Research Regarding The Protective Effect Of The Human Milk Derived Exosomes And MiRNA 145-5p From Human Breast Milk Derived Exosomes Against NEC

Background:So far,necrotizing enterocolitis remains a devastating gastrointestinal disease threatening the neonates specifically preterm infants’ life.However,the exact mechanism of the pathogenesis of NEC is still ambiguous.The causes of NEC are confirmed to be multifactorial;many clinical researches revealed that the main risk factors of NEC development are premature birth,low birth weight,perinatal asphyxia,infection and artificial feeding.Attributing to the uncertainties of its pathogenesis,subtle development and lacking specific treatment,the outcomes of NEC are dim,and the mortality is high.Breast feeding has been confirmed to be an effective way to prevent NEC from occurring,but the exact mechanism of its preventive effect is still unclear.Breast milk is enriched with exosomes which contain abundant biological response modifier(BRM)like proteins,lipids,micro RNAs and so on.These breast milk derived exosomes may participate the protection of the intestinal epithelium thus can prevent NEC from developing.Therefore,in our research,we aimed to investigate the functions of human breast milk derived exosomes including exosome derived miRNA sequencing and isolating the highest expressed miRNA for further research in order to provide some help for clinical practice and find valuable novel target for NEC treatment.Part Ⅰ:Analysis of the difference of protein content of isolated exosomes between term and preterm infants’ mother’s breast milkObjective:To isolate and confirm the exosomes from breast milk and compare the content of these isolated exosomes between term and preterm mothers’ breast milkMethods:Breast milk samples were collected,and the human body fluid exosome isolation kit was adopted to isolate the exosomes following the manufacturer’s manual and morphological observed under the electron microscope.The particle size distribution was analyzed by nanoparticle size analyzer.The specific transmembrane proteins CD63 and CD81 of exosomes were tracked by Western-blot.Breast milk derived exosome content of two different groups(term and preterm)was analyzed by BCA kit and compared.Results:54 breast milk samples were collected including 33 from term infants’ mother and 23 from preterm.The exosomes of samples were isolated by human body fluid exosome isolation kit and observed by electron microscope.Under the electron microscope,vesiclelike microvesicles were observed and through the analysis carried out by nanoparticle size analyzer we found the size distribution of these vesicles was ranged from 70nm to 150nm.Compared to the intestinal epithelial cell control group,the specific proteins CD63 and CD81 of exosomes were detected in the isolated vesicles by Western-blot which stood for these vesicles were exosomes.Exosome content from term and preterm mothers’ breast milk was assessed by BCA kit showing no significant difference between two groups(p<0.05).Conclusion:There are abundant exosomes in humans breast milk and these exosomes could be isolated by the exosome isolation kit that no need to use the conventional gradient centrifugation,which is time and labor consuming.In terms of our analysis,the exosome content from term and preterm mothers’ breast milk was identical but whether there is a functional difference between the two groups is unclear thus further research is needed.Part Ⅱ:The protective effect of breast milk derived exosomes to the integrity of the intestinal epithelial barrierObjective:To explore the protective effect of the human milk derived exosomes to the epithelial tight junction proteins of Caco2 and NCM460 cells after LPS stimulation in the intestinal tissue of mouse pups after NEC induction as well as to evaluate the serum proinflammatory cytokines’ levels of NEC mouse pups after being treated with human milk derived exosomes.Methods:① Invitro,the human intestinal epithelial cells Caco2 and NCM460 were randomly divided into five groups:(1)The control group with no any agent added;(2)pretreated with human milk derived exosomes with a protein concentration of 50μg/ml;(3)stimulated by LPS at a concentration of 10μg/ml(4)pretreated with human milk derived exosomes then stimulated by LPS;(5)pretreated with exosome-free human milk at the same protein concentration then stimulated by LPS.After being stimulated by LPS 12 hours,the expression levels and the contents of epithelial tight junction proteins claudinl,occludin and ZO1 were analyzed and compared between groups by using rt-qPCR and Western blot.②In vivo,100 C7/BL6 mouse pups were randomly allocated into four groups,25 pups in each.(1)Pups were breast fed as controls;(2)NEC induced pups;(3)pups were nasogastric fed with human milk derived exosomes at a protein volume of 40μg/g proportionate to the body weight 6 hours prior NEC induction and(4)pups fed with exosome-free human milk at the same protein amount 6 hours prior NEC induction.The establishment of NEC model was in a way of hypertonic formula feeding combined with LPS and hypoxic stimulation.Results:① Invitro,either in Caco2 cells or in NCM460 cells,cells being pretreated with human milk exosomes 6 hours prior LPS stimulation,the relative expression levels and the contents of their epithelial tight junction proteins were remarkably higher than cells being treated with exosome-free human milk for 6 hours then stimulated by LPS or cells being stimulated by LPS solely(p<0.05)whereas similar with the cells in control group through rt-qPCR and Western blot evaluation.