Bovine colostrum exosomes mediate anti-inflammatory responses in macrophages via the miR-30a-5p/Sox4 axis

Bovine colostrum is essential for neonatal immunity and development, with its exosomal cargo of microRNAs (miRNAs) 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 antiinflammatory activity.It significantly reduced the expression of key proinflammatory cytokines, namely, tumor necrosis factor- (TNF-), IL-1, 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- 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.