| Breast milk is the most important nutrient source for mammals after birth,which contains complex nutritional ingredients and can promote the growth and development of newborns.Studies indicated that milk contains miRNA,and milk-derived miRN As encapsulated in exosomes or bound to certain proteins were resistant against RNase degradation.Whether milk-derived miRN As can be absorbed by intestinal tract remains controversial.In order to verify whether milk-derived miRNAs can be absorbed by animal,in vivo and in vitro assays were designed in the present study.In addition,investigation on the effects of milk-derived exosomes on immune function in mice was conducted.The main results are as follows:(1)Bioinformatics analysis and qRT-PCR showed that six miRNAs(miR-2284 x,miR-2291,miR-7134,miR-1343,miR-2478,miR-1224)were significantly different between bovine and porcine milk whey(p<0.05)and two miRNAs(miR-500,miR-223)showed non-significance difference(p>0.05),providing that milk from different species may have various miRN A profiles.The level of four miRN As(miR-2284 x,miR-2291,miR-7134,miR-1343)showed significantly different level in piglets serum after feeding bovine or porcine milk separately(p<0.05),and another four(miR-2478,miR-1224,miR-500,miR-223)showed non-significance differences(p>0.05).(2)Bovine and porcine milk exosomes and supernatants were harvested by a series of centrifugation.q RT-PCR showed that partial milk-borne miRN As were enriched in exosomes or in supernatants and the distribution of miRNAs in two milk were different prompting that the distribution of miRNAs in milk may d iffer among species.Exosomes or supernatants from two milk were added to IPEC-J2 cells separately,finding that the higher miRN A concentration in treatment sample caused higher miRNA level in IPEC-J2 cells.In addition,immunoprecipitation and q RT-PCR of Ago2 protein,NPM1 protein and high-density lipoproteins showed that there was a certain amount of miRNAs in the three proteins.(3)qRT-PCR showed that the level of two of six miRNAs in porcine milk exosomes or supernatants-feeding weaning mouse intestine and serum were significantly higher than that in control group(p<0.05),two miRN As in experimental mouse intestine were significantly higher than that in control group(p<0.05),and another two miRN As in experimental groups serum were significantly higher than that in control group(p<0.05).(4)Detection of weaning mice serum by using ELISA showed that feeding bovine milk supernatants could significantly improve the level of IgA?TNF-??IFN-?(p<0.01;p<0.05;p<0.01),both porcine milk exosomes and bovine milk exosomes could significantly improve the level of IFN-?(p<0.01;p<0.05),and both porcine exosomes,bovine exosomes and bovine supernatants could significantly increase the ratio of IFN-?/ IL-4(p<0.01;p<0.05;p<0.01).Ileum ELISA showed that both feeding porcine exosomes and feeding bovine supernatants could significantly improve the level of IFN-?(p<0.05;p<0.05).Weaning mice ileum qRT-PCR showed that bovine supernatants could significantly improve the level of TLR4 mRN A(p<0.01).Spleen q RT-PCR showed that porcine exosomes could significantly improve the level of T-bet mRNA(p<0.05),and both bovine and porcine supernatants could significantly impair the level of GATA-3 mRNA(p<0.05;p<0.05).The results above showed that partial miRNAs in milk were absorbed into piglets' serum;miRN As from both milk-derived exosomes and supernatants could be taken up by intestinal epithelial cells;the results of IP assays showed that some miRNAs in supernatants were binding to Ago2 protein,NPM1 protein and high-density lipoproteins;milk-derived miRN As could be absorbed into mice intestine tract and some of them were further uptake into blood;Except supernatants,milk-derived exosomes could also enhance mice immune function to some extent.In this study,we provided more evidence on the absorption of milk-derived miRN As,and explored the effects of milk-derived exosomes on improving immune function in mammals. |
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