Beta-defensin-1 (BD-1) is among the class of antibacterial peptides that are rich in disulfide bonds, have direct antibacterial activity and showed enhanced expression following external stimulation. However, existing research studies only treated BD-1 to cell models without stimulation from pathogen-associated molecular patterns (PAMPs), which will further influence our understanding of the role of BD-1. In this study, we map the tissue distribution of Equus BD-1 (i.e., eBD-1, ass BD-1, and mule BD-1) and compare their expression levels in various tissues. We further characterize the three kinds of Equus BD-1 by analyzing their full-length cDNA. We showed that eBD-1, ass BD-1, and mule BD-1 have an identical (100%) open reading frame (ORF). The ORF encoding OEBD-1 expressed the ORF in the E. coli Top10 expression system. This expression system was combined with an S. aureus-infected J774A.1 macrophage cell line to determine the influence on innate immune mediator expression. Using this expression model system, it was determined that the OEBD-1 protein enhanced IL-6 and TNF-α secretion. It can also promote TLR2, IL-1β, CCL2, CCL7, CXCL10 and NF-κB p65 mRNA expression. Moreover, OEBD-1 upregulates phosphorylation of ATK, Syk and IκB-α. In addition, OEBD-1 enhances the macrophage’s ability to phagocytose S. aureus. In conclusion, Equus BD-1 was shown to play an essential role in macrophage-involved innate immune responses in an in vitro system.

Brucellosis is a zoonotic disease caused by Brucella species. Its pathogenesis is closely associated with bacterial evasion of macrophage-mediated killing, induction of a pro-inflammatory cytokine storm, and immune-mediated pathological damage. Accumulating evidence indicates that Codonopsis pilosula polysaccharides (CPPS) inhibit inflammatory signaling, enhance macrophage phagocytosis, and exert immunomodulatory effects across multiple organs. However, the therapeutic potential of CPPS in brucellosis remains largely unexplored. This study aimed to investigate the effects of CPPS on the inflammatory response induced by Brucella outer membrane protein 19 (OMP19). In vivo , CPPS significantly alleviated tissue damage and simultaneously downregulated the expression of high mobility group box 1 protein (HMGB1), E-cadherin, and paxillin. In vitro , CPPS inhibited SYK/FAK/AKT phosphorylation, PKC activation, and WNT-1 signaling pathway transduction. Additionally, CPPS modulated the cytokine profile by downregulating pro-inflammatory cytokines ( TNF-α , IL-6 ) while increasing the level of the anti-inflammatory cytokine IL-10. Furthermore, CPPS decreased the expression levels of E-cadherin and paxillin and reduced the intracellular calcium ion (Ca ²⁺ ) concentration. ATP2A1 was identified as a key differentially expressed gene through transcriptome sequencing. Knockdown experiments further confirmed that CPPS exerts anti-inflammatory effects by regulating ATP2A1 . Collectively, CPPS attenuates the inflammatory response in macrophages induced by OMP19 via regulating ATP2A1 to modulate cell adhesion and calcium signaling