基于UHPLC-Q-TOF-MS与网络药理学分析梓木草-夏枯草药对的抗炎药效物质基础及体外验证

Anti-inflammatory Pharmacodynamic Substance Basis of Lithospermum zollingeri - Prunella vulgaris Herb Pair Based on UHPLC-Q-TOF-MS and Network Pharmacology Analysis with In vitro Validation

关键词:  梓木草  夏枯草  UHPLC-Q-TOF-MS  网络药理学  RAW264.7细胞

Key words:  Lithospermi zollingeri herba  Prunellae spica  UHPLC-Q-TOF-MS  network pharmacology  RAW264.7 Cells

摘要:

目的 鉴定梓木草-夏枯草药对化学成分,利用网络药理学筛选活性成分并通过细胞实验探讨其抗炎药效物质基础。方法 采用UHPLC-TOF-MS技术鉴定梓木草-夏枯草药对的化学成分；通过Swiss ADME平台、SwissTargetPrediction、GeneCards及OMIM数据库筛选活性成分,运用STRING数据库及Cytoscape 3.10.1构建蛋白互作(PPI)网络。采用David数据库进行GO功能及KEGG通路富集分析。建立脂多糖(lipopolysaccharide, LPS)诱导的RAW264.7巨噬细胞炎症模型,通过ELISA法检测细胞上清液中COX-2、PGE2、MCP-1及细胞裂解液中COX-1含量,验证提取物及活性成分的抗炎功效并探讨其药效物质基础。结果 共鉴定出56种化合物,其中梓木草药材的UHPLC-Q-TOF-MS表征为首次报道。网络药理学筛选出桑色素、秦皮乙素、咖啡酸为重要活性成分；核心靶点包括GAPDH、TNF、ALB、AKT1、EGFR等。GO富集分析显示主要涉及外源刺激应答、细胞迁移正向调控、炎症反应等过程；KEGG富集分析提示主要富集于细胞对氮化合物反应、炎症应答及癌症通路。细胞实验表明梓木草、夏枯草、梓木草-夏枯草药对提取物及单体化合物(桑色素、秦皮乙素、咖啡酸)均能显著抑制LPS诱导的MCP-1、COX-2、PGE2产生,而对COX-1的蛋白表达量无显著影响。结论 梓木草-夏枯草药对通过多成分、多靶点、多通路协同发挥抗炎作用,为阐明其抗炎药效物质基础提供了理论依据。

Abstract:

ABSTRACT: OBJECTIVE To identify the chemical constituents of the Zimucao (Lithospermi zollingeri herba)-Xiakucao (Prunellae spica) herb pair, screen its active components using network pharmacology, and explore the material basis of its anti-inflammatory efficacy through cellular experimental verification. METHODS Ultra-high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF-MS) was employed to identify the chemical constituents of Zimucao and Xiakucao. Active components were screened via the Swiss ADME platform, SwissTargetPrediction, GeneCards, and OMIM databases. The STRING database and Cytoscape 3.10.1 were used to construct a protein-protein interaction (PPI) network. GO functional annotation and KEGG pathway enrichment analyses were performed using the David database. A lipopolysaccharide (LPS)-induced inflammatory model in RAW264.7 macrophages was established. The contents of COX-2, PGE2, and MCP-1 in cell supernatants, as well as COX-1 in cell lysates, were detected by ELISA to verify the anti-inflammatory effects of extracts and active components and explore their pharmacodynamic material basis. RESULTS A total of 56 compounds were identified, among which the UHPLC-Q-TOF-MS characterization of?Zimucao?was reported for the first time. Network pharmacology screening identified morin, esculetin, and caffeic acid as key active components; core targets included GAPDH, TNF, ALB, AKT1, and EGFR. GO enrichment analysis revealed involvement in processes such as response to external stimuli, positive regulation of cell migration, and inflammatory response. KEGG enrichment analysis indicated enrichment in pathways related to cellular responses to nitrogen compounds, inflammatory response, and cancer. Cellular experiments showed that extracts of?Zimucao,?Xiakucao, their herb pair, and the monomer compounds (morin, esculetin, caffeic acid) significantly inhibited LPS-induced production of MCP-1, COX-2, and PGE2, but had no significant effect on the protein expression of COX-1. CONCLUSION The Zimucao-Xiakucao herb pair exerts its anti-inflammatory effects through a multi-component, multi-target, multi-pathway synergistic mechanism. This study provides a theoretical foundation for elucidating the pharmacodynamic material basis underlying its anti-inflammatory efficacy. KEY WORDS: Lithospermi zollingeri herba; Prunellae spica; UHPLC-Q-TOF-MS; network pharmacology; RAW264.7 Cells