目的 基于网络药理学整合体内实验对比四物汤中4种单味药（川芎Chuanxiong Rhizoma、白芍Paeoniae Radix Alba、当归Angelicae Sinensis Radix、熟地黄Rehmanniae Radix Praeparata）治疗胆汁淤积性肝损伤（cholestatic liver injury，CLI）的药效差异，并探讨各单味药的作用机制。方法 利用TCMSP数据库获取川芎、白芍、当归、熟地黄的主要活性成分及作用靶点，通过GeneCards数据库获取CLI靶点，使用Cyctoscape 3.10.1软件分析并构建“单味药-活性成分-作用靶点”网络，借助在线网站获取各单味药与CLI之间的交集靶点，并绘制韦恩图。将靶点基因导入DAVID数据库进行基因富集分析。构建胆总管结扎（bile duct ligation，BDL）动物模型，给予各单味药水提物后，检测血清肝功能相关生化指标，并对肝组织进行病理染色对比药效作用。同时结合qRT-PCR检测CLI相关靶点的基因表达。结果 网络药理学结果显示，四物汤中川芎、白芍、当归、熟地黄与CLI之间均存在3个交集靶点[核受体亚家族3C组成员2（nuclear receptor subfamily 3 group C member 2，Nr3c2）、类视黄醇X受体（retinoid X receptor alpha，Rxra）、前列腺素内过氧化物合成酶2（prostaglandin-endoperoxide synthase 2，Ptgs2）]。通过体内验证实验表明白芍、川芎、当归、熟地黄水提物均可显著改善CLI以及相关的肝纤维化情况，且均能下调上述3个共同靶点表达（P＜0.05、0.01、0.001）。此外，白芍、川芎的体内抗CLI疗效优于当归、熟地黄提取物。结果还显示白芍与CLI有18个、川芎与CLI有4个与其他单味药不同的独立交集靶点，而当归、熟地黄与CLI之间不存在独立的交集靶点。进一步生信分析发现，白芍作用靶点基因富集于血管生成、肿瘤坏死因子（tumor necrosis factor，TNF）信号通路；川芎作用靶点富集于血管内皮、血管生成、血管生成因子（vascular endothelial growth factor，VEGF）通路。进一步验证结果显示白芍通过下调肝脏中血管细胞黏附分子（vascular cell adhesion molecule 1，Vcam1）、Tnf、有丝分裂原激活蛋白激酶（mitogen-activated protein kinase 8，Mapk8）、AKT丝氨酸/苏氨酸激酶（AKT serine/threonine kinase 1，Akt1）表达改善CLI，川芎通过下调肝脏激酶插入结构域受体（kinase insert domain receptor，Kdr）、Mapk14表达改善CLI。结论 四物汤中川芎、白芍、当归、熟地黄均能不同程度地改善胆汁淤积引起的肝损伤及纤维化，它们通过靶向Nr3c2、Rxra、Ptgs2共同改善CLI。白芍、川芎改善CLI疗效优于当归、熟地黄，前者可基于TNF信号通路而后者则是通过调控VEGF信号通路分别特异性改善CLI。

Objective To compare the therapeutic effects of the individual components of Siwu Tang (四物汤, SWT) including Chuanxiong (Chuanxiong Rhizoma), Baishao (Paeoniae Radix Alba), Danggui (Angelicae Sinensis Radix), Shudihuang (Rehmanniae Radix Praeparata) in the treatment of cholestatic liver injury (CLI) and explored the related mechanisms based on network pharmacology and experimental validation in vivo. Methods Themain active ingredients and targets of Chuanxiong Rhizoma, Paeoniae Radix Alba, Angelicae Sinensis Radix and Rehmanniae Radix Praeparata were obtained by TCMSP database. CLI targets were obtained by GeneCards database and “monomeric herb-active ingredient-target” network was analyzed and constructed by Cytoscape 3.10.1 software. Intersection targets between each monomeric herb and CLI were obtained using online resources, and Venn diagram was generated. The target genes were imported into DAVID database for gene enrichment analysis. Bile duct ligation (BDL) model was conducted, after administering the water extracts of each individual herb, serum biochemistry markers related to liver function were measured, and pathological staining was performed on liver tissue to compare the pharmacological effects. Moreover, qRT-PCR was employed to detect the gene expressions of CLI related targets. Results The results of network pharmacology revealed that Chuanxiong Rhizoma, Paeoniae Radix Alba, Angelicae Sinensis Radix and Rehmanniae Radix Praeparata in SWT shared three intersecting targets [nuclear receptor subfamily 3 group C member 2 (Nr3c2), retinoid X receptor alpha (Rxra) and prostaglandin-endoperoxide synthase 2 (Ptgs2)] with CLI. In vivo validation experiments showed that water extracts of Chuanxiong Rhizoma, Paeoniae Radix Alba, Angelicae Sinensis Radix and Rehmanniae Radix Praeparata significantly improved CLI and related liver fibrosis, and downregulated the expressions of the three common targets mentioned above (P < 0.05, 0.01, 0.001). In addition, the in vivo anti CLI efficacy of Paeoniae Radix Alba and Chuanxiong Rhizoma were better than that extracts of Angelicae Sinensis Radix and Rehmanniae Radix Praeparata. The results also showed that there were 18 independent intersection targets between Paeoniae Radix Alba and CLI, and four independent intersection targets between Chuanxiong Rhizoma and CLI that were different from other individual herbs, while there were no independent intersection targets between Angelicae Sinensis Radix, Rehmanniae Radix Praeparata and CLI. Further bioinformatics analysis revealed that the target genes of Paeoniae Radix Alba were enriched in angiogenesis and tumor necrosis factor (TNF) signaling pathways; The targets of Chuanxiong Rhizoma were enriched in vascular endothelium, angiogenesis and vascular endothelial growth factor (VEGF) pathways. Further validation results showed that Paeoniae Radix Alba improved CLI by downregulating the expressions of vascular cell adhesion molecule 1 (Vcam1),Tnf, mitogen activated protein kinase 8 (Mapk8), and AKT serine/threonine kinase 1 (Akt1) in liver, Chuanxiong Rhizoma improved CLI by downregulating the expressions of kinase insertion domain receptor (Kdr) and Mapk14 in liver. Conclusion Chuanxiong Rhizoma, Paeoniae Radix Alba, Angelicae Sinensis Radix and Rehmanniae Radix Praeparata in SWT could ameliorate liver injury and fibrosis caused by cholestasis. They collectively improve CLI by targeting Nr3c2, Rxra and Ptgs2. Paeoniae Radix Alba and Chuanxiong Rhizoma exhibit superior efficacy in improving CLI compared to Angelicae Sinensis Radix and Rehmanniae Radix Praeparata. Paeoniae Radix Alba specifically improves CLI through TNF signaling pathway, while Chuanxiong Rhizoma achieves specificity in improving CLI through VEGF signaling pathway.

国家自然科学基金面上项目（82274186）