The Role Of MFG-E8 In Improving Intestinal Permeability And Injury Repair In Necrotizing Enterocolitis

Objects:Necrotizing enterocolitis(NEC) is the most common devastating gastrointestinal disease of premature infants. Although, the etiology of NEC is unknown, it’s accepted that the stability of intestinal barrier is associated with NEC. In our previous data, it was proved that MFG-E8(lactadherin) could promote the expression of tight junction proteins and decrease the intestinal permeability in Rotavirus infected newborn rats.In this study, we tried to detect the lactadherin function on expression and location of tight junction proteins(Claudin, Occludin and ZO-1). In the meanwhile, we would identify the effect of lactadherin on the expression of intestinal injury repair protein(TLR4). Our purpose was to investigate the benifit function of lactaderin on NEC and to know its molecular mechanism.Methods:Part 1 animal experiments:(1)Animal grouping: The newborn sanitary SD rats were randomly divided into 3groups, including dam fed group(DF), NEC group(N), on the basis of NEC supplement with 100μg rh MFG-E8(N+M). Experimental NEC was induced by exposure to asphyxia, cold stress and aggressive formula feeding.(2)The small intestine morphology examinations: After 96 h of artificial feeding,macroscopic appearances of the gastrointestinal were recorded. The intestinal ileocecums were collected to determine and evaluate the small intestinal mucosal morphology by HE staining and transmission electron microscope(TEM) testing.(3)Intestinal permeability test: After 96 h of artificial feeding and before tissues collected, all the pups were fed with FITC-dextran(FD10S) for the intestinal barrier permeability test.(4)The levels and location of intestinal tight junction proteins: The levels and location of CLDN3 and Occludin in ileum were determined by using q RT-PCR, Western blot and immunohistochemistry.(5)The levels of intestinal TLR4: The levels of TLR4 in ileum were determined by using q RT-PCR, Western blot and immunohistochemistry.Part 2 cell experiments:(1)Cell grouping: There were three groups including PBS group(P group, control group), LPS group(L group, exposure to LPS 50μg/ml) and LPS+rh MFG-E8group(L+M group, on the basis of L group supplement with rh MFG-E8 2μg/ml).(2)Monolayer permeability test: The concentrations of FD10 S in lower chambers from each group were tested on the basis of monolayer cell model.(3)The levels and location of tight junction proteins in IEC-6 cell: The levels and location of CLDN3, Occludin and ZO-1 from each group were determined by using q RT-PCR, Western blot and immunofluorescence.(4)The wound assay: The distances of cells migration from each group were recorded at 0h, 6h.12 h and 24 h after the cell wounded.Results:Part 1 animal experiments:(1)Effect of MFG-E8 on symptoms of neonatal NEC rat: The symptoms of feeding intolerance(including the increasing of gastric residual and the decreasing of milk intake) and the change of stool appeared in the neonatal rat of N group firstly. With the help of rh MFG-E8, the symptoms above changed a lot.(2)Effect of MFG-E8 on morphology of intestinal ileocecum in the NEC rat model:Compared with N group, macroscopic appearances of the gastrointestinal in N+M group was much better without segmental necrosis in the intestine and was pneumatosis and edema only. The score of N+M group was significant lower than N group(P<0.05) as well as the incidence of NEC. Few apoptosis was observed in N+M group than N group.(3)Effect of MFG-E8 on intestinal permeability and the level of CLDN3, Occludin and TLR4 in the NEC rat model: Compared with N group, the permeability of intestine in N+M group was significant lower(P<0.05). The distribute of CLDN3 changed after the supplement of rh MFG-E8 in the NEC rat model. In N+M group, the level of TLR4 in intestine was significant lower than N group.Part 2 cell experiments:(1)Effect of MFG-E8 on the monolayer permeability and the level of CLDN3,Occludin and ZO-1 in LPS interfered IEC-6: Compared with L group, the apparent permeability coefficient(Papp) in L+M group was significant lower(P<0.05),approaching P group. The level of CLDN3 in L+M group was lower than L group(P<0.05),and the distribute of ZO-1 was changed in L+M group.(2)Effect of MFG-E8 on the migration in LPS interfered IEC-6: Compared with L group, the distance of the cells migration in L+M group was significant larger(P<0.05)in the point of 12 h, approaching P group.Conclusion:(1)MFG-E8 can improve the macroscopic appearances of intestinal ileocecum, NEC scoring and the ultrastructure in the NEC rat model, decreasing the incidence of NEC in neonatal rat and the severity of intestinal injury.(2)MFG-E8 can redistribute the CLDN3 protein in the epithelial cell and decrease the level of TLR4, so that reducing the permeability of intestine in NEC neonatal rat and improving the ability of injury repair.(3)MFG-E8 can decrease the level of CLDN3, redistribute the ZO-1 protein in the LPS interfered IEC-6 and decrease the paracellular permeability of FD10 S.(4)MFG-E8 can improve the ability of migration in the LPS interfered IEC-6.