S-亚硝基谷胱甘肽抑制肿瘤坏死因子-α诱导的中性粒细胞中核因子κB的激活。

S-nitrosoglutathione inhibits TNF-alpha-induced NFkappaB activation in neutrophils.

作者信息

Fortenberry J D, Owens M L, Chen N X, Brown L A

机构信息

Critical Care Division, Children's Healthcare of Atlanta at Egleston, GA 30322, USA.

出版信息

Inflamm Res. 2001 Feb;50(2):89-95. doi: 10.1007/s000110050729.

DOI:10.1007/s000110050729

PMID:11289659

Abstract

OBJECTIVE AND DESIGN

Cytokine expression is controlled by transcription factors including NFkappaB, which has recently been found to exist in human neutrophils. We previously showed that exogenous nitric oxide (NO) induces neutrophil apoptosis and hypothesized that this NO effect could be mediated by inhibition of NFkappaB activation.

MATERIALS AND METHODS

Isolated human neutrophils were incubated with or without S-nitrosoglutathione (GSNO 0.1 mM-5 mM; Sigma) for 2 h. Neutrophils were either unstimulated or stimulated with TNFalphalpha or n-formyl methionyl leucine phenylalanine (fMLP). Viability was assessed by vital dye cytotoxicity assay. After nuclear extraction and measurement of protein concentration, NFkappaB binding was determined by electrophoretic mobility shift assay. Effects of GSNO on activation of IkappaB alpha, which inhibits intranuclear translocation of NFkappaB, were measured by Western immunoblot technique. For comparison, experiments were also performed in the presence of the NFkappaB inhibitor PDTC.

RESULTS

TNFalpha increased nuclear NFkappaB activity compared to unstimulated neutrophils (p < 0.001, n = 5). GSNO (500 microM) decreased TNFalpha-induced NFkappaB activity (p<0.05) and inhibited NFkappaB activity whether given prior to or during TNFalpha exposure. IkappaB alpha was significantly degraded at 30 and 120 min of TNFalpha exposure compared to control neutrophils (p < 0.05). GSNO exposure (500 microM) inhibited IkappaB alpha degradation in the presence of TNFalpha. PDTC enhanced neutrophil cell death and DNA fragmentation, in association with decreased NFkappaB activity, similar to GSNO effects.

CONCLUSION

Neutrophils possess NFkappaB activity that is increased by stimulation with TNFalpha. GSNO inhibits NFkappaB activity in association with inhibiting TNFalpha-induced degradation of IkappaB alpha. GSNO effects are similar to those seen with NFkappaB inhibition by PDTC. Inhibition of NF kappaB could represent a potential anti-inflammatory effect of GSNO.

摘要

目的与设计

细胞因子的表达受包括核因子κB（NFκB）在内的转录因子调控，最近发现NFκB存在于人类中性粒细胞中。我们之前表明外源性一氧化氮（NO）可诱导中性粒细胞凋亡，并推测这种NO效应可能是通过抑制NFκB激活来介导的。

材料与方法

将分离出的人类中性粒细胞与或不与S-亚硝基谷胱甘肽（GSNO，0.1 mM - 5 mM；Sigma公司）孵育2小时。中性粒细胞未受刺激或用肿瘤坏死因子α（TNFα）或N-甲酰甲硫氨酰亮氨酰苯丙氨酸（fMLP）刺激。通过活体染料细胞毒性试验评估细胞活力。在进行核提取物制备和蛋白质浓度测定后，通过电泳迁移率变动分析确定NFκB结合情况。采用蛋白质免疫印迹技术测量GSNO对抑制NFκB核内转位的IκBα激活的影响。为作比较，实验也在NFκB抑制剂PDTC存在的情况下进行。

结果

与未受刺激的中性粒细胞相比，TNFα可增加核NFκB活性（p < 0.001，n = 5）。GSNO（500 μM）可降低TNFα诱导的NFκB活性（p < 0.05），并且无论在TNFα暴露之前还是期间给予，均可抑制NFκB活性。与对照中性粒细胞相比，在TNFα暴露30分钟和120分钟时，IκBα明显降解（p < 0.05）。在存在TNFα的情况下，GSNO暴露（500 μM）可抑制IκBα降解。与GSNO的作用相似，PDTC可增强中性粒细胞死亡和DNA片段化，同时伴有NFκB活性降低。