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Necrotizing enterocolitis (NEC) is a devastating intestinal disease of neonates, and clinical studies suggest the beneficial effect of probiotics in NEC prevention. Recently, we have shown that administration of Bifidobacterium bifidum protects against NEC in a rat model. Intestinal apoptosis can be suppressed by activation of cyclooxygenase-2 (COX-2) and increased production of prostaglandin E2 (PGE2). The present study investigates the effect of B. bifidum on intestinal apoptosis in the rat NEC model and in an intestinal epithelial cell line (IEC-6), as a mechanism of protection against mucosal injury. Premature rats were divided into the following three groups: dam fed, hand fed with formula (NEC), or hand fed with formula supplemented with B. bifidum (NEC + B. bifidum ). Intestinal Toll-like receptor-2 (TLR-2), COX-2, PGE2, and apoptotic regulators were measured. The effect of B. bifidum was verified in IEC-6 cells using a model of cytokine-induced apoptosis. Administration of B. bifidum increased expression of TLR-2, COX-2, and PGE2 and significantly reduced apoptosis in the intestinal epithelium of both in vivo and in vitro models. The Bax-to-Bcl-w ratio was shifted toward cell survival, and the number of cleaved caspase-3 positive cells was markedly decreased in B. bifidum -treated rats. Experiments in IEC-6 cells showed anti-apoptotic effect of B. bifidum . Inhibition of COX-2 signaling blocked the protective effect of B. bifidum treatment in both in vivo and in vitro models. In conclusion, oral administration of B. bifidum activates TLR-2 in the intestinal epithelium. B. bifidum increases expression of COX-2, which leads to higher production of PGE2 in the ileum and protects against intestinal apoptosis associated with NEC. This study indicates the ability of B. bifidum to downregulate apoptosis in the rat NEC model and in IEC-6 cells by a COX-2-dependent matter and suggests a molecular mechanism by which this probiotic reduces mucosal injury and preserves intestinal integrity.

Necrotizing enterocolitis (NEC) is a devastating intestinal disease of premature infants. Epidermal growth factor (EGF) is one of the most promising candidates in NEC prophylaxis. Autophagy regulates cell homeostasis, but uncontrolled activation of autophagy may lead to cellular injury. The aim was to evaluate the effects of EGF on intestinal autophagy in epithelial cells and in the rat NEC model and measure autophagy in NEC patients. Intestinal epithelial cells (IEC-6) and the rat NEC model were used to study the effect of EGF on intestinal autophagy. Protein levels of Beclin 1 and LC3II were measured in the intestinal epithelium in both in vivo and in vitro models. Ultrastructural changes in intestinal epithelium were studied by electron microscopy. Expression of Beclin 1, LC3II, and p62 protein was evaluated in biopsies from NEC patients. Autophagy was induced in IEC-6 cells and inhibited by adding EGF into the culture. In the rat NEC model, EGF treatment of NEC reduced expression of Beclin 1 and LC3II in ileal epithelium. Morphologically, typical signs of autophagy were observed in the epithelium of the NEC group, but not in the EGF group. A strong signal for Beclin 1 and LC3II was detected in the intestine from patients with NEC. Autophagy is activated in the intestinal epithelium of NEC patients and in the ileum of NEC rats. Supplementation of EGF blocks intestinal autophagy in both in vivo and in vitro conditions. Results from this study indicate that EGF-mediated protection against NEC injury is associated with regulation of intestinal autophagy.

Introduction Branched chain fatty acids (BCFA) are found in the normal term human newborn's gut, deposited as major components of vernix caseosa ingested during late fetal life. We tested the hypothesis that premature infants' lack of exposure to gastrointestinal (GI) BCFA is associated with their microbiota and risk for necrotizing enterocolitis (NEC) using a neonatal rat model. Methods Pups were collected one day before scheduled birth. The pups were exposed to asphyxia and cold stress to induce NEC. Pups were assigned to one of three experimental treatments. DF (dam-fed) ; Control, hand-fed rat milk substitute ; BCFA, hand-fed rat milk substitute with 20%w/w BCFA. Total fat was equivalent (11%wt) for both the Control and BCFA groups. Cecal microbiota were characterized by 16S rRNA gene pyrosequencing, and intestinal injury, ileal cytokine and mucin gene expression, interleukin-10 (IL-10) peptide immunohistochemistry, and BCFA uptake in ileum phospholipids, serum and liver were assessed. Results NEC incidence was reduced by over 50% in the BCFA group compared to the Control group as assessed in ileal tissue; microbiota differed among all groups. BCFA-fed pups harbored greater levels of BCFA-associated Bacillus subtilis and Pseudomonas aeruginosa compared to Controls. Bacillus subtilis levels were five-fold greater in healthy pups compared to pups with NEC. BCFA were selectively incorporated into ileal phospholipids, serum and liver tissue. IL-10 expression increased three-fold in the BCFA group versus Controls and no other inflammatory or mucosal mRNA markers changed. Conclusion At constant dietary fat level, BCFA reduce NEC incidence and alter microbiota composition. BCFA are also incorporated into pup ileum where they are associated with enhanced IL-10 and may exert other specific effects.

