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 the most common gastrointestinal emergency of premature infants. Previously, we showed that luminal bile acids (BAs) are increased and correlated with disease development and that the apical sodium-dependent BA transporter (ASBT), which transports BAs from the ileal lumen into enterocytes, is upregulated in rats with NEC. We hypothesized that intraenterocyte, rather than luminal, BAs are associated with NEC and that upregulation of ASBT may be a mechanism by which this occurs. Neonatal rats with or without the ASBT inhibitor SC-435, mice in which ASBT was knocked out, and mice that overproduce BAs were subjected to the NEC protocol. Disease development, ASBT, and the farnesoid X receptor protein, along with luminal and intraenterocyte BA levels, were assessed. In addition, ileal sections from premature infants with and without NEC were examined for ASBT via immunohistology and real-time PCR. When BAs were not transported into enterocytes (rats given SC-435 and ASBT knockout mice), severity and incidence of NEC were reduced. In contrast, in mice that overproduce BAs, ASBT was elevated, intraenterocyte BAs were increased, and disease development was increased. ASBT staining was more intense on the apical membrane of ileal enterocytes from premature infants with NEC than premature infants with non-NEC diagnoses. In addition, ASBT mRNA levels were significantly higher in infants with NEC. These data show that accumulation of intraenterocyte BAs contributes to disease development, elevated ASBT increases disease severity in experimental models of NEC, and ASBT is elevated in human NEC. These data confirm that BAs and upregulation of ASBT play a crucial role in NEC pathogenesis and suggest that inhibition of ASBT could be utilized as a therapeutic modality against this disease.

Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency of premature infants, but its etiology remains unclear. We have previously shown that mucin 2 (Muc2) positive goblet cells are significantly decreased in NEC. We have also shown that ileal bile acids (BAs) are significantly increased during the development of this disease. Because BAs can affect mucins, we hypothesized that elevated ileal BAs contribute to decreased Muc2 in experimental NEC. The role of Muc2 in NEC was evaluated in Winnie +/+ mice, a strain that produces aberrant Muc2. Muc2 and trefoil factor 3 (Tff3) were assessed in neonatal rats subjected to the NEC protocol when bile acids were removed, and in ileal explants from newborn and older rats cultured with and without BAs. Further, the role of active transport of BAs was determined using neonatal rats given the apical sodium dependent bile acid transporter (Asbt) inhibitor SC-435 and in neonatal Asbt knockout mice subjected to the NEC protocol. Mice with aberrant Muc2 had significantly greater incidence and severity of NEC. Using both in vivo and ex vivo techniques, we determined that BAs decrease Muc2 positive cells in neonatal but not older ileum. However, Tff3 positive cells are not decreased by BAs. In addition, active transport of BAs is required for BAs to decrease Muc2 in immature ileum. These data show that functional Muc2 plays a critical role in the prevention of NEC and BAs can potentiate the decreased Muc2 in disease development. Further, BAs have a more profound effect on Muc2 in immature versus older ileum, which may explain at least in part why NEC occurs almost exclusively in premature infants.