目的 探究加味黄芩汤（JWHQ）治疗淤胆型肝炎肝脏纤维化（CHF）的药效及作用机制。方法 应用三重四极杆高效液相色谱-质谱（QQQ-HPLC-MS）联用检测JWHQ中的活性成分与物质基础。C57BL6/J小鼠随机分为对照组、模型组、熊去氧胆酸(阳性药，UDCA,75 mg·kg^-1)和JWHQ低、中、高剂量(0.5、1.0、1.5 g·kg^-1)组，除对照组外，应用α-萘异硫氰酸酯（ANIT）诱导C57BL6/J小鼠CHF模型，JWHQ和UDCA均ig给药。通过化学试剂盒检测大鼠血清丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）、白蛋白（ALB）、碱性磷酸酶（ALP）、γ-谷氨酰转移酶（γ-GT）、总胆汁酸（TBA）、总胆红素（TBIL）、直接胆红素（DBIL）水平；通过离体肝脏状态检测和苏木素-伊红（HE）染色检测小鼠肝脏损伤程度；应用酶联免疫吸附法（ELISA）测定小鼠血清肝纤四项[透明质酸（HA）、层黏连蛋白（LN）、Ⅳ型胶原（Col-Ⅳ）和Ⅲ型前胶原氨基端肽（PⅢNP）];Masson染色和天狼星红染色评估小鼠肝脏纤维化水平；网络药理学预测JWHQ治疗CHF的关键作用靶点及通路；通过Western blotting、ELISA和分子对接验证JWHQ对关键靶点和通路的作用。结果 QQQ-HPLC-MS共检测出JWHQ中472种成分。体内实验结果表明，JWHQ可显著减少CHF小鼠的血清ALT、AST、ALP、ALB、γ-GT、TBA、TBIL和DBIL（P<0.05、0.01）；离体肝脏检测结果表明，JWHQ可改善CHF小鼠的肝脏状态，肝脏表面坏死样斑点减少；HE结果表明，JWHQ可减少CHF小鼠肝脏的局部弥漫性坏死面积和坏死程度；ELISA结果表明，中、高剂量的JWHQ干预可显著减少CHF小鼠血清中HA、LN、Col-Ⅳ和PⅢNP含量（P<0.01）;Masson和天狼星红染色结果表明，中、高剂量的JWHQ可改善CHF小鼠肝脏门管区纤维胶原沉积和假小叶面积。网络药理学结果表明，JWHQ治疗CHF的潜在作用靶点有266个，关键作用靶点为TNF、TP53、STAT3、TGFB1、NFKB1、IL1B和FN1；关键作用通路为AGE-RAGE通路、TNF信号通路和脂质与动脉粥样硬化通路，关键生物过程为对药物的反应和对炎症的反应，关键细胞结构为膜筏、膜域和膜微结构域，关键分子功能为核受体活性和配体结合的转录因子活性。Western blotting和ELISA检测结果表明，JWHQ可显著降低CHF小鼠肝脏信号转导因子和转录激活因子3（STAT3）表达、STAT3磷酸化、p53表达、核因子（NF）-κB表达、NF-κB磷酸化、白细胞介素1β（IL-1β）、白细胞介素6（IL-6）和肿瘤坏死因子α（TNF-α）、转化生长因子（TGF）-β1和Fibronectin表达水平（P<0.05、0.01）；分子对接结果表明，JWHQ 90个关键活性成分中与STAT3可形成自发结合的有82个，与Fibronectin可形成自发结合的有82个，与p53可形成自发结合的有90个，与NF-κB可形成自发结合的有89个，与TGF-β1可形成自发结合的有88个。结论 JWHQ可通过调控肝脏STAT3/p53/NF-κB信号通路抑制炎症因子旁分泌，降低肝脏细胞TGF-β1表达，阻断细胞外基质沉积，从而发挥治疗CHF作用。

