NAD⁺Ameliorates Inflammation-Induced Epithelial Barrier Dysfunction in Cultured Enterocytes and Mouse Ileal Mucosa

Abstract

In the course of other experiments, we serendipitously observed that extracellular nicotinamide adenine dinucleotide (NAD ϩ ) ameliorated the development of epithelial hyperpermeability when monolayers of Caco-2 enterocyte-like cells were incubated with cytomix, a mixture containing interferon-␥, interleukin-1␤, and tumor necrosis factor-␣. We sought to characterize the effects of NAD ϩ on inflammation-induced epithelial barrier dysfunction using Caco-2 monolayers that were exposed to cytomix in the absence or presence of NAD ϩ or other purine-containing molecules. Paracellular barrier function measured as the apical-to-basolateral passage of fluorescein isothiocyanate-conjugated dextran (mol. wt. ϳ4000) was preserved in a concentration-dependent manner when immunostimulated Caco-2 cells were exposed to extracellular NAD ϩ . Incubation with NAD ϩ prevented cytomix-induced derangements in the expression and localization of the tight junction proteins occludin and zonula occludens-1 in Caco-2 cells. Treatment of cytomix-stimulated cells with NAD ϩ also blocked nuclear factor-B (NF-B) activation, inducible nitric-oxide synthase induction, and increased production of nitric oxide (NO⅐). Ileal mucosal permeability to fluorescein isothiocyanate-dextran mol. wt. ϳ4000 was increased in mice 18 h after lipopolysaccharide (endotoxin) injection, but treatment of endotoxemic mice with NAD ϩ ameliorated the development of gut mucosal hyperpermeability. Thus, extracellular NAD ϩ seems to ameliorate inflammation-induced intestinal epithelial barrier dysfunction by inhibiting NF-B activation and increased NO⅐ production.

Enterocyte-specific epidermal growth factor prevents barrier dysfunction and improves mortality in murine peritonitis

“…Prior to the present study, claudins have not been evaluated for their roles in intestinal permeability in critical illness. However, decreased intestinal expression and altered subcellular distribution of occludin has been observed following endotoxemia (29), following bile duct ligation (55), and when intestinal epithelial cells are exposed to proinflammatory cytokines in vitro (28,30). ZO-1 expression has also been found to be altered in hemorrhagic shock (54).…”

Regulation of intestinal barrier function by signal transducer and activator of transcription 5b

“…Nuclear protein (NE) was prepared utilizing the NE-PER kit (Pierce, Rockford, Illinois, USA), and the postnuclear pellet was dissolved with SDS-dissolving buffer and kept as the membrane fraction (MP) 2 21. The EMSA used to measure DNA-binding activity was carried out using a double-stranded oligonucleotide, as previously published 22 23. Briefly, the sequence of the double-stranded NF-κB oligonucleotide was as follows: sense, 5′-AGT TGA GGG GAC TTT CCC AGG C-3′; antisense, 3′-TCA ACT CCC CTG AAA GGG TCC G-5′ (the NF-κB DNA-binding consensus sequence is underlined; Invitrogen).…”

β-Nicotinamide adenine dinucleotide attenuates lipopolysaccharide-induced inflammatory effects in a murine model of acute lung injury

“…ALI is characterized by pulmonary microvascular endothelial gaps and disarray with cellular breakdown and subsequent endothelial permeability and interstitial edema [29-31]. Recent studies suggest that in proinflammatory conditions, β -NAD released from cellular sources, including the endothelium into the extracellular fluid, is an important vascular mediator [18, 32]. Our experimental data demonstrate that β -NAD administration significantly attenuates the accumulation of protein in LPS-induced murine models of ALI (Figure 1), suggesting an improvement of barrier function via β -NAD–induced signaling.…”