Yak-milk-derived exosomes promote proliferation of intestinal epithelial cells in an hypoxic environment

Abstract

Intestinal epithelial cells (IEC) are an important part of the intestinal barrier. Barrier function was disrupted under hypoxia, but milk-derived exosomes can regulate the intestinal barrier function. However, the mechanisms underlying the association between yak milk exosomes and hypoxia in IEC remain poorly understood. In this follow-up study, we proposed an effective optimization method for purifying yak-milk-derived exosomes. The Western blot analyses indicated that the expression of the proteins of the endosomal sorting complexes required for transport (TSG101), proteins of the tetraspanin family (CD63), and heat shock protein 70 (Hsp-70) proteins from yak-milk-derived exosomes were significantly higher than those in cow-milk-derived exosomes. Flow cytometry analysis showed that yak milk had 3.7 times the number of exosomes compared with cow milk. Moreover, we explored whether yak milk exosomes could facilitate intestinal cell survival under hypoxic conditions in vitro. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide results showed that yak-milk-derived exosomes significantly increased survival of IEC-6 cells with rates of up to 29% for cells incubated in hypoxic conditions for 12 h, compared with those of cow-milk-derived exosomes posttreatment (rates of up to 22% for cells incubated in hypoxic conditions for 12 h). Confocal microscopy revealed that the IEC-6 cells uptake more yak-milk-derived exosomes than cow milk in hypoxic conditions. Furthermore, the Western blot analyses indicated that yak-milk-derived exosomes significantly promote oxygen-sensitive prolyl hydroxylase (PHD)-1 expression and decrease the expression of hypoxia-inducible factor-α and its downstream target vascular endothelial growth factor (VEGF) in the IEC-6 cells. Further, yak-milk-derived exosomes significantly inhibited p53 levels. In conclusion, our findings demonstrate that yak-milk-derived exosomes more effectively activate the hypoxia-inducible factor signaling pathway, thus promoting IEC-6 cell survival, which may result in higher hypoxia tolerance than cow-milk-derived exosomes.