Abstract :
[en] Small extracellular vesicles (sEVs), as important mediators of intercellular communication, carry functional molecules such as miRNAs, mRNAs, and lncRNAs. The composition of sEVs is highly dynamic and can be significantly influenced by different nutritional statuses and disease conditions. For instance, changes in diet, including the supplementation of functional ingredients, can alter the contents of sEVs. This alteration can further modulate immune responses, cellular metabolism, and tissue repair. In addition, under various pathological conditions, such as subclinical mastitis (SCM) or inflammation, sEVs can carry molecular signals that not only reflect the state of disease but also actively participate in regulating immune responses and other physiological processes. Previous studies have demonstrated that inulin supplementation can alleviate SCM and improve milk yield and quality in dairy cows, possibly by modulating microbial composition and metabolites. However, the underlying molecular mechanisms by which inulin affects the composition of sEVs, especially regarding their RNA contents, remain unclear. Moreover, sEVs are not only carriers of information but can also reverse regulatory effects on the organism, influencing processes like immune response, metabolism, and cellular differentiation. Therefore, this study aimed to investigate the effects of inulin supplementation on sEVs-derived miRNAs and other RNA molecules in bovine serum and milk, and to explore the potential signaling pathways involved, providing insights into the molecular basis of inulin's effects.
Firstly, we profiled sEVs-derived miRNAs in serum from both control and inulin groups (n=5 for each group), and found that inulin supplementation significantly altered the expression of multiple miRNAs, including 23 known miRNAs and 21 novel miRNAs (adjusted p-value < 0.05). Gene ontology (GO) enrichment analysis indicated that the differentially expressed (DE) miRNAs were primarily involved in biological processes such as signal transduction, cell differentiation, cell adhesion, apoptotic processes, actin cytoskeleton organization, and DNA-binding transcription factor activity, highlighting their roles in immune regulation and cellular responses. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that these miRNAs were significantly enriched in immune- and metabolism-related pathways, including the NF-κB signaling pathway, MAPK signaling pathway, natural killer cell-mediated cytotoxicity, PPAR signaling pathway, and insulin signaling pathway.
Secondly, the expression analysis of sEVs-derived miRNAs in milk from both control and inulin groups (n=7 for each group), revealed that inulin supplementation also modulated miRNAs associated with local mammary immune regulation. Nine DE miRNAs were identified between the two groups (adjusted p-value < 0.05). GO enrichment analysis showed that the DE miRNAs were involved in biological processes such as transmembrane signaling receptor activity, signaling receptor activity, molecular transducer activity, and ion channel activity, highlighting their roles in cell signaling and immune responses. KEGG pathway analysis identified significant enrichment in pathways such as the thyroid hormone signaling pathway, regulation of actin cytoskeleton, Ras signaling pathway, Rap1 signaling pathway, and inflammatory mediator regulation of TRP channels, all of which are essential for immune regulation and cellular responses.
Furthermore, we analyzed other RNA profiles in bovine milk-sEVs and identified 542 DE lncRNAs and 1300 DE mRNAs (adjusted p-value < 0.05). The GO analysis of DE lncRNAs revealed that they were mainly involved in processes such as tRNA binding, peptide binding, DNA-binding transcription factor activity, cysteine-type deubiquitinase activity, and chromatin binding. These lncRNAs may play significant roles in regulating gene expression. KEGG pathways suggesting that they may play important roles in immune regulation, metabolic balance, and cell survival, including sphingolipid metabolism, retinol metabolism, protein processing in the endoplasmic reticulum, lysosome function, and apoptosis-multiple species. The GO analysis of DE mRNAs revealed their involvement in several crucial biological processes, such as regulation of triglyceride biosynthesis, DNA replication, cell growth, and mRNA metabolism. Additionally, these mRNAs were associated with processes like chromatin remodeling, cellular response to xenobiotic stimulus, and activation of cysteine-type endopeptidase activity involved in apoptosis. These findings suggest that the differentially expressed mRNAs may play significant roles in lipid metabolism, cell cycle regulation, and apoptosis, as well as in responses to external stimuli. KEGG pathway analysis revealed significant enrichment of DE mRNAs in pathways such as Taurine and hypotaurine metabolism, NF-κB signaling pathway, and Inositol phosphate metabolism, which are essential for immune regulation, metabolic balance, and cellular signaling. Furthermore, DE mRNAs were associated with pathways involved in endocrine resistance, cell cycle regulation, biosynthesis of nucleotide sugars, and amino sugar and nucleotide sugar metabolism, highlighting their potential roles in cellular growth, stress response, and metabolic control.
In conclusion, inulin supplementation significantly altered the expression profiles of sEVs-derived miRNAs, mRNAs, and lncRNAs in both bovine serum and milk. These RNA molecules were associated with critical immune regulation and metabolic pathways, including immune signaling, cellular processes, and metabolic regulation. These findings suggest that inulin supplementation could help restore immune balance, reduce inflammation, and support metabolic homeostasis.
