Impact of mare milk-derived small extracellular vesicles on proliferation, phagocytosis, and migration in RAW264.7 macrophage.

Mare milk is rich in nutrients and bioactive compounds, suggesting that its Small extracellular vesicles may possess immunomodulatory properties. In this study, we developed an optimized ultracentrifugation method to isolate Mare Milk-Derived Small Extracellular Vesicles (MM-sEVs) from mare milk, achieving higher yield and purity compared to conventional approaches. The isolated MM-sEVs were characterized using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting. Proteomic and microarray analyses revealed that MM-sEVs are enriched with immune-related proteins (e.g., RHOA) and microRNAs (e.g., miR-155). When co-cultured with RAW264.7 macrophages, MM-sEVs significantly enhanced phagocytic activity, reduced cell migration, upregulated IL-10 expression, and modulated the expression of pro-inflammatory cytokines such as IL-1β and IL-6, etc., while IFN-γ levels remained unchanged. These findings highlight the immunomodulatory potential of MM-sEVs, particularly in regulating macrophage functions, and provide novel evidence supporting their prospective applications in health-related fields. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1038/s41598-026-38285-1.