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Respiratory Research

, 3:16

First Online: 05 September 2002Received: 22 November 2001Revised: 04 April 2002Accepted: 06 June 2002

Abstract

BackgroundChronic lung disease CLD of prematurity is a major problem of neonatal care. Bacterial infection and inflammatory response have been thought to play an important role in the development of CLD and steroids have been given, with some benefit, to neonates with this disease. In the present study, we assessed the ability of lipopolysaccharide LPS to stimulate rat alveolar macrophages to produce nitric oxide NO, express inducible nitric oxide synthase iNOS and activate nuclear factor-κB NF-κB in vitro. In addition, we investigated the impact of dexamethasone and budesonide on these processes.

MethodsGriess reaction was used to measure the nitrite level. Western blot and a semi-quantitative RT-PCR were performed to detect iNOS expression. Electrophoretic mobility shift assay EMSA was performed to analyze the activation of NF-κB.

ResultsWe found that LPS stimulated the rat alveolar macrophages to produce NO in a dose ≥10 ng-ml and time dependent manner p < 0.05. This effect was further enhanced by IFN-γ ≥10 IU-ml, p < 0.05, but was attenuated by budesonide 10–10 M and dexamethasone 10–10 M p < 0.05. The mRNA and protein levels of iNOS were also induced in response to LPS and attenuated by steroids. LPS triggered NF-κB activation, a mechanism responsible for the iNOS expression.

ConclusionOur findings imply that Gram-negative bacterial infection and the inflammatory responses are important factors in the development of CLD. The down-regulatory effect of steroids on iNOS expression and NO production might explain the beneficial effect of steroids in neonates with CLD.

KeywordsChronic lung disease LPS macrophage nitric oxide nuclear factor-κB AbbreviationsCLDchronic lung disease

DTTdithiothreitol

EMSAelectrophoretic mobility shift assay

HEPESHCO3-free N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid

IFN-γgamma interferon

iNOSinducible nitric oxide synthase

LPSlipopolysaccaride

NF-κBnuclear factor-κB

NOnitric oxide

RT-PCRreverse transcription – polymerase chain reaction

PMSFphenylmethysulfonyl fluoride

TAFtracheobronchial aspirate fluid

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Autor: Ying-Hua Li - Zhong-Qun Yan - Annelie Brauner - Kjell Tullus

Fuente: https://link.springer.com/







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