Intraperitoneal BSN-37 activates peritoneal macrophages in Balb/c mice and reshapes the circRNA–mRNA transcriptome

Our preliminary work indicates that the antimicrobial peptide BSN-37 exhibits immunostimulatory activity; however, its in vivo molecular mechanisms remain insufficiently defined. Here, using male Balb/c mice, we profiled mRNA and circRNA expression in peritoneal macrophages under optimized dosing and timing conditions. By integrating these in vivo datasets with our in vitro RAW264.7 results, we aimed to identify key circRNAs involved in BSN-37–mediated immune enhancement and to construct putative circRNA–miRNA–mRNA regulatory networks.Male Balb/c mice (6–8 weeks old) were randomly assigned to a control group or a BSN-37–treated group. To enrich peritoneal macrophages, mice received an intraperitoneal injection of 3% thioglycollate broth three days prior to sampling. The control group was then injected intraperitoneally with PBS, whereas the treatment group received BSN-37 (48 μg/mL, 0.2 mL per mouse). Twelve hours later, peritoneal lavage fluid was collected for macrophage isolation. Total RNA was extracted and subjected to quality control. CircRNA and mRNA transcriptomes were obtained using the Mouse circular RNA Array V2.0 and the Arraystar Mouse LncRNA Expression Microarray V3.0, respectively. Differentially expressed molecules were identified using the criteria of P < 0.05 and fold change > 2, followed by hierarchical clustering, GO/KEGG enrichment analyses, and ceRNA network construction. To reduce false positives, multiple prediction algorithms were applied and only intersecting targets were retained. Network visualization was performed using Cytoscape. Compared with the control group, a total of 149 differentially expressed mRNAs were identified in peritoneal macrophages from the BSN-37–treated group (104 upregulated and 45 downregulated), together with 20 differentially expressed circRNAs (6 upregulated and 14 downregulated). Volcano plots and hierarchical clustering heatmaps demonstrated a clear separation between the two groups at the transcriptomic level. Functional enrichment analyses indicated that these differentially expressed molecules were involved in multiple biological processes and signaling pathways related to immune regulation. By further integrating in vivo and in vitro datasets, we constructed a circRNA–miRNA–mRNA tripartite regulatory network comprising 5 circRNAs, 6 miRNAs, and 50 mRNAs, providing an omics-based foundation for prioritizing key candidate circRNAs and subsequent mechanistic validation. BSN-37 markedly reshapes the mRNA/circRNA expression landscape of peritoneal macrophages in vivo and establishes immune regulation–associated molecular networks. The candidate circRNAs and ceRNA network identified here offer important clues for elucidating the immunostimulatory mechanisms of BSN-37 and for screening critical regulatory targets.