Abstract
Bovine colostrum is essential for neonatal immunity and development, with its exosomal cargo of microRNAs (miRNA) playing a key role in intercellular communication. However, the specific immunomodulatory functions of these exosomes and their temporal variation remain unclear. This study aimed to isolate and characterize exosomes from bovine colostrum at 2 h (2h-Exo) and 24 h (24h-Exo) postpartum, evaluate their effects on cell proliferation and inflammation, and identify the underlying miRNA-mediated mechanisms. Exosomes were successfully isolated using an optimized method combining rennet enzyme digestion, filtration, and differential ultracentrifugation. Small RNA sequencing revealed 69 differentially expressed miRNAs between 2h-Exo and 24h-Exo (22 upregulated, 47 downregulated). Functionally, in an in vitro LPS-induced inflammatory model using macrophages, 2h-Exo demonstrated superior anti-inflammatory activity. It significantly reduced the expression of key proinflammatory cytokines, namely TNF-α, IL-1β, and IL-6, as well as IL-10, compared with 24h-Exo. Bioinformatic and functional analyses identified bta-miR-30a-5p as a critical, temporally enriched miRNA in 2h-Exo. Mechanistically, dual-luciferase reporter assays confirmed that bta-miR-30a-5p directly targets the 3'-untranslated region of the transcription factor SOX4. Furthermore, miR-30a-5p mimic transfection recapitulated the anti-inflammatory effect by downregulating SOX4 and attenuating the expression of TNF-α and IL-6. This study demonstrates that bovine colostrum exosomes, particularly those harvested immediately postpartum, possess potent immunomodulatory capacity. The anti-inflammatory effect is mediated, at least in part, by the exosomal delivery of bta-miR-30a-5p and its subsequent targeting of SOX4 mRNA. These findings highlight early colostrum as a valuable source of therapeutic exosomes and elucidate a specific miRNA-mRNA axis for regulating inflammation.