Bovine milk-derived extracellular vesicles prevent gut inflammation by regulating lipid and amino acid metabolism.

Inflammatory bowel disease (IBD) is a global health problem in which metabolite alteration plays an important pathogenic role. Bovine milk-derived extracellular vesicles (mEVs) have been shown to regulate nutrient metabolism in healthy animal models. This study investigated the effect of oral mEVs on metabolite changes in DSS-induced murine colitis. We performed metabolomic profiling on plasma samples and measured the concentrations of lipids and amino acids in both fecal samples and colonic tissues. Plasma metabolome analysis found that mEVs significantly upregulated 148 metabolite levels and downregulated 44 metabolite concentrations (VIP > 1, and p < 0.05). In the fecal samples, mEVs significantly increased the contents of acetate and butyrate and decreased the levels of tridecanoic acid (C13:0), methyl cis-10-pentadecenoate (C15:1) and cis-11-eicosenoic acid (C20:1). Moreover, the concentrations of eicosadienoic acid (C20:2), eicosapentaenoic acid (C20:5), and docosahexaenoic acid (C22:6) were decreased in colonic tissues with mEV supplementation. In addition, compared with the DSS group, mEVs significantly increased the content of L-arginine, decreased the level of L-valine in the fecal samples, and also decreased the levels of L-serine and L-glutamate in the colonic tissues. Collectively, our findings demonstrated that mEVs could recover the metabolic abnormalities caused by inflammation and provided novel insights into mEVs as a potential modulator for metabolites to prevent and treat IBD.

Supplementation with Milk-Derived Extracellular Vesicles Shapes the Gut Microbiota and Regulates the Transcriptomic Landscape in Experimental Colitis.

Harboring various proteins, lipids, and RNAs, the extracellular vesicles (EVs) in milk exert vital tissue-specific immune-protective functions in neonates via these bioactive cargos. This study aims to explore the anti-inflammatory effects of bovine milk-derived EVs on a dextran sulfate sodium (DSS)-induced colitis model and to determine the underlying molecular mechanisms. Sixty C57BL/6 mice were divided into the NC group (normal control), DSS group (DSS + PBS), DSS + LOW group (DSS + 1.5 × 108 p/g EVs), DSS + MID group (DSS + 1.5 × 109 p/g EVs), and DSS + HIG group (DSS + 1.0 × 1010 p/g EVs). Histopathological sections, the gut microbiota, and intestinal tissue RNA-Seq were used to comprehensively evaluate the beneficial functions in mitigating colitis. The morphology exhibited that the milk-derived EVs contributed to the integrity of the superficial epithelial structure in the intestine. Additionally, the concentrations of IL-6 and TNF-α in the colon tissues were significantly decreased in the EVs-treated mice. The abundances of the Dubosiella, Bifidobacterium, UCG-007, Lachnoclostridium, and Lachnospiraceae genera were increased in the gut after treatment with the milk-derived EVs. Additionally, the butyrate and acetate production were enriched in feces. In addition, 1659 genes were significantly down-regulated and 1981 genes were significantly up-regulated in the EVs-treated group. Meanwhile, 82 lncRNAs and 6 circRNAs were also differentially expressed. Overall, the milk-derived EVs could attenuate colitis through optimizing gut microbiota abundance and by manipulating intestinal gene expression, implying their application potential for colitis prevention.

Effects of oral milk extracellular vesicles on the gut microbiome and serum metabolome in mice.

Milk extracellular vesicles (EVs) are rich in abundant bioactive macromolecules, such as glycoconjugates, proteins, lipids and nucleic acids, and these vesicles might transmit signals to human consumers. However, it remains to be determined whether milk EVs import new pathogens to humans or are beneficial for human health. Here, C57BL/6 female and male mice were randomly divided into 4 EV dose levels (0, 1.5 × 109 p g-1, 1.0 × 1010 p g-1 and 1.5 × 1010 p g-1). Based on the alterations in body weight, the control group (0 p g-1, PBS) and the middle treatment group (1.0 × 1010 p g-1) were chosen for further analysis of the effects of EVs on the gut microbiota and blood metabolites in mice, by 16S rRNA gene sequencing and untargeted metabolomics, respectively. We found that milk EVs increased the abundance of "beneficial" microbes such as Akkermansia, Muribaculum and Turicibacter, while decreased the level of "harmful" bacteria Desulfovibrio. Serum metabolites showed that EVs mainly changed the lipid and amino acid metabolism, and especially increased several serum anti-inflammatory factors, which might be beneficial for inflammation and other metabolic diseases. The results of KEGG analysis suggested that the enriched pathways were the intestinal immune network for IgA production, retinol metabolism, and D-glutamine and D-glutamate metabolism. Taken together, the positive effect of milk EVs on serum nutrient metabolism without promoting "harmful" bacterial colonization in female and male mice may indicate that they are safe bioactive molecules, and some of the changes they induce may provide protection against certain diseases.

Characterization of Sheep Milk Extracellular Vesicle-miRNA by Sequencing and Comparison with Cow Milk.

