Necrotizing enterocolitis (NEC) is a major cause of morbidity and death in premature infants. NEC is associated with increased levels of pro-inflammatory cytokines in plasma and tissues that are regulated by the transcription factor nuclear factor-B (NF-B). It remains unknown, however, whether NF-B mediates injury in neonatal NEC. We therefore examined the activation status of NF-B perinatally in the small intestine and in a neonatal rat model of NEC. We found that intestinal NF-B is strongly activated at birth and, in dam-fed newborn rats, is down-regulated within a day. In contrast, NF-B remains strongly activated at both d 1 and d 2 in stressed animals, and this is accompanied by a significant decrease in the levels of the endogenous NF-B inhibitor protein IB␣ and IB␤ at d 2. To determine the importance of elevated NF-B activity in intestinal injury in NEC, we administered the NEMO-binding domain (NBD) peptide that selectively inhibits the critical upstream IB kinase (IKK). NBD but not a control peptide decreased mortality and bowel injury in this model, supporting the hypothesis that bowel injury in NEC results from elevated NF-B activity. Our findings therefore lead us to conclude that selective NF-B inhibition represents a promising therapeutic strategy for NEC.

SummaryCXCL2 (macrophage inflammatory protein-2 (MIP-2)), a critical chemokine for neutrophils, has been shown to be produced in the rat intestine in response to platelet-activating factor (PAF) and to mediate intestinal inflammation and injury. The intestinal epithelium, constantly exposed to bacterial products, is the first line of defence against micro-organisms. It has been reported that enterocytes produce proinflammatory mediators, including tumour necrosis factor (TNF) and PAF, and we showed that lipopolysaccharide (LPS) and TNF activate nuclear factor (NF)-jB in enterocytes. However, it remains elusive whether enterocytes release CXCL2 in response to LPS and TNF via a NF-jB-dependent pathway and whether this involves the endogenous production of TNF and PAF. In this study, we found that TNF and LPS markedly induced CXCL2 gene expression in IEC-6 cells, TNF within 30 min, peaking at 45 min, while LPS more slowly, peaking after 2 hr. TNF-and LPS-induced CXCL2 gene expression and protein release were completely blocked by pyrrolidine dithiocarbamate (PDTC) and helenalin, two potent NF-jB inhibitors. NEMO-binding domain peptide, a specific inhibitor of inhibitor protein jB kinase (IKK) activation, a major upstream kinase mediating NF-jB activation, significantly blocked CXCL2 gene expression and protein release induced by LPS. WEB2170 (PAF antagonist) and anti-TNF antibodies had no effect on LPS-induced CXCL2 expression. In conclusion, CXCL2 gene is expressed in enterocytes in response to both TNF and LPS. LPS-induced CXCL2 expression is dependent on NF-jB activation via the IKK pathway. The effect of LPS is independent of endogenous TNF and PAF.

Consumption of the flavonoid quercetin exerts beneficial effects on many chronic diseases. The mechanisms involved in the vasorelaxant effect of quercetin remain uncertain. In the present study, we examined the role of quercetin in vasodilation and rapid endothelial nitric oxide synthase (eNOS) activity in endothelial cells. Quercetin induced a rapid, dose-dependent phosphorylation of eNOS at serine 1179. PKA, Akt and ERK1/2 were all quickly phosphorylated in the process too, but not AMPK and CaMK II. The specific kinase inhibitors for Akt or ERK1/2 could not abolish the quercetin-induced eNOS phosphorylation at Ser1179, which, however, was significantly abolished by H89, an inhibitor of PKA. Concomitantly, intracellular cAMP production was quickly increased by quercetin stimulation and an adenylate cyclase activator, forskolin, also induced eNOS phosphorylation at Ser1179. Quercetin enhanced nitric oxide (NO) production, which was abolished by an eNOS inhibitor, L-NAME or H89. Quercetin exerted a vasodilatory effect on rings with an intact endothelium but not on endothelium-deprived rings, and also inhibited vascular contractility induced by angiotensin II or phenylephrine in rat aortic rings. We conclude that quercetin quickly phosphorylates eNOS at Ser1179 via an Akt-independent, cAMP/PKA-mediated pathway to enhance the production of NO and to promote vasodilation.

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