Effect And Mechanism Of Human Breast Milk Derived-Exosomes In Ameliorating Neonatal Necrotizing Enterocolitis

Neonatal necrotizing enterocolitis,a serious threat to neonatal health,is one of the most common intestinal inflammatory diseases in preterm infants.Currently,symptomatic treatment in NEC includes withholding of enteral feedings,gastric decompression,broadspectrum antibiotics,parenteral nutrition,and surgery to remove the necrotic intestine if necessary.Children with severe NEC may suffer from failure to thrive,strictures of the intestine,short bowel syndrome,and neurodevelopmental delay.Therefore,it’s beneficial for infants,especially premature ones,to find effective strategies to prevent and treat NEC which is significant to improve the survival rate and the long-term prognosis of NEC children.The Preventive effect of breast-feeding on NEC has been widely recognized,mainly due to the abundant bioactive substances in it,but the specific mechanism remains unclear.With the successful extraction of exosomes from human milk,more and more studies have focused on the function of the protective effect of human milk derived-exosomes(HM-exos)on the damaged intestinal barrier.Exosomes are membranous vesicles secreted by cells,the diameter of which is generally 40-160nm.Exosomes in human milk are mainly derived from mammary epithelial cells,stem cells,immune cells,etc.HM-exos have the advantages of stable bioactivity,low immunogenicity,and cross-species tolerance.As drug delivery vectors,HM-exos have a broad prospect in the prevention and treatment of NEC.The composition of breast milk changes dynamically in milk secretion time,and the macronutrient content of colostrum and mature milk is different.In view of this,this study focuses on intestinal injury repair and compares the effect of colostrum derived exosomes(C-exos)and mature milk derived exosomes(M-exos)in NEC mouse model.Further elucidate the mechanism of HM-exos promoting intestinal injury repair by regulating the migration and proliferation of intestinal epithelial cells.Thus,we provide a base for new strategies in the prevention and treatment of NEC.Part Ⅰ.The comparison of protective effects of C-exos and M-exos against intestinal injury in NECObjective:To verify the repair effects of C-exos and M-exos on intestinal injury in NEC mice,and to compare the different effects of them.Methods:Exosomes were extracted and identified from colostrum and mature milk by ultracentrifugation.Establish NEC mouse model as following.Forty 6-day-old neonatal C57BL/6 mice were randomly divided into 4 groups with 10 mice in each:(1)control group(CON group);(2)NEC model group(NEC group);(3)The NEC model group was treated with C-exos(C-exos group);(4)The NEC model group was treated with M-exos(M-exos group).The general condition of mice was observed during modeling,the changes in body weight of mice were recorded,and the survival curve was drawn.The mice were sacrificed after 96h.Duodenum,jejunum and ileum tissues were taken for HE staining and intestinal histological score.The expression levels of zonula occludens-1(ZO-1),E-cadherin(E-cad),leucine-rich repeat containing G protein-coupled receptor 5(Lgr5)and interleukin-6(IL-6)were compared in ileum immunohistochemistry.Plasma IL-6 concentration was detected by ELISA kit and mRNA levels of IL-6 and Lgr5 in mouse intestinal tissue were detected by qPCR.Results:1.Vesicles with double-layer membrane structure were observed by transmission electron microscopy,and the average particle size was between 40 and 160nm.The exosome surface markers CD63,CD81 and TSG101 were identified by Western Blot.2.There was no significant difference between C-exos group and M-exos group in body weight and survival curve.3.Compared with CON group,pathological scores of duodenums,jejunum and ileum in NEC group were significantly increased,and the ileum segment had the highest pathological score.C-exos or M-exos intervention could reduce the pathological score,and there was no statistical difference between them.4.Compared with CON group,the immunohistochemical average optical density(AOD)for ZO-1 and E-cad in NEC group was significantly decreased,which was increased by C-exos and M-exos intervention,and the effect of C-exos was better.5.The AOD and