DataSheet1_Mechanism of Paeoniflorin on ANIT-Induced Cholestatic Liver Injury Using Integrated Metabolomics and Network Pharmacology.ZIP

Background: Paeoniflorin (PF), the major active compound isolated from the roots of Paeonia lactiflora Pall., has been used in the treatment of severe hepatic diseases for several decades and displays bright prospects in liver protective effect. However, its biological mechanism that regulates bile acid metabolism and cholestatic liver injury has not been fully elucidated. Our study aims to investigate the mechanism by which PF in the treatment of cholestatic liver injury using a comprehensive approach combining metabolomics and network pharmacological analysis.
Methods: The hepatoprotective effect of PF against cholestasis liver injury, induced by α-naphthylisothiocyanate (ANIT), was evaluated in rats. The serum biochemical indices including ALT, AST, TBA, TBIL, ALP, ALB, and the pathological characteristics of the liver were analyzed. Moreover, UHPLC-Q-TOF was performed to explore the feces of rats with ANIT-induced cholestatic liver injury treated with PF and the potential biomarkers were screened by metabolomics. The targets for the regulation of potential biomarkers by PF were screened by network pharmacology, and then the relevant key targets were verified by immunohistochemical and western blotting methods.
Results: PF significantly improved serum indexes and alleviated liver histological damage. Metabolomics analyses showed that the therapeutic effect of PF is mainly associated with the regulation of 13 metabolites involved in 16 metabolic pathways. The “PF-targets-metabolites” interaction network was constructed, and then five key targets including CDC25B, CYP2C9, MAOB, mTOR, and ABCB1 that regulated the potential biomarkers were obtained. The above five targets were further verified by immunohistochemistry and western blotting, and the results showed that PF significantly improved the expression of key proteins regulating these biomarkers.
Conclusion: Our study provides direct evidence for the modulatory properties of PF treatment on ANIT-induced cholestatic liver injury using metabolomics and network pharmacology analyses. PF exhibits favorable pharmacological effect by regulating related signal pathways and key targets for biomarkers. Therefore, these findings may help better understand the complex mechanisms and provide a new and effective approach to the treatment of cholestatic liver injury.

背景：芍药苷（Paeoniflorin, PF）是从芍药（Paeonia lactiflora Pall.）根中分离得到的主要活性成分，数十年来被用于治疗重症肝脏疾病，在保肝领域展现出良好的应用前景。然而其调控胆汁酸代谢与胆汁淤积性肝损伤的生物学机制尚未完全阐明。本研究旨在通过结合代谢组学（metabolomics）与网络药理学分析（network pharmacological analysis）的综合策略，探究PF治疗胆汁淤积性肝损伤的作用机制。
方法：本研究在大鼠模型中评估了PF对α-萘异硫氰酸酯（α-naphthylisothiocyanate, ANIT）诱导的胆汁淤积性肝损伤的保肝效应。检测了血清生化指标，涵盖丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）、总胆汁酸（TBA）、总胆红素（TBIL）、碱性磷酸酶（ALP）与白蛋白（ALB），并分析了肝脏组织的病理形态特征。此外，采用超高效液相色谱-四极杆飞行时间质谱（UHPLC-Q-TOF）技术，对经PF治疗的ANIT诱导胆汁淤积性肝损伤大鼠的粪便样本进行检测，通过代谢组学筛选潜在生物标志物。通过网络药理学分析筛选PF调控潜在生物标志物的作用靶点，并通过免疫组化（immunohistochemistry）与蛋白质印迹法（western blotting）验证相关关键靶点。
结果：PF可显著改善血清生化指标，减轻肝脏组织病理损伤。代谢组学分析显示，PF的治疗效应主要与调控16条代谢通路中的13种差异代谢物相关。构建了“PF-靶点-代谢物”相互作用网络，筛选得到5个调控潜在生物标志物的核心靶点，分别为CDC25B、CYP2C9、MAOB、mTOR与ABCB1。通过免疫组化与蛋白质印迹法对上述5个靶点进行验证，结果显示PF可显著改善调控这些生物标志物的关键蛋白的表达水平。
结论：本研究通过代谢组学与网络药理学分析，为PF调控ANIT诱导的胆汁淤积性肝损伤的作用特性提供了直接实验证据。PF通过调控相关信号通路与生物标志物的核心靶点展现出优良的药理活性。因此，本研究结果有助于深入阐明其复杂的作用机制，并为胆汁淤积性肝损伤的治疗提供全新的有效策略。