Systemic Pharmacology Reveals the Potential Targets and Signaling Mechanisms in the Adjuvant Treatment of Brucellosis with Traditional Chinese Medicine

Abstract

Human brucellosis is one of the world's most common zoonoses, caused by Brucella infection and characterized by induced inflammation, which in severe cases can lead to abortion and sterility in humans and animals. There is growing evidence that traditional Chinese medicine (TCM) is beneficial as an adjunct to the treatment of brucellosis. However, its specific targets of action and molecular mechanisms remain unclear. In this study, a systematic pharmacological approach was applied to demonstrate pharmacological targets, biological functions, and signaling pathways of TCM as an adjunct to the treatment of brucellosis (TCMTB). The results of network pharmacology were further verified by in vitro experiments. Network analysis revealed that 133 active ingredients and 247 targets were screened in TCMTB. Further data analysis identified 21 core targets and 5 core compounds in TCMTB, including beta-sitosterol, quercetin, kaempferol, luteolin, and paeoniflorin. Gene ontology and the Kyoto Encyclopedia of Gene and Genome analysis showed that TCMTB might actively treat brucellosis by regulating inflammatory response, enhancing immune function, and targeting signaling pathways such as tuberculosis and TNF. Molecular docking results showed that multiple compounds could bind to multiple targets. Further, in vitro experiments confirmed that quercetin, among the active compounds screened, induced the strongest immunomodulatory and pro-inflammatory cytokine production during Brucella abortus infection. Further, quercetin induced nitric oxide production, which attenuated the ability of B. abortus to internalize THP-1 cells as well as intracellular survival. This study reveals the mechanism by which TCMTB aids in the treatment of brucellosis through a synergistic multicomponent, multipathway, and multitarget action. The contribution of quercetin treatment to B. abortus infection was demonstrated for the first time, which may be related to the quercetin-induced production of nitric oxide and immunomodulatory and inflammatory cytokines. These predictions of the core compounds and targets may be used in the future for the clinical treatment of brucellosis.

Figure 1

Workflow diagram. The figure indicates the role and mechanism of TCM in the treatment of Brucellosis using bioinformatics and network pharmacology analysis methods shown.

Figure 2

Compound-target network diagram of TCMTB. Nodes with regulatory relationships in the network are connected to each other with gray connecting lines, and the magnitude of the degree value is indicated by the size of the node. TCMTB: Gan Cao (GC), Dang Gui (DG), Fu Ling (FL), Chen Pi (CP), Bai Shao (BS), Chuan Xiong (CX), Bai Zhu (BZ), Huang Qi (HQ), Dang Shen (DS), Di Huang (DH).

Figure 3

Potential target genes and PPI network map of TCMTB therapy for brucellosis. (A) Venny results of potential target genes of TCMTB therapy for brucellosis. (B) PPI network map of 21 target genes.

Figure 4

Construction and analysis of the TCMTB–brucellosis–potential target gene. (A) Sankey diagram showing the relationship between TCMTB, compounds, and target genes. (B) Network analysis of core TCMTB–compound–brucellosis potential target gene: pink represents brucellosis, blue represents the target gene, orange represents the compound, and light green represents TCM.

Figure 5

Functional characterization of TCMTB against brucellosis intersecting genes. (A) GO analysis of the top 18 significantly enriched potential target genes of TCMTB in brucellosis. (B) KEGG analysis of potential TCMTB target gene signaling pathways in the top 20 significantly enriched brucellosis. (C, D) Suggested KEGG signaling pathways for tuberculosis and TNF. The red rectangular node represents the core gene.

Figure 6

(A–J) 3D and 2D maps of active compound binding to target proteins. The blue color indicates the active compound, the green dashed line indicates the hydrogen bond, and the brown color indicates the region where the active compound binds to the target protein.

Figure 7

TCMTB core active compound inhibits B. abortus-induced inflammation in THP1 cells. (A) PMA induces differentiation of THP1 monocytes into macrophages. (B) Effect of quercetin on cell viability of THP-1 macrophages. Values are expressed as mean ± SEM of three independent experiments. (C, D) Expression of mRNA and protein levels of immunity and inflammation-related targets after active compound treatment of B. abortus-infected THP1 macrophages. (E) B. abortus was incubated with different concentrations of quercetin, and bacterial survival rates were determined. (F) Cells were infected with B. abortus for 1 h. Quercetin was optionally added or not, and NO accumulation was detected after 2, 24, and 48 h of incubation. (G) Bacterial internalization efficiency. After 1 h of infection with B. abortus, cells were incubated with or without quercetin for 20 and 60 min. (H) Bacterial intracellular survival efficiency. *p < 0.05, **p < 0.01.