目的 整合网络毒理学与代谢组学技术探究梣酮的潜在肝毒性机制。方法 采用网络毒理学方法预测梣酮的化学成分靶点和肝损伤疾病潜在靶点，并利用AutoDock分子对接验证其结合能力。建立梣酮诱导的小鼠肝损伤模型，通过苏木素–伊红（HE）染色观察肝脏组织病理学变化；酶联免疫吸附法（ELISA）检测血清丙氨酸氨基转移酶（ALT）和天门冬氨酸氨基转移酶（AST）、γ-谷氨酰转移酶（GGT）水平，评估肝功能损伤程度；利用代谢组学技术对肝组织代谢谱进行分析，筛选差异代谢物及相关代谢通路；整合代谢组学与网络毒理学结果进行联合分析，并通过蛋白质印迹法（Western blotting）检测相关通路蛋白的表达。结果 网络毒理学分析共鉴定出182个交集靶点，其中信号转导与转录激活因子3（STAT3）、肿瘤蛋白p53（⁺TP53）、肿瘤坏死因子（TNF）、白细胞介素（IL）-6等为关键靶点，KEGG通路富集分析显示IL-17信号通路、Toll样受体信号通路、细胞凋亡等显著富集。动物实验结果显示，与对照组比较，梣酮组可显著升高血清ALT、AST、GGT水平（P＜0.01），诱导肝细胞坏死和炎症浸润。代谢组学鉴定出39个肝脏差异代谢物，13个血清差异代谢物，主要涉及甘油磷脂代谢、嘌呤代谢、牛磺酸和次牛磺酸代谢等22条相关代谢途径；多组学联合分析揭示花生四烯酸代谢为梣酮致肝损伤的关键通路，且IL-6、谷胱甘肽过氧化物酶4（GPX4）、IL-1β、丝裂原活化蛋白激酶14（MAPK14）与花生四烯酸代谢物具有显著相关性，分子对接证实了梣酮与这些肝损伤相关靶点具有显著的结合活性，提示梣酮通过调节IL-6、GPX4、IL-1β、MAPK14进而调控花生四烯酸、甘油磷脂代谢及嘌呤代谢代谢途径进而诱发小鼠肝损伤。Western blotting验证进一步表明，梣酮可显著上调IL-6、IL-1β、MAPK14的蛋白表达，下调GPX4蛋白表达（P＜0.001）。结论 梣酮通过多靶点网络机制诱导肝损伤，其潜在作用机制可能与激活IL-6、GPX4、IL-1β、MAPK14介导的炎症信号，并调控甘油磷脂代谢、嘌呤代谢、牛磺酸和次牛磺酸代谢、花生四烯酸代谢等通路密切相关。

Objective To integrate network toxicology and metabolomics techniques to preliminarily explore the potential liver toxicity mechanism of fraxinellone. Methods The chemical component targets and potential liver injury disease targets of fraxinellone were predicted by network toxicology methods, and the binding ability was verified by AutoDock molecular docking. A mouse liver injury model induced by fraxinellone was established, and the pathological changes of liver tissues were observed by HE staining; the levels of serum ALT, AST, and GGT were detected by ELISA, to evaluate the degree of liver function damage. Liver tissue metabolic profile was analyzed by metabolomics technology to screen differential metabolites and related metabolic pathways, the combined analysis of metabolomics and network toxicology results was conducted, and the expression of related pathway proteins was detected by Western blotting. Results A total of 182 intersection targets were identified by network toxicology analysis, among which STAT3, TP53, TNF, IL-6, etc. were key targets. KEGG pathway enrichment analysis showed that the IL-17 signaling pathway, Toll-like receptor signaling pathway, apoptosis, etc. were significantly enriched. The animal experiment results showed that compared with control group, the fraxinellone administration group were significantly increased the levels of serum ALT, AST, and GGT (P < 0.01), induce hepatocyte necrosis and inflammatory infiltration. Metabolomics identified 39 liver differential metabolites and 13 differential serum metabolites, mainly involving glycerophospholipid metabolism, purine metabolism, taurine and secondary taurine metabolism, etc. 22 related metabolic pathways. Integrated multi-omics analysis revealed that arachidonic acid metabolism was the key pathway of fraxinellone-induced liver injury, and IL-6, GPX4, IL-1β, MAPK14 were significantly correlated with arachidonic acid metabolites. Molecular docking confirmed that fraxinellone had significant binding activity with these liver injury-related targets. Fraxinellone induces liver injury in mice by regulating IL-6, GPX4, IL-1β, and MAPK14, which in turn modulate the metabolic pathways of arachidonic acid, glycerophospholipids, and purine metabolism. Western blotting verification further indicated that fraxinellone could significantly upregulate the protein expression of IL-6, IL-1β, and MAPK14, and downregulate the expression of GPX4 (P < 0.001). Conclusion Fraxinellone induces liver injury through a multi-target network mechanism. Its potential mechanism may be related to the activation of IL-6, GPX4, IL-1β, MAPK14-mediated inflammatory signals and the regulation of glycerophospholipid metabolism, purine metabolism, taurine and secondary taurine metabolism, arachidonic acid metabolism, etc.

R99

国家重点研发计划中医药现代化重点专项项目（2022YFC3502104）