目的 整合网络药理学与代谢组学，探究草苁蓉纳拉苷抗肝纤维化的作用机制。方法 通过网络药理学预测草苁蓉纳拉苷干预肝纤维化的潜在作用靶点与通路； SD大鼠随机分为对照组、模型组、水飞蓟素（50 mg·kg^-1）组和草苁蓉纳拉苷高、中、低剂量（270、135、68 mg·kg^-1）组，采用四氯化碳（CCl4）诱导肝纤维化大鼠模型，造模完成后，连续ig给药4周，试剂盒法检测血清丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）、透明质酸（HA）、层黏连蛋白（LN）、碱性磷酸酶（ALP）、白蛋白（ALB）、羟脯氨酸（HYP）水平和肝脏肿瘤坏死因子（TNF）-α、白细胞介素（IL）-6、IL-1β、超氧化物歧化酶（SOD）、丙二醛（MDA）、谷胱甘肽过氧化物酶（GSH-Px）水平；苏木精-伊红（HE）染色检测大鼠肝脏病理变化；利用UPLC/Q-TOF-MS技术对血清和肝脏代谢轮廓进行分析；最后整合网络药理学与代谢组学结果，构建“代谢物-酶-基因”网络，筛选核心调控靶点。通过Western blotting验证关键蛋白己糖激酶抗体（HK） II、缺氧诱导因子-1α（HIF1A）、蛋白激酶B（Akt）、p-Akt、磷脂酰肌醇激酶（PI3K）、p-PI3K表达。结果 网络药理表明草苁蓉纳拉苷抗肝纤维化核心靶点包括GAPDH、EGFR、GSK3B、MAPK1、HRAS，可能通过HIF-1信号通路发挥作用；药效学表明草苁蓉纳拉苷剂量相关性地减缓肝脏病理损伤，与模型组比较，水飞蓟素与草苁蓉纳拉苷均可显著降低肝纤维化大鼠血清AST、ALT、HA、LN、ALP水平（P＜0.05、0.01），升高ALB水平（P＜0.01）；显著降低肝脏中TNF-α、IL-6、IL-1β水平（P＜0.01），显著降低肝脏中MDA水平（P＜0.05、0.01），显著升高SOD水平（P＜0.01）；水飞蓟素组及草苁蓉纳拉苷高、中剂量组GSH-Px水平显著升高（P＜0.05、0.01）。代谢组学共鉴定出33个潜在生物标志物，涉及糖酵解、糖异生代谢、醚脂代谢以及谷胱甘肽代谢等代谢途径；联合分析进一步表明草苁蓉纳拉苷主要通过调控糖酵解与糖异生代谢途径维持稳态。Westernblotting显示，草苁蓉纳拉苷可显著下调肝纤维化模型升高的p-Akt、p-PI3K、HIF-1α及HKII蛋白表达（P＜0.05、0.01）。结论 草苁蓉纳拉苷可以调控糖酵解与糖异生等关键代谢通路，发挥多靶点、多途径的抗肝纤维化作用。

Objective To integrate network pharmacology and metabolomics to investigate the mechanism of action of Boschniakia rossica boschnaloside (from B. rossica) against hepatic fibrosis (HF). Methods Network pharmacology was used to predict potential targets and pathways involved in B. rossica boschnaloside’s intervention in liver fibrosis. SD rats were randomly divided into control, model, silymarin (50 mg·kg^-1), and three B. rossica boschnaloside groups (high, medium, low doses: 270, 135, 68 mg·kg^-1). A carbon tetrachloride (CCl₄)-induced liver fibrosis rat model was established. After modeling, animals received intragastric administration for four consecutive weeks. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), hyaluronic acid (HA), laminin (LN), alkaline phosphatase (ALP), albumin (ALB), and hydroxyproline (HYP), as well as hepatic tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β, superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH-Px) were measured using enzyme-linked immunosorbent assay kits. Hematoxylin-eosin (HE) staining was performed to evaluate pathological changes in the liver. Serum and liver metabolic profiles were analyzed using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS). Finally, integrating results from network pharmacology and metabolomics, a “metabolite-enzyme-gene” network was constructed to identify core regulatory targets. Western blotting was used to validate the expression of key proteins including hexokinase II (HKII), hypoxia-inducible factor-1α (HIF1A), protein kinase B (Akt), phosphorylated Akt (p-Akt), phosphoinositide 3-kinase (PI3K), and phosphorylated PI3K (p-PI3K). Results Network pharmacology analysis revealed that the core targets of B. rossica boschnaloside in anti-hepatic fibrosis include GAPDH, EGFR, GSK3B, MAPK1, and HRAS, suggesting its potential involvement via the HIF-1 signaling pathway. Pharmacological studies showed that B. rossica boschnaloside dose-dependently alleviated liver pathological damage. Compared with the model group, both silymarin and B. rossica boschnaloside significantly reduced serum levels of AST, ALT, HA, LN, and ALP (P<0.05, 0.01) and increased ALB levels (P<0.01). They also significantly decreased TNF-α, IL-6, and IL-1β, and MDA levels in the liver (P<0.01), while increasing SOD levels (P<0.01). The GSH-Px levels in the silymarin group and high- and medium-dose B. rossica boschnaloside groups were significantly elevated (P<0.05, 0.01). Metabolomics identified 33 potential biomarkers associated with glycolysis, gluconeogenesis, ether lipid metabolism, and glutathione metabolism. Integrated analysis further indicated that B. rossica boschnaloside primarily regulates homeostasis by modulating glycolysis and gluconeogenesis pathways. Western blotting demonstrated that B. rossica boschnaloside significantly downregulated the upregulated expressions of p-Akt, p-PI3K, HIF-1α, and HKII proteins in the liver fibrosis model (P<0.05, 0.01). Conclusion B. rossica boschnaloside exerts anti hepatic fibrosis effects through multi target and multi pathway mechanisms, notably by regulating key metabolic pathways such as glycolysis and gluconeogenesis.

R965

2024年度黑龙江省重点研发计划项目（2024ZXDXB62）