TIGHT JUNCTIONS FORMATION IN EXPERIMENTAL MODEL OF NECROTIZING ENTEROCOLITIS - EFFECT OF EGF TREATMENT

Abstract

Background: Necrotizing enterocolitis (NEC) is the most common intestinal disease predominately of formula fed, premature babies. Epidermal growth factor (EGF) reduces the incidence of disease in a neonatal rat model of NEC. Claudin and occludin are the major structural and functional components of the tight junction barrier. However, the role of tight junctions in NEC pathogenesis and EGF treatment is currently unknown. Aim: The aim of this study was to determine if EGF affects expression of tight junction genes and proteins in the ileum during the development of NEC. Methods: Neonatal rats, either dam fed (DF), milk formula fed (NEC), or fed with formula plus 500 ng/ml EGF (NEC + EGF) were exposed to asphyxia and cold stress to develop NEC. After 96 hours, ileal expression of claudin-3 and occludin were evaluated using Realtime-PCR, Western blot and immunohistochemistry. Results: Claudin-3 and occludin mRNA levels in the ileum were significantly increased 2-4 fold in NEC animals compared to DF animals (p < 0.0001). Supplementation with EGF normalized mRNA expression to DF levels. Histological localization of claudin-3 and occludin proteins was disturbed in animals with NEC compared to DF animals. NEC animals had upregulated expression and disorganization of the tight junction proteins. EGF treatment resulted in localization of the proteins at the tight junction complex. Conclusion: The ability of EGF to normalize expression and localization of tight junction proteins may be one mechanism by which integrity of the mucosal barrier is maintained, thereby protecting the intestinal mucosa against NEC. The increased expression of mRNA and disorganization of protein in animals with NEC may represent a compensation for the inability to form functional tight junctions at the mucosal barrier. Supported by the APS Porter Physiology Fellowship (to J.C.) and NIH Grant HD-39657 (to B.D.)