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-milkderived 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-milkderived 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 hypoxiainducible 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.

Background: Pigmentation is controlled by complex mechanisms. Evidence suggests that miRNAs can regulate pigmentation. However, the mechanism has not been fully elucidated. Objective In this study, we revealed a novel mechanism that regulates pigmentation involving exosomes, miRNAs and the crosstalk between keratinocytes and melanocytes. Methods: The expression and localization of exosome specific marker TSG101 in keratinocytes and melanocytes; Changes of melanin content in melanocytes after co-culture of exosome and melanocytes; Expression changes of target gene TYR and its related genes and inhibitory effect of miR-330-5p on pigmentation were studied by using various molecular biological techniques. Results: In this experiment, we used miR-330-5p in keratinocytes to verify the effect of keratinocyte derived exosome on melanocyte pigmentation. First, we found that keratinocytes secrete exosomes carrying miR-330-5p; moreover, greater miR-330-5p expression was found in exosomes derived from keratinocytes that overexpressed miR-330-5p. Second, we found that exosomes derived from keratinocytes with overexpression of miR-330-5p caused a significant increase in miR-330-5p in melanocytes. Finally, exosomes derived from keratinocytes that overexpressed miR-330-5p induced a significant decrease in the production of melanin and expression of TYR in melanocytes. Meanwhile, we overexpressed miR-330-5p in melanocytes, which also proved the inhibitory effect of miR-330-5p on pigmentation. Conclusion: These findings suggest that keratinocytes crosstalk with melanocytes in the epidermal melanin unit via exosomal miRNAs. These studies reveal an important role of exosomes in melanocyte pigmentation, which opens a new pathway of melanogenesis.

Melatonin, an amine hormone highly conserved during evolution, has a wide range of physiological functions in animals and plants. It is involved in plant growth, development, maturation, and aging, and also helps ameliorate various types of abiotic and biotic stresses, including salt, drought, heavy metals, and pathogens. Melatonin-related growth and defense responses of plants are complex, and involve many signaling molecules. Among these, the most important one is nitric oxide (NO), a freely diffusing amphiphilic biomolecule that can easily cross the cell membrane, produce rapid signal responses, and participate in a wide variety of physiological reactions. NO-induced S-nitrosylation is also involved in plant defense responses. NO interacts with melatonin as a long-range signaling molecule, and helps regulate plant growth and maintain oxidative homeostasis. Exposure of plants to abiotic stresses causes the increase of endogenous melatonin levels, with the consequent up-regulation of melatonin synthesis genes, and further increase of melatonin content. The application of exogenous melatonin causes an increase in endogenous NO and up-regulation of defense-related transcription factors, resulting in enhanced stress resistance. When plants are infected by pathogenic bacteria, NO acts as a downstream signal to lead to increased melatonin levels, which in turn induces the mitogen-activated protein kinase (MAPK) cascade and associated defense responses. The application of exogenous melatonin can also promote sugar and glycerol production, leading to increased levels of salicylic acid and NO. Melatonin and NO in plants can function cooperatively to promote lateral root growth, delay aging, and ameliorate iron deficiency. Further studies are needed to clarify certain aspects of the melatonin/NO relationship in plant physiology.