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.
International audienceHeat treatment is one of the essential operations widely used in most dairy processes, and heat stability is one of the essential properties of milk. Casein micelles are the major component in milk responsible for the heat stability of milk during processing. This study assessed the effects of heat treatment temperature and duration on the average size, turbidity, polydispersity index and heat stability of casein micelles in yak skim milk and distilled water. The results showed that whey protein had an important role in influencing the heat stability of casein micelles. The average size, polydispersity index and turbidity of micelles in skim milk were higher than those of micelles in distilled water in all cases while the heat stability of casein micelles in skim milk was lower than those in distilled water. As a result of the heat treatment, the size of micelles in skim milk increased due to complex of casein/whey protein formed via covalent bonds, whereas it decreased in distilled water attributed to the change of hydrophobicity in micelles. The size distribution of particles broadened with increasing heating temperature, resulting in the increase in turbidity and polydispersity index of casein micelles both in skim milk and distilled water. The micelles in skim milk combined with whey protein during heating. These findings will help processors design appropriate heating conditions for yak milk and yak casein products and help identify new opportunities for product development
International audienceYak caseins are special materials which has attracted more and more attention of researchers due to its increasing. Succinylation is a useful method of improving the functional properties of protein significantly. This study assessed the effects of succinylation on the functional properties of yak and cow caseins, including solubility, emulsifying property, water and oil absorption capacity, foaming property, and digestibility. Six succinylated levels of caseins were prepared by adding different amounts of succinic anhydride to caseins. The succinylated degree of yak and cow caseins was different when similar amounts of succinic anhydride were added. Succinylation improved solubility, emulsifying activity, water and oil absorption capacity, and reduced foam stability and digestibility of yak and cow native caseins. However, succinylation decreased foaming capacity and emulsion stability of cow caseins while increased them of yak caseins. The functional properties of caseins changed irregularly with increasing levels of succinylation. The findings of this study revealed that succinylation had different effects on the functional properties of different sources of caseins. The results will provide the basic data of functional properties of yak native caseins and succinylated yak caseins
We studied the effect of iron saturation level on the osteogenic activity of lactoferrin (LF) in vitro and in vivo. Different iron saturation levels of LF (1.0, 9.0, 38, 58, and 96%) were prepared as the following samples: apo-LF, LF-9, LF-38, LF-58, and holo-LF. Using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, we observed that the stimulating osteoblast proliferation activity of LF in vitro decreased with increasing iron saturation level at 100 and 1,000 μg/mL. In vivo, 4-wk-old ICR Swiss male mice were randomly divided into 4 groups: blank control (physiological saline), negative control (BSA), apo-LF, and holo-LF. Four groups of mice were injected subcutaneously with physiological saline, BSA, apo-LF, or holo-LF over the calvarial surface twice a day for 5 consecutive days at a dose of 4 mg/kg per day. Bone histomorphometry showed that new bone formation (assessed using tetracycline-HCl labels) tended to be stronger with apo-LF than with holo-LF. Using fluorescence spectroscopy and circular dichroism measurements, we found that exposure of tryptophan increased, α-helix content increased, but β-structure content decreased with increasing iron saturation level. These findings indicated that the osteogenic activity of LF decreases with increasing iron saturation level in vitro and in vivo, which may be related to conformational changes in LF.
Intestinal epithelial cells (IEC) are important parts of the mucosal barrier, whose function can be impaired upon various injury factors such as lipopolysaccharide. Although food-derived exosomes are preventable against intestinal barrier injuries, there have been few studies on the effect of yak milk-derived exosomes and the underlying mechanism that remains poorly understood. This study aimed to characterize the effect of exosomal proteins derived from yak and cow milk on the barrier function of IEC-6 treated with lipopolysaccharide and the relevant mechanism involved. Proteomics study revealed 392 differentially expressed proteins, with 58 higher expressed and 334 lower expressed in yak milk-derived exosomes than those in cow exosomes. Additionally, the top 20 proteins with a relatively consistent higher expression in yak milk exosomes than cow milk exosomes were identified. Protein CD46 was found to be a regulator for alleviating inflammatory injury of IEC-6. In vitro assay of the role of yak milk exosomes on survival of IEC-6 in inflammation by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay confirmed the effectiveness of yak milk exosomes to increase IEC-6 survival up to 18% for 12 h compared with cow milk exosomes (up to 12%), indicating a therapeutic effect of yak milk exosomes in the prevention of intestinal inflammation. Furthermore, yak and cow milk exosomes were shown to activate the PI3K/AKT/C3 signaling pathway, thus promoting IEC-6 survival. Our findings demonstrated an important relationship between yak and cow milk exosomes and intestinal inflammation, facilitating further understanding of the mechanisms of inflammationdriven epithelial homeostasis. Interestingly, compared with cow milk exosomes, yak milk exosomes activated the PI3K/AKT/C3 signaling pathway more to lower the incidence and severity of intestine inflammation, which might represent a potential innovative therapeutic option for intestinal inflammation.
Intestinal epithelial cells (IEC) act as an important intestinal barrier whose function can be impaired upon induction by hypoxia. Although intestinal barrier injuries are preventable by milk-derived exosomal micro-RNAs (miRNAs), the underlying mechanism remains poorly understood. This study aimed to characterize the effect of yak and cow milk-derived exosomal miRNA on the barrier function of IEC-6 under hypoxic conditions, and explore the mechanism of yak milk exosomal miRNA to relieve the hypoxia stress. First, by Illumina HiSeq 2500 (Illumina Inc., San Diego, CA) sequencing, the miRNA expression was systematically screened, and differential expression of 130 miRNAs was identified with 51 being upregulated and 79 downregulated in yak and cow milk-derived exosomes. Furthermore, the top 20 miRNAs that had a relatively consistent high expression in yak milk exosome were identified, and bta-miR-34a was found to be an effective regulator for alleviating hypoxic injury of IEC-6. In vitro assay of the role of bta-miR-34a on survival of IEC-6 in hypoxia by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) confirmed its effectiveness to significantly increase IEC-6 survival up to 13% for 12 h, and up to 9.5% for 24 h. Investigation on the regulatory relationship between bta-miRNA-34a and the hypoxia-inducible factor/apoptosis signaling pathway provided insights into the possible mechanisms by which bta-miR-34a activated the hypoxia-inducible factor and apoptosis signaling pathway, thus promoting IEC-6 survival. The results of this study suggest an important relationship between miRNA expression and intestine barrier integrity, which facilitated further understanding of the physiological function of yak and cow milk exosomal miRNAs, as well as mechanisms of hypoxia-driven epithelial homeostasis.
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