Milk can mediate maternal-neonatal signal transmission by the bioactive component extracellular vesicles (EVs), which select specific types of miRNA to encapsulate. The miRNA profiling of sheep milk EVs was characterized by sequencing and compared with that of cow milk. Nanoparticle tracking analysis revealed that the concentration of sheep milk EVs was 1.3 ± 0.09 × 1012 particles/mL and the diameter was peaked at 131.2 ± 0.84 nm. Sheep milk EVs contained various small RNAs, including tRNA, Cis-regulatory element, rRNA, snRNA, other Rfam RNA, and miRNA, which held about 36% of all the small RNAs. In total, 84 types of miRNA were annotated with Ovis aries by miRBase (version 22.0) in sheep milk EVs, with 75 shared types of miRNAs in all samples. The miR-26a, miR-191, let-7f, let-7b and miR-10b were highly expressed both in cow and sheep milk EVs, and 14 sheep milk EV-miRNAs in the top 20, occupying 98% of the total expression, were immune-related. Although pathway analysis showed different potential functions of cow and sheep milk EV-miRNAs, there were still some shared points: lipid metabolism (phospholipase D, glycerophospholipid and glycosylphosphatidylinositol), calcium metabolism, and nerve conduction (axon guidance and synapse). This study provides reference for the bioactive components in the milk of different species.

Replacement of forage fiber with non-forage fiber sources in dairy cow diets changes milk extracellular vesicle-miRNA expression.

Milk is a dynamic source of nutrients and bioactive factors, varying with the nutrition status of the cattle. We partly replaced alfalfa hay with whole cotton seed and soybean hull (non-forage fiber source, NFFS) in the feed formula of treated cows and evaluated the effects on milk extracellular vesicles (EVs). The NFFS supplement did not affect the shape of milk EVs observed using a transmission electron microscope. Nanoparticle tracking analysis revealed that the EV concentration increased significantly in treated cows (P = 0.019), with the peak diameter unaffected by the treatment. The EV-RNA concentration and small RNA content, particularly rRNAs and tRNAs, significantly increased in the treated cows (P < 0.05). The other small RNAs, i.e. miRNAs, cis-regulatory elements, snRNAs, and other Rfam RNAs showed no significant difference between the two groups. Totally 276 milk EV-miRNAs were identified. Thirteen miRNAs, accounting for 76%, in the highly expressed top 20, were immune-related. In addition, 9 differently expressed miRNAs (4 up-regulated and 5 down-regulated) were identified (P < 0.05). Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the differently expressed miRNAs were related to the citrate cycle, fat digestion and absorption process, taurine and hypo-taurine metabolism, and glycosphingolipid biosynthesis. This study documents the milk nutrition assessment from macromolecules, especially EVs.

Different Diets Change the Expression of Bovine Serum Extracellular Vesicle-miRNAs.

Cells can communicate with neighboring or distant cells using extracellular vesicles (EVs), mainly attributed to their containing miRNAs. Given that diets can change host circulatory miRNA profiling, and EVs are the major miRNA carriers in serum, we hypothesized that different diets could change bovine circulating EV-miRNA expression. We partly replaced alfalfa hay with whole cotton seed and soybean hull in the feed formula of the tested cows. Blood EVs were isolated using a polyethylene glycol precipitation kit. Particle size analysis revealed exosomes were dominant in bovine serum EVs. Small RNAs were enriched in bovine serum EVs, including miRNAs, snRNAs, tiRNAs, Cis-regulatory elements, piRNAs, etc. In total, 359 types of Bos taurus miRNAs were identified by Solexa sequencing. Each cow in the control group contained about 244 types of serum EV-miRNAs, compared to 246 types in the tested group. There were 15 immune-related miRNAs in the top 20 serum EV-miRNAs, accounting for about 80% of the total. Seven differently expressed known miRNAs were detected in responding to different diets. An analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) showed differently expressed miRNAs were related to hormone signal pathways and protein metabolism. Bovine serum EVs are abundant with miRNAs, most of which are immune-related. Different diets eventually change the miRNA profiling of bovine serum EVs.

Blood amino acids profile responding to heat stress in dairy cows.

OBJECTIVE: The objective of this experiment was to investigate the effects of heat stress on milk protein and blood amino acid profile in dairy cows. METHODS: Twelve dairy cows with the similar parity, days in milk and milk yield were randomly divided into two groups with six cows raised in summer and others in autumn, respectively. Constant managerial conditions and diets were maintained during the experiment. Measurements and samples for heat stress and no heat stress were obtained according to the physical alterations of the temperature-humidity index. RESULTS: Results showed that heat stress significantly reduced the milk protein content (p<0.05). Heat stress tended to decrease milk yield (p = 0.09). Furthermore, heat stress decreased dry matter intake, the concentration of blood glucose and insulin, and glutathione peroxidase activity, while increased levels of non-esterified fatty acid and malondialdehyde (p<0.05). Additionally, the concentrations of blood Thr involved in immune response were increased under heat stress (p<0.05). The concentration of blood Ala, Glu, Asp, and Gly, associated with gluconeogenesis, were also increased under heat stress (p<0.05). However, the concentration of blood Lys that promotes milk protein synthesis was decreased under heat stress (p<0.05). CONCLUSION: In conclusion, this study revealed that more amino acids were required for maintenance but not for milk protein synthesis under heat stress, and the decreased availability of amino acids for milk protein synthesis may be attributed to competition of immune response and gluconeogenesis.