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Stress and corticotropin releasing factor (CRF) promote necrotizing enterocolitis in a formula-fed neonatal rat model.

ABSTRACT: The etiology of necrotizing enterocolitis (NEC) is not known. Alterations in gut microbiome, mucosal barrier function, immune cell activation, and blood flow are characterized events in its development, with stress as a contributing factor. The hormone corticotropin-releasing factor (CRF) is a key mediator of stress responses and influences these aforementioned processes. CRF signaling is modulated by NEC's main risk factors of prematurity and formula feeding. Using an established neonatal rat model of NEC, we tested hypotheses that: (i) increased CRF levels-as seen during stress-promote NEC in formula-fed (FF) newborn rats, and (ii) antagonism of CRF action ameliorates NEC. Newborn pups were formula-fed to initiate gut inflammation and randomized to: no stress, no stress with subcutaneous CRF administration, stress (acute hypoxia followed by cold exposure-NEC model), or stress after pretreatment with the CRF peptide antagonist Astressin. Dam-fed unstressed and stressed littermates served as controls. NEC incidence and severity in the terminal ileum were determined using a histologic scoring system. Changes in CRF, CRF receptor (CRFRs), and toll-like receptor 4 (TLR4) expression levels were determined by immunofluorescence and immunoblotting, respectively. Stress exposure in FF neonates resulted in 40.0% NEC incidence, whereas exogenous CRF administration resulted in 51.7% NEC incidence compared to 8.7% in FF non-stressed neonates (p<0.001). Astressin prevented development of NEC in FF-stressed neonates (7.7% vs. 40.0%; p = 0.003). CRF and CRFR immunoreactivity increased in the ileum of neonates with NEC compared to dam-fed controls or FF unstressed pups. Immunoblotting confirmed increased TLR4 protein levels in FF stressed (NEC model) animals vs. controls, and Astressin treatment restored TLR4 to control levels. Peripheral CRF may serve as specific pharmacologic target for the prevention and treatment of NEC.

SUBMITTER: Bell RL

PROVIDER: S-EPMC8191945 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Inflammation in the premature intestine is a key factor that leads to the development of necrotizing enterocolitis (NEC). Activation of nuclear factor kappa B (NF-?B) and subsequent inflammation increases the severity of NEC. The aim of this study was to investigate the early temporal expression of inflammatory markers and activation of NF-?B in a neonatal rat model of NEC.Pre- and full-term newborn Sprague-Dawley rat pups were sacrificed at birth, 1.5, 4, 8, and 24 hours after receiving their first feed. Control pups were vaginally delivered and mother fed; NEC was induced by a combination of gavage feeding formula, hypoxia, and enteral lipopolysaccharide (LPS); and formula fed pups were fed every 4 hours with infant formula. Ileal tissue was collected for immunohistochemistry, real-time polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay. Serum was collected for cytokine content. Fold change of expression of inducible nitric oxide synthase (iNOS), interleukin (IL)-1?, IL-6, tumor necrosis factor-? (TNF-?), IL-10, NF-?B p65, and I?B? used RT-PCR. Data were analyzed by paired two-tailed t test, expressed as mean ± standard error of the mean, and p ? 0.05 considered significant.No histologic injury was evident in ileal sections. At 1.5 h, iNOS expression increased twofold over control in NEC pups (2.1 vs. 1.0, p ? 0.05) and remained elevated at 24 h (0.7 vs. 9.4, p ? 0.05). IL-1? and IL-6 reached a peak at 24 h in NEC tissue compared with control. IL-10 expression rose in NEC pups after 4 h of insult and remained elevated in formula and NEC stressed pups. Coincident with an increase in p65 translocation into the nucleus and a reduction of I?B? detected in the cytoplasm, increased transcription of I?B? occurs.These findings suggest that NF-?B activation initiates inflammation early in the course of NEC resulting in increased proinflammatory protein expression, underscoring the importance of the inflammatory response in this NEC model, which precedes evidence of histological injury.

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Stress and corticotropin releasing factor (CRF) promote necrotizing enterocolitis in a formula-fed neonatal rat model.

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