Abstract
Antimicrobial peptides (AMPs) possess vaccine adjuvant activity; however, their specific targets and molecular mechanisms remain incompletely understood, which hinders their clinical application. This study aimed to elucidate the key targets and pathways through which the antimicrobial peptide BSN-37 modulates immune responses in macrophages, providing evidence for its potential clinical translation. In this investigation, Balb/c mice were administered BSN-37 for 12 h, after which total RNA was extracted from peritoneal macrophages to assess the mRNA expression levels of cytokines and key molecules on the cell surface, followed by transcriptomic sequencing. The results demonstrated that BSN-37 significantly upregulated the mRNA expression of these molecules and cytokines. A total of 228 differentially expressed long non-coding RNAs (lncRNAs) (121 upregulated, 107 downregulated) and 149 differentially expressed mRNAs (104 upregulated, 45 downregulated) were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed significant enrichment of differentially expressed mRNAs in immune response pathways, PI3K-Akt signaling, and NOD-like receptor signaling. Differentially expressed lncRNA target genes were associated with T cell receptor signaling, PD-1 checkpoint regulation, and other immune regulatory pathways. Protein-protein interaction network analysis identified core genes such as CCchemokine receptor 1 (CCR1) and Toll Like Receptor 8 (TLR8). Molecular docking studies confirmed that BSN-37 exhibited strong binding affinity to TLR8 and CCR1, with binding energies less than - 5 kcal/mol. RT-qPCR validation confirmed the reliability of the sequencing data. These findings indicate that BSN-37 activates multiple immune response pathways in macrophages by targeting immune-related genes such as TLR8 and CCR1, offering theoretical support for the development of novel immune adjuvants.