mRNA relative expression of Lgr5 in ileum of NEC group were significantly decreased,while those of C-exos group and M-exos group were increased.6.The expression of IL-6 in intestinal tissue and plasma were significantly increased.in NEC group.However,both C-exos and M-exos intervention could reduce those,while there was no statistical difference between the two groups.Conclusions:NEC caused pathological injury in all sections of intestinal tissue,and the ileal segment was the most serious.C-exos and M-exos intervention both improved the general condition of NEC mice,alleviated intestinal pathological injury,and relieved systemic and local intestinal inflammation,but there was no difference between them.Meanwhile,C-exos was better than M-exos in increasing the expression of intestinal connexin.Part Ⅱ.Comparing the effects of C-exos and M-exos on intestinal epithelial cell damage repair in vitroObjective:To verify the protective effect of HM-exos on intestinal epithelial cells in vitro,to explore and compare the regulatory effects of C-exos and M-exos on the proliferation and migration of intestinal epithelial cells.Methods:IEC-6 and IEC-18 cells were divided into 4 groups:(1)control group(CON group);(2)LPS group;(3)LPS combined with C-exos intervention group(C-exos group);(4)LPS combined with M-exos intervention group(M-exos group).Then migration test,scratch healing test and CCK-8 proliferation test were used to compare and analyze the cell proliferation and migration ability of each group.Results:(1)The migration experiment showed that the number of migrating cells in LPS group was significantly lower than that in CON group,which could be reversed by Cexos or M-exos intervention.Especially the reversal effect of C-exos was better.(2)Scratch experiment showed that LPS stimulation resulted in impaired scratch healing ability of cells.Both C-exos and M-exos intervention could promote the healing of scratch cells,and C-exos had better effect.(3)The proliferation experiment showed that cell proliferation was inhibited after LPS stimulation,and C-exos and M-exos intervention could promote cell proliferation,but there was no statistical difference between C-exos group and M-exos group.Conclusion:LPS stimulation inhibits the proliferation and migration of intestinal epithelial cells,leading to intestinal epithelial repair dysfunction after injury.Both C-exos and M-exos can promote the repair of intestinal epithelial cells by regulating cell proliferation and cell migration,and C-exos works better.Part Ⅲ.The mechanism of HM-exos promoting intestinal epithelial repair after injuryObjective:On the basis of clarifying the repair effects of HM-exos on intestinal epithelial injury,the mechanism of HM-exos-mediated intestinal epithelial injury repair was further investigated by RNA sequencing of mouse intestinal tissue.Methods:The differentially expressed genes between the NEC and CON group were screened and Igf1 gene was identified.Then the expression of IGF-1 protein in HM-exos was detected.Subsequently,the downstream signaling molecules related to IGF-1,the phosphorylation levels of Stat3,Akt and Erk,were analyzed by Western Blot to identify the key signaling molecules.Finally,IEC-6 cells were divided into 5 groups:(1)control group(CON group);(2)LPS group;(3)LPS combined with C-exos intervention group(C-exos group);(4)LPS combined with GSK143 intervention group(GSK143 group);(5)LPS+GSK143+C-exos intervention group(GSK143+C-exos group).We analyze the expression of p-Erk/Erk protein and the changes of cell migration ability in each group.Results:The results of RNA sequencing indicated that Igf1 gene was low expressed in the intestinal tissue of NEC mice,while IGF-1 protein was detected in HM-exos.Using TSG101 as the internal reference,the amount of IGF-1 protein in C-exos was more than that in M-exos.C-exos or GSK143 intervention inhibited LPS-induced phosphorylation of Erk,and the combination of both had stronger inhibitory effect.GSK143 or C-exos could promote IEC-6 migration,and the combination of GSK143 and C-exos could resume IEC-6 migration ability to normal level.Conclusion:HM-exos acts on Erk signaling pathway to promote intestinal epithelium proliferation and migration.IGF-1 may play a key protective role in HM-exos.