Milk-derived exosomes were identified as a novel mechanism of mother-to-child transmission of regulatory molecules, but their functions in intestinal tissues of neonates are not well-studied. Here, we characterized potential roles of porcine milk-derived exosomes in the intestinal tract. In vitro, treatment with milk-derived exosomes (27 ± 3 ng and 55 ± 5 ng total RNA) significantly promoted IPEC-J2 cell proliferation by MTT, CCK8, EdU fluorescence and EdU flow cytometry assays. The qRT-PCR and Western blot analyses indicated milk-derived exosomes (0.27 ± 0.03 μg total RNA) significantly promoted expression of CDX2, IGF-1R and PCNA, and inhibited p53 gene expression involved in intestinal proliferation. Additionally, six detected miRNAs were significantly increased in IPEC-J2 cell, while FAS and SERPINE were significantly down-regulated relative to that in control. In vivo, treated groups (0.125 μg and 0.25 μg total RNA) significantly raised mice’ villus height, crypt depth and ratio of villus length to crypt depth of intestinal tissues, significantly increased CDX2, PCNA and IGF-1R’ expression and significantly inhibited p53′ expression. Our study demonstrated that milk-derived exosomes can facilitate intestinal cell proliferation and intestinal tract development, thus giving a new insight for milk nutrition and newborn development and health.

Breast milk is the most important nutrient source for newborn mammals. Studies have reported that milk contains microRNAs (miRNAs), which are potential regulatory components. Currently, existing functional and nutritional two competing hypotheses in milk field though little date have been provided for nutritional hypothesis. In this study, we used the qRT-PCR method to evaluated whether milk miRNAs can be absorbed by newborn piglets by feeding them porcine or bovine milk. The result showed that miRNA levels (miR-2284×, 2291, 7134, 1343, 500, 223) were significantly different between bovine and porcine milk. Four miRNAs (miR-2284×, 2291, 7134, 1343) were significantly different in piglet serum after feeding porcine or bovine milk. After separated milk exosomes by ultracentrifugation, the results showed the selected milk miRNAs (miR-2284×, 2291, 7134, 1343) were present in both exosomes and supernatants, and the miRNAs showed the coincidental expression in IPEC-J2 cells. All our founding suggested that the milk miRNAs can be absorbed both in vivo and in vitro, which will building the foundation for understanding whether these sort of miRNAs exert physiological functions after being absorbed and provided additional evidence for the nutritional hypotheses. Breast milk is the first and most important source of nutrition for newborn mammals 1. By differential centrifugation, milk can be divided into milk fat, whey, casein, cells, and debris and further separated by ultra-centrifugation into extracellular vesicles (EVs) and supernatant 2. Exosomes, which are EVs of 30-100 nm in diameter and of endocytic origin, are released by numerous cells and are present in several body fluids, including saliva 3,4 , plasma 5 , urine 6 , amniotic fluid 7 , malignant ascites 8 , bronchoalveolar lavage fluid 9 , and synovial fluids 10. Studies have reported that exosomes contain lipids, proteins, mRNA, and microRNA (miRNA) 11-14 and that they serve as novel vehicles in cell-to-cell communication 15,16. Just like other body fluids, milk contains EVs 2,17. Hata et al. detected the presence of mRNA and miRNA in bovine milk-derived vesicles 18. MiRNAs represent a class of endogenous non-coding RNAs of approximately 22 nucleotides in length that are widely distributed in eukaryotes. The biological function of miRNAs is to destabilize mRNAs or halt mRNA translation 19,20. Studies have reported that 12 body fluids contain miRNAs, and milk has the highest concentration of total RNA that is rich in miRNAs 21. Milk components that contain miRNAs include milk fat globules 22 , whey 23,24 , and exosomes 11,15. Interestingly, Izumi et al. suggested that miRNAs were also present in the supernatant of ultra-centrifuged bovine raw milk 25. Furthermore, Zhou et al. confirmed the presence of 452 pre-miRNAs in human milk exosomes, which lead to 602 mature miRNAs 26. Chen et al. reported the presence of 245 miRNAs in bovine milk 27 , and Kosaka et al. detected 281 of 723 known human miRNAs in human milk by microarray technology 28. Porcine milk exosomes c...