Study On Lactadherin In Promoting Polarity Formation Of Intestinal Epithelial Cells In Prevention And Treatment Of Neonatal Necrotizing Enterocolitis

Objective:Neonatal necrotizing enterocolitis(NEC)is a serious cause of premature infant death.There is no effective treatment to change its outcome,the key is “prevention”.On the basis of our previous studies,this study used NEC animal model to explore the specific mechanism of MFG-E8 in maintaining intestinal barrier integrity,promoting polarity generation of intestinal epithelial cells,regulating damage repair of intestine.In order to provide a new target for the prevention of NEC with effective ingredients in breast milk.Methods:1.The neonatal rat NEC model was intervened by MFG-E8.Observe the changes of NEC disease,mortality and histopathological score of newborn rats in each group after adminstion of MFG-E8.The effect of MFG-E8 on the ultrastructure of intestinal epithelial cells and the permeability of single layer intestinal epithelial cells was studied by transmission electron microscopy.Real-time PCR,western blot and immune-fluorescence were used to detect the expression level and distribution of tight junction protein and adhesive junction protein,respectively,and Co-IP immunoprecipitation was used to analyze the interaction between different junction proteins.The gene expression network was constructed based on gene correlation analysis to predict the possible influence pathway of MFG-E8.2.After the pre-administration of MFG-E8 and N-terminal domain peptides in NEC animal models,incidence of NEC were evaluated.The activity of alkaline phosphatase and the expression and distribution of polar protein PAR3 in intestinal tissues were detected.LPS was used to interfere with IEC-6 cell line to establish a cell inflammatory model in vitro,and the migration ability of intestinal epithelial cells was observed by wound-healing.The expression and distribution of EGFR,a key regulator of cell polarity formation,were further detected in the small intestine.After blocking the EGFR receptor,the phosphorylation ratio of EGFR and the activation degree of downstream Akt signal in intestinal epithelial cells of each experimental group were observed.The expression of related signaling pathways regulated proteins was analyzed according to TPM value.Genetic correlation analysis may provide data support for further research on specific signal pathways.Results:1.The incidence and mortality of NEC in neonatal rats fed with formula containing MFG-E8 was significantly lower than that in the group fed with formula only,the course of disease was shortened,the pathological histological score was decreased,and the intestinal inflammation injury was milder.And under TEM,the junctions between adjacent cells is tight and the gap is narrow in the MFG-E8 intervention group.MFG-E8 can promote the expression of ZO-1,claudin 3,JAM-A and E-cadherin,and recruit them to their normal functional location.Then promoting the interaction inside AJC between ZO-1 and occludin.Gene network analysis revealed that Cdc42,an important regulatory signal of cell junction,was possible regulated by MFG-E8.2.Pre-administration of MFG-E8 and its N-terminal domain can significantly lower the incidence and mortality of NEC compare to NEC group.In group MFG-E8 the activity of alkaline phosphatase increased,the expression and distribution of polar protein PAR3 increased.In vitro,MFG-E8 can promote the intestinal epithelial cells migrate to the injured site,and the expression and distribution of EGFR receptor on the surface of intestinal epithelial cells increased.Meanwhile the level of phosphorylation EGFR and the activation ratio of Akt increased.Whole transcriptome gene sequencing revealed that the level of Vav,a signaling protein affecting cytoskeletal formation,in the EGFR pathway was up-regulated after the intervention of MFG-E8.Conclusion:1.MFG-E8 can prevent the occurrence and development of NEC and reduce the mortality of NEC.2.By activating the Cdc42 related signaling pathway,MFG-E8 can up-regulate the expression of cellular connection protein,anchor it at the normal functional position,promote the interaction between ZO-1 and occludin,assemble AJC,maintain the stability of intestinal epithelial cell connection,and play a role in protecting the integrity of intestinal barrier.Cdc42 signaling molecule may be one of the targets of MFG-E8 to improve the formation of intercellular junction proteins in intestinal epithelial cells3.MFG-E8 may activate the downstream Akt signal by acting on the EGFR receptor on the surface of intestinal epithelial cells,thereby affecting the expression of polar protein PAR3,further affecting the polarity formation of intestinal epithelial cells,restoring the orderly polar arrangement,so as to forming effective connections between adjacent cells,promoting the injury and repair of intestinal epithelial cells,and maintaining the functional homeostasis of the intestinal tract.