Neonatal necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in premature infants. Oral administration of probiotics has been suggested as a promising strategy for prevention of NEC. However, little is known about the mechanism(s) of probiotic-mediated protection against NEC. The aim of this study was to evaluate the effects of Bifidobacterium bifidum treatment on development of NEC, cytokine regulation, and intestinal integrity in a rat model of NEC. Premature rats were divided into three groups: dam fed (DF), hand fed with formula (NEC), or hand fed with formula supplemented with 5 × 106CFU B. bifidum per day ( B. bifidum ). All groups were exposed to asphyxia and cold stress to develop NEC. Intestinal injury, mucin and trefoil factor 3 (Tff3) production, cytokine levels, and composition of tight junction (TJ) and adherens junction (AJ) proteins were evaluated in the terminal ileum. B. bifidum decreased the incidence of NEC from 57 to 17%. Increased levels of IL-6, mucin-3, and Tff3 in the ileum of NEC rats was normalized in B. bifidum treated rats. Reduced mucin-2 production in the NEC rats was not affected by B. bifidum . Administration of B. bifidum normalized the expression and localization of TJ and AJ proteins in the ileum compared with animals with NEC. In conclusion, administration of B. bifidum protects against NEC in the neonatal rat model. This protective effect is associated with reduction of inflammatory reaction in the ileum, regulation of main components of mucus layer, and improvement of intestinal integrity.

Necrotizing enterocolitis (NEC) is a devastating disease of premature babies. Previously, we have shown that epidermal growth factor (EGF) reduces NEC and that overproduction of hepatic TNF-α is associated with intestinal damage. Leakage of TNF-α may be a consequence of epithelial hepatic cellular junction dysfunction. The aim of this study was to investigate changes in the composition of hepatic tight junctions (TJs) and adherens junctions (AJs). Using an established rat model of NEC, animals were divided into the following groups: dam fed (DF); formula fed (NEC); or fed with formula supplemented with EGF (EGF). Serum EGF and histological localization of major TJ and AJ proteins were evaluated. Distribution patterns of hepatic TJ and AJ proteins were significantly altered in the NEC group compared to DF or EGF groups. Cytoplasmic accumulation of occludin, claudin-2, and ZO-1 with reduction of claudin-3 signal was detected in the liver of NEC rats. Localization of β-catenin was associated with the hepatocyte membrane in EGF and DF groups, but diffused in the NEC group. These data show that hepatic cellular junctions are significantly altered during NEC pathogenesis. EGF-mediated reduction of experimental NEC is associated with protection of hepatic integrity and structure.

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease of prematurely born infants. Epidermal growth factor (EGF) and heparin-binding EGF-like growth factor (HB-EGF) have protective effects against intestinal injury. The aim of this study was to compare the effect of oral administration of HB-EGF, EGF, or both on the incidence of NEC in a neonatal rat model.Premature rats were fed by hand and exposed to asphyxia and cold stress to develop NEC. Four diets were used: formula (NEC), formula supplemented with 500 ng/mL HB-EGF (HB), 500 ng/mL EGF (EGF), or a combination of both (E+HB). Ileal injury, endogenous HB-EGF production, expression of EGF receptors, goblet cell density, and expression of apoptotic proteins were evaluated.Oral administration of either EGF or HB-EGF significantly reduced the incidence of NEC; however, EGF provided better protection in physiologically relevant doses. Simultaneous administration of both growth factors did not result in any synergistic protective effect against NEC. There were no significant differences between treatment groups in ileal gene expression of EGF receptors or HB-EGF. However, the balance of apoptotic proteins in the ileum was shifted in favor of cell survival in EGF-treated rats. This mechanism may be responsible for the higher efficiency of EGF protection against NEC.These data suggest that a physiological dosage of EGF or a pharmacological dosage of HB-EGF could be used for prevention of NEC.

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease predominantly of prematurely born infants, characterized in its severest from by extensive hemorrhagic inflammatory necrosis of the distal ileum and proximal colon. Proinflammatory cytokines have been implicated in the development of NEC, and we have previously shown that IL-18 is significantly elevated in the well-established neonatal rat model of NEC. To determine whether IL-18 contributes to intestinal pathology in NEC, we subjected IL-18 knockout mice to the protocol used to develop experimental NEC in newborn rats. Newborn B6.129P2- Il18tm1Aki/J (NEC IL-18−/−) and wild-type (NEC WT) mice were hand fed every 3 h with cow's milk-based formula and exposed to asphyxia and cold stress twice daily. After 72 h, animals were killed and distal ileum and liver were removed. Disease development was determined via histological changes in the ileum as scored by a blinded evaluator. The number of TNF-α-, IL-12-, and IL-1β-positive cells and macrophages were determined in both ileum and liver via immunohistology. IκB-α and IκB-β were determined from protein extracts from both ileum and liver using Western blot analysis. The incidence and severity of NEC was significantly reduced in NEC IL-18−/−mice compared with NEC WT. Furthermore, mean ileal macrophages and hepatic IL-1β were significantly reduced in IL-18−/−mice subjected to the NEC protocol. There were no statistically significant changes in Kupffer cells, hepatic TNF-α, ileal IL-1β, or IL-12. IκB-α and IκB-β were significantly increased in NEC IL-18−/−mice ileum and liver, respectively. These results confirm that IL-18 plays a crucial role in experimental NEC pathogenesis.