基于网络药理学和UPLC-Q-TOF/MS结合动物实验探究蓍草预防急性肝损伤的作用机制

Mechanism of action of Achillea alpina L. in the prevention of acute liver injury based on UPLC-Q-TOF/MS combined with network pharmacology and animal experiments

目的 通过UPLC-Q-TOF/MS定性分析和网络药理学预测并结合动物实验验证初步探究蓍草保护急性肝损伤(ALI)大鼠肝脏的作用机制。方法 采用UPLC-Q-TOF/MS法鉴定蓍草中的化学成分,利用TCMSP、CTD和Similarity Ensemble Approach等数据库筛选这些成分的作用靶点,同时在GeneCards、OMIM和NCBI数据库筛选ALI相关靶点,将成分作用靶点与疾病靶点的交集靶点导入STRING构建蛋白质-蛋白质相互作用(PPI)网络,并使用Cytoscape3.8.2软件进行网络拓扑分析筛选核心靶点,通过DAVID数据库对关键靶点进行GO和KEGG富集分析。通过肝组织HE染色,大鼠血清中肝功能指标、炎症因子水平以及肝组织中氧化应激水平的测定对蓍草防治ALI的药效进行评价,并利用免疫组化的方法对预测的核心靶点及KEGG富集通路结果进行验证。结果 经UPLC-Q-TOF/MS法鉴定出20个成分,网络药理学方法筛选得到上述成分预防治疗ALI的核心靶点104个,GO功能主要与炎症反应、氧化还原酶活性和蛋白酶结合等生物过程相关,KEGG结果主要涉及IL-17、TNF及Toll样受体等信号通路;动物实验结果显示,蓍草预防性给药后能够缓解大鼠肝脏的损伤,显著降低模型组大鼠血清中ALT、AST、TNF-α、IL-6、IL-1β水平和肝组织中MDA的含量,且SOD和CAT在肝组织中的活性显著增强;此外还发现蓍草给药后可上调大鼠肝脏组织中Nfr2蛋白的表达,下调TLR-4、NF-κBp65、keap1和HO-1蛋白的表达。结论 蓍草对四氯化碳诱导的ALI大鼠肝脏具有保护作用,该作用可能与其调节keap1/Nrf2/HO-1和Toll样受体信号通路有关。

Objective To determine the mechanism of action of Achillea alpina L. in protecting against acute liver injury(ALI) in rats by UPLC-Q-TOF/MS qualitative analysis and network pharmacological prediction combined with animal experimental validation. Methods Firstly, the chemical components in Achillea alpina L. were identified by UPLC-Q-TOF/MS, and the targets of these components were screened with TCMSP, CTD and Similarity Ensemble Approach databases,while the targets related to ALI were screened in GeneCards, OMIM and NCBI databases. The intersecting targets of the components and the disease targets were imported into STRING to construct protein-protein interaction(PPI) network. The core targets were screened by network topology analysis with Cytoscape 3.8.2 software, and the key targets were enriched by GO and KEGG analysis through DAVID database. The efficacy of Achillea alpina L. in preventing against ALI was further determined by HE staining of the liver tissues, liver function parameters, inflammatory factors and oxidative stress levels in the liver tissues of rats. Results Twenty components were identified by UPLC-Q-TOF/MS, and 104 core targets of the above components for the prophylactic treatment of ALI were obtained by screening with network pharmacology. GO functions were mainly related to the biological processes such as inflammatory response, oxidoreductase activity and protease binding, and KEGG results mainly involved IL-17, TNF and Toll-like receptors signaling pathways. The animal experiments showed that Achillea alpina L. alleviated the liver injury in rats, and significantly reduced the serum levels of ALT, AST, TNF-α, IL-6, IL-1β and the content of MDA in the liver tissues of rats in the model group, and the activities of SOD and CAT in the liver tissues were enhanced. Achillea alpina L. upregulated the expression of Nfr2 protein in rat liver tissues, and downregulated TLR-4,NF-κBp65, keap1 and HO-1 protein expression. Conclusion Achillea alpina L. has a protective effect on rats with ALI induced by carbon tetrachloride, and this effect may be related to the regulation of keap1/Nrf2/HO-1 and Toll-like receptor signaling pathways.