Bovine Milk-Derived Extracellular Vesicles Ameliorate Steatohepatitis by Restoring Gut Barrier in CDA-HFD-Fed Mice

Gut barrier dysfunction and portal endotoxemia contribute to metabolic dysfunction-associated steatohepatitis (MASH) progression through activation of hepatic inflammatory signaling. Milk-derived extracellular vesicles (EVs) contain bioactive microRNAs and have recently attracted attention as modulators of intestinal homeostasis. This study investigated the effect of bovine milk-derived extracellular vesicles (B-mEVs) in ameliorating MASH by improving intestinal barrier function. C57BL/6J mice fed a choline-deficient amino acid-defined high-fat diet (CDA-HFD) were orally treated with B-mEVs, and therapeutic effects were evaluated. Liver histology, fibrosis, portal lipopolysaccharide (LPS) levels, intestinal permeability, and gut microbiota composition were evaluated. The direct effects of B-mEVs on intestinal barrier function were assessed using palmitic acid-stimulated Caco-2 cells. Small RNA sequencing and microRNA enrichment analyses were performed to characterize B-mEV cargo. B-mEV treatment attenuated hepatic steatosis, inflammation, and fibrosis in CDA-HFD-fed mice and reduced serum aminotransferase levels, portal LPS concentrations, hepatic macrophage accumulation, and hepatic TLR4/NF-κB signaling activation. Meanwhile, B-mEVs restored intestinal tight junction proteins, including ZO-1, occludin, and claudin-1, and improved intestinal permeability in vivo. In Caco-2 cells, B-mEVs attenuated palmitic acid-induced barrier dysfunction and suppressed myosin light chain kinase expression. Gut microbiota analysis revealed partial restoration of Akkermansia abundance after B-mEV administration. Furthermore, to explore the molecular basis of these protective effects, small RNA sequencing demonstrated enrichment of regulatory microRNAs, including let-7a-5p, and pathway analyses identified associations with intestinal barrier and inflammatory signaling pathways. B-mEVs ameliorated CDA-HFD-induced steatohepatitis by restoring intestinal barrier integrity and suppressing gut-derived LPS/TLR4 inflammatory signaling. These findings suggested that milk EVs may represent a novel gut–liver axis-targeted therapeutic strategy for MASH.