Evaluation of the mechanism of Gong Ying San activity on dairy cows mastitis by network pharmacology and metabolomics analysis

Abstract

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.

Fig 1. Identification of chemical components of GYS by UHPLC-QE-MS.

Total ion chromatography in positive (A) and negative (B) ion modes.

Fig 2. Venn diagram showing the intersection of targets of GYS active components with potential mastitis-associated targets.

Fig 3. GO and KEGG enrichment analysis for identifying mechanism of the anti-mastitis activity of GYS.

(A) Distribution of GO elements in biological processes, molecular functions, and cellular composition (for FDR<0.05). The x-axis shows counts of enriched targets, while the y-axis shows the GO category of the target gene. (B) The top 20 KEGG pathways (FDR<0.05). The color corresponds to p-value threshold, and the dot size indicates the number of genes for each term. The x-axis shows the enrichment score, and the y-axis gives the main pathways.

Fig 4. Construction of PPI network and core targets.

(A) PPI network showing the relationship among common GYS targets associated with the anti-inflammatory activity. (B) TSV files were obtained from the STRING dbase and imported into Cytoscape 3.8.0 for visualization. The colors represent the importance of the network from most (red) to least (yellow).

Fig 5. Compound-Disease-Target network and GO, KEGG association network.

(A) Network of specific compounds-targets-diseases of the anti-inflammatory ingredients of GYS. The orange nodes show the potential therapeutic targets of GYS against mastitis, and the blue nodes stand for the network’s compounds. The green square nodes show the TCM ingredients, and the red nodes are those associated with mastitis. (B) Interaction networks showing core biotargets, pharmacological functions, and signaling pathways of GYS compounds against mastitis. The yellow nodes show the hub targets of GYS against mastitis, and the nodes in grey are the enriched BPs from hub targets. The yellow ellipse nodes show the enriched CCs and the blue nodes are enriched MFs. The violet nodes show the KEGG enrichment analysis of GYS against mastitis.

Fig 6. The protein ligands of the docking simulation.

(A) Docking of quercetin with TNF. (B) Docking of quercetin with IL-6. (C) Docking of luteolin with TNF. (D) Docking of luteolin with IL-6.

Fig 7

Time-dependent bacteriostatic growth curve of GYS on (A) S. aureus ATCC 29740, (B) S. agalactiae CVCC 3940 and (C) E. coli CVCC 1450.

Fig 8. Antioxidant activity of the GYS extract in vitro.

(A) DPPH radical scavenging assay. Assays were run in triplicate in three independent experiments. (B) The •OH-scavenging activity of GYS extract compared with Vc (vitamin C) as positive control.