| In large-scale dairy goat production,early weaning of lambs is an important part of lamb breeding.However,the implementation of early weaning often results in problems such as intestinal inflammation and diarrhea in lambs.Studies have shown that adequate intake of breast milk significantly reduces the rate of diarrhea in lambs and that the exosomes within breast milk act as an important component in reducing intestinal inflammation in lambs.At present,most of the studies on goat breast milk at home and abroad focus on nutrients,immune antibodies and other substances,and there are few studies on milk exosomes,and few studies on the differences of milk exosomes in different lactation periods have been reported.In this study,exosomes were isolated from colostrum and mature milk of dairy goats.We also evaluated the effect of their different milk exosomes on intestinal inflammation by mouse and intestinal epithelial cell inflammation models,and analyzed the miRNA expression profiles of colostrum and mature milk exosomes by small RNA sequencing,with the aim of explaining the role of goat milk exosomes in suppressing intestinal inflammation from the miRNA level.The main findings are as follows:(1)Isolation and identification of Colostrum exosomes(C-Exo)and Mature milk exosomes(M-Exo)from dairy goats.Dairy goat C-Exo and M-Exo were isolated using isoelectric point assisted combined with differential ultracentrifugation.The Bradford method was used to detect the protein content of exosomes in two kinds of milk.The results showed that the isolated C-Exo and M-Exo protein concentrations were 2.58±0.14 mg/m L and1.98±0.13 mg/m L,respectively.The protein concentration of C-Exo was significantly higher than that of M-Exo(p < 0.01).Two exosomes were identified using transmission electron microscopy,nanoparticle size,and WB techniques.The results showed that the morphological structures of the two exosomes showed a teato-like bilayer structure;the particle sizes were all distributed between 30～150 nm,with the main peak of C-Exo at 77.65 nm and M-Exo at70.74 nm;both expressed the exosome marker proteins TSG101 and Alix.The above results indicate that C-Exo and M-Exo were successfully isolated in this study.(2)Inhibitory effects of C-Exo and M-Exo of dairy goats on intestinal inflammation induced by LPS.Using Kunming white mice as the object,LPS induced intestinal inflammation model in mice;The effects of C-Exo and M-Exo on intestinal inflammation induced by LPS in mice were evaluated.The results revealed that both C-Exo and M-Exo alleviated the LPS-induced weight loss in mice;using H&E staining to observe small intestinal villi injury,it was found that both C-Exo and M-Exo improved LPS-induced small intestinal villi injury;using the MPO activity assay kit to detect MPO activity in small intestinal tissue,it was found that both C-Exo and M-Exo significantly inhibited the LPS-induced increase in MPO activity in mice small intestinal tissue,and the effect of C-Exo was more significant(p< 0.05).Using ELISA and RT-q PCR to detect changes of pro-inflammatory factors(IL-1β,IL-6 and TNF-α)in mouse serum and small intestinal tissues,respectively,we found that CExo and M-Exo significantly inhibited LPS-induced elevation of pro-inflammatory factors,and the effect of C-Exo on IL-1β and IL-6 was more significant(p < 0.05).Subsequently,this study evaluated the effects of C-Exo and M-Exo on LPS-induced inflammatory responses in IEC-6 cells at the cellular level.Fluorescence microscopy was used to observe the uptake of C-Exo and M-Exo by IEC-6 cells and found that C-Exo and M-Exo could be taken up by IEC-6 cells.LDH activity in cell supernatants was measured using the LDH Cytotoxicity Assay Kit,and it was found that C-Exo and M-Exo significantly inhibited the LPS-induced increase in LDH content,and the effect of C-Exo was more significant(p < 0.05).Using ELISA and RT-q PCR to detect changes of pro-inflammatory factors in cell supernatant and cell,respectively,we found that C-Exo and M-Exo significantly inhibited the elevation of proinflammatory factors induced by LPS,and C-Exo had more significant effects on IL-6 and TNF-α at the protein level(p < 0.05)and on TNF-α at the m RNA level(p < 0.05).Using WB to detect NF-κB signaling pathway-related protein expression,the results showed that C-Exo and M-Exo could alleviate the inflammatory response of IEC-6 cells by inhibiting NF-κB signaling pathway,and the effect of C-Exo was more significant(p < 0.05).The nuclear translocation of p-NF-κB was observed using fluorescence microscopy,and the results showed that both C-Exo and M-Exo were able to inhibit the LPS-induced nuclear translocation of pNF-κB.The above results showed that both C-Exo and M-Exo were able to alleviate the LPSinduced intestinal inflammatory response,and C-Exo was more effective in inhibiting the intestinal inflammatory response.(3)Analysis of C-Exo and M-Exo miRNA expression profiles in dairy goats.A total of371 miRNAs(356 in C-Exo and 242 in M-Exo)were detected by miRNA sequencing analysis of C-Exo and M-Exo,and the 22 miRNAs co-highly expressed accounted for 68.97% and76.83% of the total reads of C-Exo and M-Exo.GO and KEGG analysis of the co-highly expressed miRNA potential target genes showed that the co-highly expressed miRNA potential target genes could be significantly enriched in immune response-related signaling pathways.Differential miRNA screening was performed with a threshold of |log2(FC)|>=1 and p <0.05;the results showed that there were 70 differential miRNAs in C-Exo and M-Exo,of which 58 miRNAs were up-regulated in C-Exo and 12 miRNAs were up-regulated in M-Exo.GO and KEGG analysis of potential target genes of miRNAs upregulated in C-Exo showed that the potential target genes of miRNAs upregulated in C-Exo could be significantly enriched in immune response-related pathways.Finally,the relative expression levels of 12 randomly selected differentially expressed miRNAs were verified using RT-q PCR,and the changes of the relative expression levels of these 12 miRNAs were found to be consistent with the sequencing results,indicating that the sequencing results of this study were reliable.In conclusion,C-Exo and M-Exo were successfully isolated from dairy goats using isoelectric point assisted combined with differential ultracentrifugation,and both C-Exo and M-Exo were found to have anti-inflammatory effects through mouse and intestinal epithelial cell inflammatory response models,and C-Exo is more effective in suppressing intestinal inflammation.C-Exo and M-Exo co-highly expressed miRNAs were able to significantly enrich to immune-related pathways,and miRNAs significantly upregulated by C-Exo were also involved in the immune response process. |
