Exploring The Attenuation Mechanisms Of Bovine Milk Small Extracellular Vesicles In Colitis

Bovine milk has abundant nutrients,which play an important role in human diet.Small extracellular vesicles（sEVs）contain bioactive molecules,including proteins,miRNAs as well as lipids,and these vesicles might avoid gastrointestinal degradation and be transmitted to human consumers by blood to regulate host gene expression at transcriptional and post-transcriptional levels.However,it’s still remains uncertain whether bovine milk-derived sEVs（BM-sEVs）can import new pathogens to human or are beneficial for human health.Inflammatory bowel disease（IBD）,which mainly includes ulcerative colitis（UC）and Crohn’s disease（CD）,is a chronic and non-specific inflammation.The incidence and prevalence of IBD are still increasing worldwide,and seriously affects the health of patients.Therefore,alleviating colitis is important for human health.We conducted the following studies:1.Effect of BM-sEVs on serum metabolites and gut microbiome in miceMale and female SPF C57BL/6 mice were randomly divided into 4 groups with 12 mice in each group,including control group（0 p/g,PBS）,high-dose BM-sEVs group(1.5×10¹⁰ p/g),middle-dose BM-sEVs group(1.0×10¹⁰ p/g)and low-dose BM-sEVs group（1.5×10⁹ p/g）.We recorded their body weight and activity daily during the study.Based on the alterations in body weight,the control group（0 p/g,PBS）and the middle group（BM-sEVs）were chosen for further analysis of the effects of BM-sEVs on gut microbiota and blood metabolites in mice,by 16S rRNA gene sequencing and untargeted metabolomics,respectively.We found that BM-sEVs increased the abundance of“beneficial”microbes such as Akkermansia,Muribaculum and Turicibacter,and decreased the level of“harmful”bacteria Desulfovibrio.Serum metabolites showed that BM-sEVs 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 Ig A production,retinol metabolism,as well as D-glutamine and D-glutamate metabolism.Taken together,the positive affect of BM-sEVs 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 benefit to prevent certain diseases.2.Effect of BM-sEVs on chronic colitisSPF male C57BL/6 mice were randomly divided into 5 groups with 12 mice in each group,including normal control group（CON）,DSS,BM-sEVs high-dose group（DSS+HIG）,BM-sEVs medium-dose group（DSS+MID）and BM-sEVs low-dose group（DSS+LOW）.CON group was given normal diet and water without any treatment.DSS group was treated with 2.5%DSS solution instead of water to induce chronic colitis.Compared with DSS group,DSS+LOW and DSS+MID groups could relieve body weight loss,increase colon length,reduce inflammatory cell infiltration,and restore epithelial cell structure.The result of Lef Se was following:Dubosiella and Bifidobacterium were significantly enriched in the DSS+MID group,while UCG-007 was significantly enriched in the DSS+LOW group.Lachnoclostridium and Lachnospiraceae＿fcs020＿group were significantly enriched in the DSS+HIG group.Meanwhile,compared with DSS group,acetic acid level in DSS+LOW and DSS+MID groups were significantly increased（P<0.05）,and medium dose of BM-sEVs significantly increased the level of butyric acid（P<0.05）.RNA-seq analysis of DSS and DSS+MID groups showed that 1,659 genes were significantly down-regulated and 1,981 genes were significantly up-regulated in the BM-sEVs-treated group.Meanwhile,82 lncRNAs and 6 circRNAs were also differentially expressed.Overall,BM-sEVs could attenuate colitis through optimizing gut microbiota abundance and manipulating intestinal gene expression,implying their application potential for colitis prevention.3.Effects of BM-sEVs on acute colitisSPF male C57BL/6 mice were randomly divided into 3 groups with 12 mice in each group,including normal control group（CON）,DSS group and DSS+BM-sEVs group（3.0×10⁹ p/g）.CON group was given normal diet and water without any treatment.DSS group was treated with 3.5%DSS solution to induce acute colitis.BM-sEVs supplementation mitigated the severity of acute enteritis by reducing body weight loss,DAI index,inflammatory cell infiltration,mucosal damage,and overall histological score.RNA-seq analysis showed that the proinflammatory cytokines（IL-1β,IL-6,IL-17A,and IL-33）,chemokine ligands（CXCL1,CXCL2,CXCL3,CXCL5,CCL3,and CCL11）and chemokine receptors（CXCR2 and CCR3）in the colonic tissues were significantly reduced in response to BM-sEVs.KEGG analysis of significantly down-regulated genes revealed that the enriched pathways were mainly related to inflammation,such as IL-17 signaling pathway,TNF signaling pathway,NF-κB signaling pathway and No D-like receptor signaling pathway.Supplementation of BM-sEVs significantly up-regulated 109proteins and down-regulated 150 proteins in the colonic tissues.The differentially expressed proteins were mainly concentrated on amino acid metabolism and lipid metabolism,such as the degradation of valine,leucine and isoleucine,tryptophan metabolism,arginine and proline metabolism,histidine metabolism,pyruvate metabolism and fatty acid degradation.Plasma metabolome analysis showed that BM-sEVs significantly up-regulated the levels of 148 metabolites,including phosphatidylcholine（PC）,phosphatidic acid（PA）,sphingomyelin（SM）and lysophospholipid（Lyso PE）concentrations,specifically:PC（20:3（8Z,11Z,14Z）/20:4（5Z,8Z,11Z,14Z））,PC（P-15:0/0:0）,PC（22:2（13Z,16Z）/22:2（13Z,16Z））,PC（22:5（4Z,7Z,10Z,13Z,16Z）/16:0）,PA（22:4（7Z,10Z,13Z,16Z）/14:0）,PA（16:0/22:2（13Z,16Z））,SM（d18:2（4E,14Z）/24:0）,Lyso PE（18:3（9Z,12Z,15Z）/0:0）,and Lyso PE（0:0/20:3（5Z,8Z,11Z））,down-regulated the concentrations of 44 metabolites.TCA cycle,acetaldehyde and dicarboxylic acid metabolism and pyruvate metabolism were significantly affected pathways by BM-sEVs treatment.In conclusion,BM-sEVs can promote the colonization of intestinal beneficial bacteria and reduce the colonization of harmful bacteria,which is a beneficial bioactive substance.BM-sEVs can effectively improve inflammation and intestinal health in mice with chronic and acute experimental colitis,and this study provided objective evidence on transcriptome,proteome,metabolome and microbiome levels.This study not only provides a new idea for the nutritional value of bovine milk,but also provides scientific references for the development of dietary supplements to regulate human intestinal health and attenuate colitis.