Yak milk-derived exosomes alleviate lipopolysaccharide-induced intestinal inflammation by inhibiting PI3K/AKT/C3 pathway activation.

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 inflammation-driven 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.

Yak milk-derived exosomal microRNAs regulate intestinal epithelial cells on proliferation in hypoxic environment.

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 microRNAs (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.

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

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.

Effect of pH on dissociation of casein micelles in yak skim milk.

The dissociation of yak casein (CN) micelles was evaluated by scanning electron microscopy, particle size, fluorescence properties, and soluble mineral and CN molecule content at pH 4.6 to 8.2. The results showed that the size of CN micelles remained constant with decreasing pH from 8.2 to 5.8 but sharply increased at pH ≤5.4. Casein micelles began to aggregate at pH 5.4, and the serum magnesium, potassium, iron, zinc, copper, and manganese levels had their minimum values at this pH level. During acidification, colloidal calcium phosphate dramatically disassociated from yak CN micelles, but the soluble CN monomer content decreased slightly. During alkalization, the soluble calcium and phosphorus content decreased below pH 6.8 but increased with pH increases from 6.8 to 8.2. However, the soluble CN content increased markedly during alkalization. The emission wavelength of 8-anilino-1-naphthalenesulfonic acid sodium salt fluorescence decreased during both acidification and alkalization from pH 6.6, whereas the opposite was found for intrinsic fluorescence.

Chemical composition of yak colostrum and transient milk.

The objective of this study was to assess the changes in the chemical composition of yak colostrum and transient milk. Samples were collected from 12 yaks on days 1, 2, 3, 4, 5, 6 and 7 post-partum (PP). The gross composition, nitrogen fraction distribution, amino acid (AA) profile and fatty acid (FA) profile were analysed. All the components decreased rapidly during the first 3 days except lactose which increased. The ratio of whey protein to casein protein decreased from 46:54 to 17:83 during the first 7 days PP. The content of all the AAs decreased, while the percentages of eight essential AAs in protein of samples increased during the study period. Monounsaturated FAs and polyunsaturated FAs decreased in the first 7 days PP, whereas saturated FAs increased. In conclusion, the changes in chemical composition were remarkable during the first 3 days. The slight variations, happened during the transient period, are not negligible, which also should be taken into account in the development of yak colostrum supplements.

Effect of iron saturation level of lactoferrin on osteogenic activity in vitro and in vivo.

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 ß-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.

Microscopic Characterization of Membrane Transporter Function by In Silico Modeling and Simulation.

Membrane transporters mediate one of the most fundamental processes in biology. They are the main gatekeepers controlling active traffic of materials in a highly selective and regulated manner between different cellular compartments demarcated by biological membranes. At the heart of the mechanism of membrane transporters lie protein conformational changes of diverse forms and magnitudes, which closely mediate critical aspects of the transport process, most importantly the coordinated motions of remotely located gating elements and their tight coupling to chemical processes such as binding, unbinding and translocation of transported substrate and cotransported ions, ATP binding and hydrolysis, and other molecular events fueling uphill transport of the cargo. An increasing number of functional studies have established the active participation of lipids and other components of biological membranes in the function of transporters and other membrane proteins, often acting as major signaling and regulating elements. Understanding the mechanistic details of these molecular processes require methods that offer high spatial and temporal resolutions. Computational modeling and simulations technologies empowered by advanced sampling and free energy calculations have reached a sufficiently mature state to become an indispensable component of mechanistic studies of membrane transporters in their natural environment of the membrane. In this article, we provide an overview of a number of major computational protocols and techniques commonly used in membrane transporter modeling and simulation studies. The article also includes practical hints on effective use of these methods, critical perspectives on their strengths and weak points, and examples of their successful applications to membrane transporters, selected from the research performed in our own laboratory.

Structure-activity relationship of caffeic acid analogues on xanthine oxidase inhibition.

Caffeic acid has been reported to have activity on xanthine oxidase inhibition which is related to several diseases, e.g. gout, hepatitis and tumors. Based on this study, the alpha, beta-unsaturated COOH moiety in the molecule of caffeic acid plays a very important role on the xanthine oxidase inhibition because hydrocaffeic acid was inactive and the activities of coniferyl aldehyde and coniferyl alcohol were reduced as compared with ferulic acid. Moreover, chlorogenic acid showed a weaker activity than caffeic acid. On the other hand, the phenolic OH group present in the molecule of caffeic acid makes an important contribution to the activity, e.g. transcinnamic acid in which the absence of the phenolic OH group in the structure reduced its activity as compared with caffeic acid. Ferulic acid, isoferulic acid and 3,4-dimethoxy cinnamic acid also had reduced activity due to the methoxy groups replacing the phenolic OH group in the structures. However, m-coumaric acid displayed the strongest activity (IC50 = 63.31 microM) and induced uncompetitive inhibition with respect to the substrate xanthine (Ki = 21.568 microM). Caffeic acid (IC50 = 74.6 microM) showed the second strongest activity, followed by p-coumaric acid (IC50 = 111.09 microM).

Fatty acid composition of 12 fish species from the Black Sea.

The long-chain polyunsaturated fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are healthful to humans, particularly in promoting growth and cognitive development in infants and young children, and in reducing the risk of cardiovascular disease. Though the populations who inhabit the Trabzon province of Turkey include seafood from the Black Sea in their diet, knowledge of the fatty acid composition and content of these fish is scant. Fatty acid analysis was performed on freeze-dried muscle tissue of 12 species of fish purchased in markets in Trabzon. The fat content varied from 0.2% (garfish) to 12% (shad) of dry weight. The highest DHA and DHA plus EPA contents were found in horse mackerel 16.1 and 20.6 mg/g dry weight, respectively. Only in sea bass and sea bream did the essential fatty acid linoleic acid account for more than 10% of the fatty acid total. For all 12 species, arachidonic acid accounted for 0.09% to 7.64% of the fatty acid total. Oleic acid varied greatly from 0.14% (garfish) to 32.7% (shad). The omega-3/omega-6 fatty acid ratio ranged from 0.8 to 25. A 100 g serving of fresh horse mackerel would contribute 586 mg of DHA to the diet, which exceeds the recommended daily intake of 200 to 300 mg of DHA for pregnant and lactating women. These data indicate that some, but not all, of the 12 fish species from the Black Sea fish we studied could contribute significantly to satisfy the DHA and EPA needs of the inhabitants of the eastern Black Sea region of Turkey.