胞（Caco-2）的细胞间通透性及类药性分级设定为限制条件。通过TCMSP数据库查询荜茇的相关靶基因，同时利用基因卡片（GeneCards）数据库筛选LF的相关靶点。使用Cytoscape 3.7.1构建相互作用网络。将上述数据导入STRING数据库进行蛋白质-蛋白质相互作用（PPI）网络分析。利用生物导体（Bioconductor）数据库中的基因本体论（GO）富集分析和通路分析（KEGG）来标注荜茇相关靶点的功能。最后，通过体外实验对核心靶点和通路进行初步验证。研究了荜茇主要活性成分胡椒碱（PL）对大鼠肝星状细胞（HSC-T6）增殖及α平滑肌肌动蛋白（α-SMA）和Ⅰ型胶原表达的影响。检测了肿瘤坏死因子（TNF）通路的主要因子TNF-α以及LF过程中NF-κB p65、白细胞介素-6（IL-6）蛋白的表达。通过分析生物利用度和类药性质，共获得12种如PL的活性化合物，推断出48个靶点。GO功能富集分析得到1240个GO条目，主要涉及生物学过程和分子功能。KEGG通路富集分析共富集到99条信号通路，包括TNF信号通路、环磷酸鸟苷-蛋白激酶G（cGMP-PKG）信号通路、钙信号通路。细胞计数试剂盒-8（CCK-8）检测显示，PL抑制转化生长因子-β1（TGF-β1）诱导的HSC-T6细胞增殖。蛋白质免疫印迹分析发现，PL处理可抑制HSC-T6细胞中α-SMA、Ⅰ型胶原、TNF-α和p65的蛋白表达。酶联免疫吸附测定（ELISA）显示，PL抑制HSC-T6细胞培养上清中TNF-α和IL-6的表达。综上所述，PL可能通过调节TNF/NF-κB信号通路发挥抗肝纤维化作用。本研究为荜茇及其主要活性成分抗LF作用提供了机制依据，可能有助于对荜茇作用机制和关键靶点的进一步研究。
