Platelet-activating factor induces the processing of nuclear factor-κB p105 into p50, which mediates acute bowel injury in mice

Abstract

Platelet-activating factor (PAF), an endogenous proinflammatory phospholipid, when injected intravascularly to rats and mice, causes shock, acute bowel injury, and a rapid activation of NF-kappaB p50-p50 with upregulation of the chemokine CXCL2 in the intestine. In this study, we investigate the mechanism of NF-kappaB activation and the role of the NF-kappaB p50 subunit in PAF-induced shock and acute bowel injury. NF-kappaB p50-deficient mice and wild-type mice were anesthetized and tracheotomized, and their carotid artery was cannulated for blood pressure monitoring, blood sampling, and PAF administration. For determination of bowel injury, shock, and survival, PAF (2.2 microg/kg, intra-arterially, i.a.) was injected. Two hours later, animals were euthanized, and their small intestines were removed for histological examination. For biochemical studies, PAF (1.5 microg/kg i.a.) was administered and the small intestine removed after 15-60 min. We found that PAF induced an increase in p105 processing within 30 min, but there were no changes in the levels of the NF-kappaB inhibitory proteins IkappaBalpha and beta. NF-kappaB p50-deficient mice were protected against PAF-induced mortality, shock, intestinal hypoperfusion, and injury compared with wild-type animals. We also found that p50-deficient mice had decreased gene expression of CXCL2 and TNF and a decrease in CXCL2 protein production compared with wild-type mice. Our study suggests that PAF increases the processing of NF-kappaB p105 into p50, with upregulation of proinflammatory cytokines, which leads to PAF-induced systemic inflammatory response and acute bowel injury.

Trinitrobenzene sulfonic acid-induced intestinal injury in neonatal mice activates transcriptional networks similar to those seen in human necrotizing enterocolitis

“…The cellular inflammatory response was comprised of monocyte/macrophage-rich infiltrates and also resembled NEC (6); (c) TNBS did not affect germ-free mice, indicating that the mucosal injury occurred only in the presence of gut bacteria (similar to NEC) and was not due to chemical/corrosive action of TNBS (6); (d) TNBS-mediated enterocolitis can be used to investigate the temporal evolution of neonatal intestinal injury (from the time of instillation of TNBS), unlike the hypothermia-hypoxia model where individual animals develop bowel injury at different times during the NEC protocol (14, 15); (e) comparison of pups vs . adult mice treated with weight-normalized doses of TNBS can provide insights into the developmental aspects of NEC; (f) unlike NEC models involving splanchnic ischemia-reperfusion or the administration of platelet-activating factor where the animals need to be euthanized within a few hours (16, 17), the evolution of TNBS-enterocolitis can be observed over longer periods; (g) because all pups in a cohort are affected predictably, fewer animals are needed (compared to the hypoxia-hypothermia model where bowel injury may occur only in 40–70% mice); and (h) the use of 10-day-old pups allows enough time to test prophylactic interventions; it was convenient that the rodent intestine at birth resembles the preterm human intestine and takes 3 weeks to reach the structural/functional maturity of the term human neonate (18, 19). …”

Intestinal Vascular Endothelial Growth Factor Is Decreased in Necrotizing Enterocolitis

“…To measure intestinal VEGF protein, whole neonatal mouse intestines were homogenized on ice and tissue lysates processed for Western blot analysis with anti-VEGF antibody (SC Biotechnology, Santa Cruz, CA) using a previously described protocol[15]. Results were expressed as VEGF/β-actin band density ratio.…”

“…After tissue homogenization over ice, total RNA was purified using a Qiagen RNeasy Kit (Qiagen, Valencia, CA) and real-time RT-PCR performed as previously described [15] using the following primers: VEGF164 (VEGF-A) Forward, 5'-GCA GGC TGC TGT AAC GAT GA-3'; VEGF Reverse, 5'-GCA TGA TCT GCA TGG TGA TGT T-3'; GAPDH Forward, 5'-CTT CAC CAC CAT GGA GAA GGC-3'; GAPDH reverse, 5'-GGC ATG GAC TGT GGT CAT GAG-3'. VEGF gene expression was normalized to GAPDH (ΔCT=GAPDH-VEGF) as internal control.…”

Gut mucosal injury in neonates is marked by macrophage infiltration in contrast to pleomorphic infiltrates in adult: evidence from an animal model

“…Although this model is a useful screening tool for the investigation of inflammatory signaling, its short duration limits its value in the study of leukocyte trafficking (20). Intestinal ischemia-reperfusion injury, induced by superior mesenteric artery occlusion, is another short-duration model (2-3 h) with similar limitations (23).…”