S2 Data -

Objectives
The goal of this investigation was to identify the main compounds and the pharmacological mechanism of the traditional Chinese medicine formulation, Gong Ying San (GYS), by infrared spectral absorption characteristics, metabolomics, network pharmacology, and molecular-docking analysis for mastitis. The antibacterial and antioxidant activities were determined in vitro.
Methods
The chemical constituents of GYS were detected by ultra-high-performance liquid chromatography Q-extractive mass spectrometry (UHPLC-QE-MS). Related compounds were screened from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP, http://tcmspw.com/tcmsp.php) and the Encyclopedia of Traditional Chinese Medicine (ETCM, http://www.tcmip.cn/ETCM/index.php/Home/) databases; genes associated with mastitis were identified in DisGENT. A protein-protein interaction (PPI) network was generated using STRING. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment screening was conducted using the R module. Molecular-docking analyses were performed with the AutoDockTools V1.5.6.
Results
Fifty-four possible compounds in GYS with forty likely targets were found. The compound-target-network analysis showed that five of the ingredients, quercetin, luteolin, kaempferol, beta-sitosterol, and stigmasterol, had degree values >41.6, and the genes TNF, IL-6, IL-1β, ICAM1, CXCL8, CRP, IFNG, TP53, IL-2, and TGFB1 were core targets in the network. Enrichment analysis revealed that pathways associated with cancer, lipids, atherosclerosis, and PI3K-Akt signaling pathways may be critical in the pharmacology network. Molecular-docking data supported the hypothesis that quercetin and luteolin interacted well with TNF-α and IL-6.
Conclusions
An integrative investigation based on a bioinformatics-network topology provided new insights into the synergistic, multicomponent mechanisms of GYS’s anti-inflammatory, antibacterial, and antioxidant activities. It revealed novel possibilities for developing new combination medications for reducing mastitis and its complications.

研究目标
本研究旨在通过红外光谱吸收特征、代谢组学、网络药理学及分子对接分析，明确中药方剂公英散（Gong Ying San, GYS）治疗乳腺炎的主要活性成分及其药理作用机制，并通过体外实验测定其抗菌与抗氧化活性。

研究方法
采用超高效液相色谱-四极杆提取质谱（UHPLC-QE-MS）检测公英散的化学成分；从中药系统药理学数据库与分析平台（TCMSP, http://tcmspw.com/tcmsp.php）及中药百科全书（ETCM, http://www.tcmip.cn/ETCM/index.php/Home/）数据库中筛选相关化合物；从DisGENT数据库中筛选与乳腺炎相关的致病基因；借助STRING数据库构建蛋白质相互作用（PPI）网络；采用R语言模块开展基因本体（GO）及京都基因与基因组百科全书（KEGG）通路富集分析；使用AutoDockTools V1.5.6进行分子对接实验。

研究结果
本研究共鉴定出公英散中54种潜在活性成分与40个潜在作用靶点。化合物-靶点网络分析显示，槲皮素、木犀草素、山奈酚、β-谷甾醇及豆甾醇这5种成分的节点度值均大于41.6；而TNF、IL-6、IL-1β、ICAM1、CXCL8、CRP、IFNG、TP53、IL-2及TGFB1为该网络的核心靶点。富集分析结果表明，癌症通路、脂质代谢通路、动脉粥样硬化通路及PI3K-Akt信号通路可能是该药理网络的关键通路。分子对接实验数据证实，槲皮素与木犀草素可与TNF-α及IL-6实现良好结合。

研究结论
本研究基于生物信息学与网络拓扑学的整合分析，为阐明公英散抗炎、抗菌及抗氧化的多成分协同作用机制提供了新的视角，同时为开发用于治疗乳腺炎及其并发症的新型复方药物提供了全新的可能性。