The Relieve Effects Of Porcine Milk Exosomes On Intestinal Injury And The Mechanism Exploration

The death rate of weaning piglet caused by diarrhea account for about 39%.The main causes of diarrhea in piglets are multifarious,such as deoxynivalenol(DON),lipopolysaccharide(LPS)and piglet epidemic diarrhea virus(PEDV).Studies have shown that adequate intake of colostrum can significantly reduce diarrhea of newborn piglets.On the one hand,this protective effect due to colostrum contains rich nutrients and large amounts of antibodies.On the other hand,colostrum rich in exosomes,which are small membrane vesicles that are 40 to 100 nm in diameter formed by endocytosis by cells.Exosomes that enclose m RNA,miRNA,protein and other substances play an important role in communication between cells.It has been reported that human milk exosomes regulated gene expression in the body and ultimately affected the maturation and development of multiple systems when they are taken up by infant intestinal epithelial cells.We have separated porcine milk exosomes and sequencing the complete miRNAs of these exosome.On the base of the above scientific problems and our results,in this study,we used DON,LPS and PEDV to construct the intestinal damage and diarrhea model to elucidate the function and mechanism of porcine milk exosomes in promoting intestinal development and resisting intestinal damage.Therefore,this paper has carried out the following four aspects of research: Experiment 1,the piglet intestinal epithelial cell line IPEC-J2 and 18-day-old Kunming mice were used as research objects.The effects of exosomes on IPEC-J2 proliferation were elucidated by MTT and Edu.Secondly,bioinformatics analysis,q PCR,western blot(WB)and dual luciferase reporter gene assays were used to detect proliferationrelated gene expression and to analyze exosome miRNAs effect on cell proliferation by regulating gene expression in IPEC-J2.Finally,exosomes were administered to mice,and the effects of exosomes on the development of small intestine villi were determined by intestinal tissue section observation,q PCR and WB.The results showed that exosomes could significantly promote the proliferation of IPEC-J2 cells and promote the expression of proliferation related genes Cdx2,PCNA and IGF-1R,and inhibit the expression of Tp53,which is negatively correlated with proliferation.Further analysis revealed that exosome miR-769-3p and miR-181 a target SERPINE1 and Fas genes,respectively,inhibit Tp53 activation and promote cell proliferation.In addition,in vivo experiments in mice showed that exosomes promote the development of mice small intestine villi,and promote the expression of related genes Cdx2,PCNA and IGF-1R,and inhibit the expression of Tp53.Experiment 2,Kunming mice and pig intestinal epithelial cell line IPEC-J2 were used as research objects.First,IPEC-J2 cells were treated by adding basal medium(control)and porcine milk exosomes to the cells for 24 hours,and then DON treatment was performed on the cells,and then MTT and Edu methods were used to determine whether exosomes alleviate the cytotoxicity and inhibition of proliferation caused by DON.Secondly,bioinformatics analysis,dual luciferase reporter system,q PCR and WB were used to detect the expression of tight junction-associated proteins,apoptosis and cell proliferation-related genes,and to elucidate the molecular mechanism of exosomes to alleviate DON toxicity.the mice were divided into four groups,respectively,saline group(control),pig milk The exosome group(Exos),DON challenge group(Saline+DON)and treatment group(Exos+DON)that were used to construct a model of small intestinal mucosal injury induced by DON.Morphological observation and WB confirmed whether exosomes resistance to intestinal damage caused by DON in mice.The results showed that Exosomes and DON treated cells,exosomes alleviated the toxicity of DON on cell proliferation,and reversed the damaging effect of DON on tight junctions between epithelial cells.Exosome miR-30 c and miR-365-5p inhibit DON-induced apoptosis by inhibiting the expression of their target genes SERPINE1 and Tp53,respectively.Mice gavage with exosomes could significantly protect the small intestine villi integrity from DON toxicity,and exosomes could reverse the inhibitory effect of DON on proliferation-differentiation genes ?-catenin,CCND1 and Akt,and inhibit the expression of p53 and p21 associated with apoptosis.Experiment 3,IPEC-J2 cells and 18-day-old Kunming mice were also studied.The mice were divided into three groups: saline group(control),LPS challenge group(LPS)and treatment group(Exos+LPS).LPS-induced small intestinal mucosal injury model was established.The effects of pig exosome on LPS-induced small intestinal villus injury and pro-inflammatory factors in serum and intestinal mucosa were determined by small intestine morphology observation and ELISA.Secondly,exosomes and LPS were used to treat IPEC-J2 cells,and the effects of exosomes and LPS treatment on cytokine secretion and apoptosis were determined by immunofluorescence,q PCR,WB and ELISA.Finally,bioinformatics analysis,immunofluorescence,dual luciferase reporter gene,q RT-PCR,WB and flow cytometry were used to elucidate the molecular mechanism by which exosomes miRNAs block LPS-induced apoptosis induced by cellular inflammation.The results showed that exosomes can alleviate intestinal villus damage and inhibit the increase of inflammatory factors caused by LPS.After exosomes treatment,the levels of miR-4334 and miR-219 in IPEC-J2 cells were increased,and miR-4334 and miR-219 were targeted to the cell receptor TLR4 of LPS and the downstream protein My D88,respectively,and finally inhibited NF-?B signal activation and blocked the secretion of pro-inflammatory factors caused by LPS.Mi R-338 inhibits Tp53 gene expression and blocks pro-inflammatory factor-induced apoptosis by targeting the Tp53 gene.More interestingly,we found that compared with the function of a single miRNA in anti-inflammatory or anti-apoptotic effects,the three miRNAs have a synergistic effect on blocking the secretion of inflammatory factors and cell apoptosis induced by LPS.Experiment 4,the small intestinal epithelial cell lines IPEC-J2 and Vero E6,and the male newly born Landrace piglets were used as the research object.First,crystal violet staining and MTT staining on the intestinal epithelial cell line were used to determine exosomes effects on the PEDV infection intestinal epithelial cells.Secondly,it was confirmed by absolute quantitative PCR and WB on exosomes affect the replication of PEDV in intestinal epithelial cells.Finally,PEDV challenge was performed in the newborn piglets after oral administration of exosomes.Through tissue sectioning,intestinal immunofluorescence,absolute quantitative PCR,WB and ELISA tests,it was confirmed whether exosomes could block the replication of PEDV in the intestine and the intestine damage caused by PEDV.The results indicate that exosomes can inhibit the replication and infection of PEDV in epithelial cells IPEC-J2 and Vero E6.Secondly,exosome treatment can promote the development of intestinal villi in piglets,inhibit the replication of PEDV in the intestinal mucosa and inhibit damage to intestinal villi caused by PEDV,and play a critical role in inhibiting diarrhea caused by PEDV.Finally,we detected the presence of PEDV-specific antibodies in porcine milk exosomes,suggesting that exosomes functioning to inhibit PEDV replication and infect intestinal epithelial cells may be partially achieved by their inclusion of PEDV-specific antibodies.In summary,exosomes promote epithelial cell proliferation,inhibits inflammation and apoptosis through regulate intestinal epithelial cell inflammation and apoptosis-related signaling pathway genes expression through their miRNAs.Finally,exosomes promote the development of intestinal villi,and protects the intestinal tract against intestinal villus damage and diarrhea caused by harmful factors DON,LPS and PEDV.These results of this study not only compensated for the gap in milk exosomes on pig intestinal development,but also further explored the function and mechanism of exosomes and their miRNAs in regulating piglets' small intestinal epithelial cells development.Our results also provide experimental basis for future development of exosome functional products that reduce diarrhea in newborn piglets.