体外研究严重早发性 COPD 患者与健康不吸烟对照者中性粒细胞的激活：抗氧化治疗的影响。

Neutrophil activation in severe, early-onset COPD patients versus healthy non-smoker subjects in vitro: effects of antioxidant therapy.

机构信息

Research Unit, University General Hospital Consortium, and Department of Medicine, Faculty of Medicine, University of Valencia, Valencia, Spain.

出版信息

Respiration. 2012;83(2):147-58. doi: 10.1159/000332834. Epub 2011 Nov 17.

DOI:10.1159/000332834

PMID:22094549

Abstract

BACKGROUND

Neutrophils and oxidative stress have been implicated in the pathogenesis of COPD. Severe, early-onset COPD is characterized by a rapid decline in the lung function at an early age; however, nothing is known about neutrophil activation in COPD patients.

OBJECTIVES

The aim of this study was to evaluate peripheral blood neutrophil activation in severe, early-onset COPD patients versus healthy non-smokers and the effect of N-acetyl-L-cysteine (NAC) on neutrophil activation in vitro.

METHODS

Neutrophils were isolated from 15 severe, early-onset COPD patients and 15 age-matched healthy subjects and stimulated with N-formyl-Met-Leu-Phe (fMLP) in the presence or absence of NAC (10 μM to 10 mM). Neutrophil chemotaxis, elastase release, reactive oxygen species (ROS), intracellular thiols and apoptosis were measured by Boyden chamber, spectrofluorometry, CMFDA and H2DCF-DA dyes and by annexin V-FITC, respectively.

RESULTS

Chemotaxis of peripheral blood neutrophils from COPD patients in response to fMLP was 30% more increased than that observed in healthy subjects. Elastase release in response to fMLP was 2-fold higher in neutrophils from COPD patients versus healthy subjects. Intracellular thiol levels were 30% lower in COPD and ROS was approximately 30% higher in COPD versus healthy neutrophils. Spontaneous apoptosis showed no differences in both groups of patients and fMLP-induced apoptosis was higher in COPD. Pre-treatment with the antioxidant NAC effectively attenuated neutrophil chemotaxis, elastase release and ROS as well as effectively increased thiol levels in COPD.

CONCLUSIONS

Neutrophils in severe, early-onset COPD patients are highly activated and this is alleviated by NAC in vitro.

摘要

背景

中性粒细胞和氧化应激与 COPD 的发病机制有关。严重的早发性 COPD 的特征是在年轻时肺功能迅速下降；然而，目前尚不清楚 COPD 患者中性粒细胞的激活情况。

目的

本研究旨在评估严重早发性 COPD 患者与健康非吸烟者外周血中性粒细胞的激活情况，以及 N-乙酰-L-半胱氨酸（NAC）对体外中性粒细胞激活的影响。

方法

从 15 名严重早发性 COPD 患者和 15 名年龄匹配的健康受试者中分离中性粒细胞，并在存在或不存在 NAC（10 μM 至 10 mM）的情况下用 N-甲酰基-L-甲硫氨酰-L-亮氨酰-苯丙氨酸（fMLP）刺激。通过 Boyden 室、分光荧光法、CMFDA 和 H2DCF-DA 染料分别测量中性粒细胞趋化性、弹性蛋白酶释放、活性氧（ROS）、细胞内巯基和细胞凋亡，并用 annexin V-FITC 进行检测。

结果

与健康受试者相比，COPD 患者外周血中性粒细胞对 fMLP 的趋化性增加了 30%。COPD 患者中性粒细胞对 fMLP 的弹性蛋白酶释放增加了 2 倍。COPD 患者的细胞内巯基水平降低了 30%，ROS 增加了约 30%。两组患者的自发性凋亡均无差异，而 fMLP 诱导的凋亡在 COPD 中更高。抗氧化剂 NAC 的预处理可有效减轻 COPD 中性粒细胞的趋化性、弹性蛋白酶释放和 ROS，同时有效增加细胞内巯基水平。