② In vivo,the degree of pathological impairment of the intestinal tissue of the pups being pretreated with human milk derived exosomes then induced with NEC was notably milder than pups being treated with exosome-free human milk then induced with NEC or pups induced with NEC only(p<0.05),meanwhile,their abundances of the intestinal epithelial tight junction proteins:claudin1,occludin and ZO1 were much higher than aforementioned pups(p<0.05)and similar with the pups being fed with breast milk evaluated by Western blot.Moreover,after immunofluorescence assessment,the fluorescence intensity of claudinl of intestinal epithelial tissue from NEC pups being pretreated with human milk derived exosomes was also stronger than NEC pups being pretreated with exosome-free human milk and NEC pups and even mild stronger than breastfed pups.During the period of NEC induction,the survival time of breast-fed pups,NEC+exosomes pretreated pups,NEC pups and NEC+exosome free milk pretreated pups were 92.8±4.6h,82.5 ± 2.7h,74.9 ± 9.2h and 72.8±6.5h respectively.The increment of body weight of breast-fed pups,NEC+ exosomes pretreated pups,NEC pups and NEC+exosome free milk pretreated pups were 2.1 ±0.42g,0.9±0.23g,-0.88±0.17g and-0.97±0.26g respectively.Through the ELISA test,the level of the proinflammatory cytokines IL-1β and TNF-α of NEC+exosomes pretreated pups were 0.85±0.33pg/ml and 18.22±4.4pg/ml respectively,which were significantly lower than NEC pups and NEC+exosome free milk pretreated pups(p<0.05)whereas similar to the breast-fed pups.Conclusion:Human milk derived exosomes are able to protect the intestinal epithelial tight junction proteins from being destructed by LPS and reduce the levels of serum proinflammatory cytokines,maintain the integrity of the intestinal epithelial barrier thereby prevent NEC from occurring.Part Ⅲ:The analysis of the mechanisms of human milk exosomes-derived miR145-5p in NEC preventionObjective:To find out the differentially expressed miRNAs in human milk derived exosomes,then predict its downstream target genes and validate the function of the miRNAmRNA interaction,in order to explore its mechanisms in NEC prevention.Methods:① 4 samples of human milk derived exosomes and 4 samples of exosome free milk were analyzed by Illumina next generation sequencing and in terms of a criteria of p<0.05 and |logFC|>1,the differentially expressed miRNAs were identified.Then,the most significant differentially expressed miRNAs were selected as the research objects.Furthermore,based on our previous pilot trail,among these selected miRNAs,miR-145-5p showed more powerful protective effect on intestinal epithelial barrier through pathohistological scoring,so we decided to employ it for our further research.Three miRNA databases(TargetScan,Mirdb and TarBase)were employed for target genes prediction and intersected to obtain the genes that in all databases.Then,the GO term and KEGG enrichment analysis were performed and the protein-protein interaction network was constructed by String database and the results yielded from it showed TLR4 was the highest score by the coexpression scoring system,a plug-in of String database.Moreover,TLR4 is well known strongly related to inflammation in many previous studies.Therefore,in present study,we focused on the regulation of miR-145-5p on TLR4.②40 C57/BL6 mouse pups were randomly allocated into two groups,20 pups in each:(1)NEC induction group and(2)breast-fed group.After NEC induction accomplished(96 hours),pups were sacrificed and the intestinal tissue was harvested.The expression levels of miR-145-5p and TLR4 in intestinal tissue were assessed through rt-qPCR and compared between groups.Through lentivirus packaging,viruses with miR-145-5p overexpression(OE),miR-145-5p inhibition(inhibition)and non-biological modification(empty vector)were established.According to different viral transfection and intervention,human intestinal epithelial cells Caco2 and NCM460 were categorized into five groups:(1)vector+LPS group;(2)OE+LPS group;(3)inhibition+LPS group;(4)normal cells+LPS group and(5)TLR4 inhibitor TAK242+LPS group.The concentration of LPS being used in this study was l0ug/ml.12 hours after LPS stimulation,the poptotic rate of cells was analyzed by flowctytometry(FCM)and the expression and protein content of TLR4 as well as its downstream proinflammatory cytokines IL-1β and TNF-α were analyzed by rt-qPCR,Western blot and ELISA,then compared between groups.Results:① After Illumina next generation sequencing,three most significant differentially expressed miRNAs were screened out,they were miR-146a-5p,miR-145-5p and miR-222-3p.And miR-145-5p were locked down for consequent research(The reason is addressed in the methods part).After target genes prediction via the intersection of aforementioned three miRNA database,77 potential target genes were determined.After GO term and KEGG enrichment analysis,we found that these genes were mainly enriched in signaling pathways as follows:Rap1 signaling pathway,tight junction pathway,Wnt pathway,MAPK pathway and so on.Through the protein-protein interaction network being constructed by String database and analyzed by Cytoscape software,TLR4 was confirmed as the most significant gene among aforementioned 77 genes in terms of the coexpression scoring system,a plug-in of Cytoscape.What’s more,TLR4 is well known related to inflammatory diseases specifically in infectious diseases.②The results of rt-qPCR showed that in the intestinal tissue of NEC pups,the expression level of miR-145-5p was remarkably lower than breast-fed pups(p<0.05)while the expression level of TLR4 was much higher than breast-fed pups(p<0.05).After cellular experiment,the survival rate of the OE+LPS group was 96.5%while the inhibition+LPS group,normal cells+LPS group and vector+LPS group were 70.2%,80.1%and 81.7 respectively through FCM evaluation.The mRNA expression level and the protein contents of TLR4 and its downstream proinflammatory cytokines IL-1β and TNF-α were much higher in vector group,inhibition+LPS groupand normal cells+LPS group compared to OE+LPS group(p<0.05)while the TLR4 expression level of TAK242+LPS group was mildly up-regulated compared to inhibition+LPS group and normal cells+LPS group but the proinflammatory cytokines were significantly down-regulated and similar to OE+LPS group.Conclusion:Human breast milk exosome derived miR-145-5p is capable of inhibiting the expression of TLR4 thus suppress the production of its downstream proinflammatory cytokines thereby alleviate inflammatory insult of intestine and prevent NEC from occurring.