Objective To explore the efficacy and mechanism of action of JWHQ（JWHQ） in the treatment of cholestatic hepatitis fibrosis（CHF）. Methods The active components and material basis of JWHQ were detected by triple quadrupole ultra-performance liquid chromatography-mass spectrometry（QQQ-HPLC-MS）. C57 BL6/J mice were randomly divided into the control group, model group, ursodeoxycholic acid(positive drug, UDCA, 75 mg·kg^-1) group and JWHQ low, medium and high dose(0.5, 1.0, 1.5 g·kg^-1) groups. Except for the control group, the CHF model of C57 BL6/J mice was induced by α-naphthyl isothiocyanate（ANIT）. JWHQ and UDCA were ig administered. The levels of alanine aminotransferase（ALT）, aspartate aminotransferase（AST）, albumin（ALB）, alkaline phosphatase（ALP）, γ-glutamyl transferase（γ-GT）, total bile acid（TBA）, total bilirubin（TBIL）, and direct bilirubin（DBIL） in rat serum were detected by chemical reagent kits. The degree of liver injury in mice was detected by in vitro liver state detection and hematoxylin-eosin（HE） staining. The levels of liver fibrosis four items [hyaluronic acid（HA）, laminin（LN）, type IV collagen（Col-Ⅳ）, and type III procollagen amino-terminal peptide（PⅢNP）] in mouse serum were determined by enzyme-linked immunosorbent assay（ELISA）. The levels of liver fibrosis in mice were evaluated by Masson staining and Sirius red staining. Network pharmacology was used to predict the key targets and pathways of JWHQ in the treatment of CHF. The effects of JWHQ on key targets and pathways were verified by Western blotting, ELISA and molecular docking. Results A total of 472 components were detected in JWHQ by QQQ-HPLC-MS. The in vivo experimental results showed that JWHQ could significantly reduce the levels of serum ALT, AST, ALP, ALB, γ-GT, TBA, TBIL and DBIL in CHF mice（P ＜0.05, 0.01）. The in vitro liver test results indicated that JWHQ could improve the liver condition of CHF mice, with a reduction in the number of necrotic-like spots on the liver surface. The HE results demonstrated that JWHQ could reduce the area and degree of local diffuse necrosis in the liver of CHF mice. The ELISA results showed that the medium and high doses of JWHQ intervention could significantly reduce the levels of HA, LN, Col-Ⅳ and PⅢNP in the serum of CHF mice（P ＜0.01）. The Masson and Sirius Red staining results indicated that the medium and high doses of JWHQ could improve the fibrous collagen deposition in the portal area and the area of pseudolobules in the liver of CHF mice. The network pharmacology results suggested that there were 266 potential targets for JWHQ in the treatment of CHF, with the key targets being TNF, TP53, STAT3, TGFB1, NFKB1, IL1B and FN1. The key pathways were the AGE-RAGE pathway, TNF signaling pathway and lipid and atherosclerosis pathway. The key biological processes were the response to drugs and the response to inflammation. The key cellular structures were lipid rafts, membrane domains and membrane microdomains. The key molecular functions were nuclear receptor activity and ligand-binding transcription factor activity. The Western blotting and ELISA results indicated that JWHQ could significantly reduce the expression of signal transducer and activator of transcription 3（STAT3）, phosphorylated STAT3, p53, nuclear factor（NF）-κB, phosphorylated NF-κB, interleukin 1β（IL-1β）, interleukin 6（IL-6）, tumor necrosis factor α（TNF-α）, transforming growth factor（TGF）-β1 and Fibronectin in the liver of CHF mice（P ＜0.05, 0.01）. The molecular docking results showed that among the 90 key active components of JWHQ, 82 could spontaneously bind to STAT3, 82 could spontaneously bind to Fibronectin, 90 could spontaneously bind to p53, 89 could spontaneously bind to NF-κB, and 88 could spontaneously bind to TGF-β1. Conclusion JWHQ can inhibit inflammatory factors by regulating the STAT3/p53/NF-κB in the liver, reduce the expression of TGF-β1 in liver cells, and block the deposition of extracellular matrix to treat CHF.

R285.5

江苏省卫生健康委医学科研项目(2022119); 江苏省高校“青蓝工程”优秀教学团队培养对象资助项目(苏教师[2020]42号)