二异丙基氟磷酸酯对细胞凋亡的调控及中性粒细胞氧化爆发的引发作用。

Regulation of apoptosis and priming of neutrophil oxidative burst by diisopropyl fluorophosphate.

机构信息

Interdepartmental Division of Critical Care, University of Toronto, Toronto, Canada.

出版信息

J Inflamm (Lond). 2010 Jul 7;7:32. doi: 10.1186/1476-9255-7-32.

DOI:10.1186/1476-9255-7-32

PMID:20609247

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC2913997/

Abstract

BACKGROUND

Diisopropyl fluorophosphate (DFP) is a serine protease inhibitor that is widely used as an inhibitor of endogenous proteases in in vitro neutrophil studies. Its effects on neutrophil function are unclear. We sought to determine the biological effects of DFP on human neutrophil apoptosis and oxidative burst.

METHODS

We isolated neutrophils from healthy volunteers, incubated them with DFP (2.5 mM), and evaluated neutrophil elastase (NE) activity, neutrophil degranulation, apoptosis as reflected in hypodiploid DNA formation and exteriorization of phosphatidylserine (PS), processing and activity of caspases-3 and -8, oxidative burst activity and hydrogen peroxide release.

RESULTS

Consistent with its activity as a serine protease inhibitor, DFP significantly inhibited NE activity but not the degranulation of azurophilic granules. DFP inhibited constitutive neutrophil apoptosis as reflected in DNA fragmentation, and the processing and activity of caspases-3 and -8. DFP also inhibited priming of neutrophils for oxidative burst activity and hydrogen peroxide release. However, DFP enhanced the exteriorization of PS in a dose-dependent manner.

CONCLUSION

We conclude that DFP exerts significant effects on neutrophil inflammatory function that may confound the interpretation of studies that use it for its antiprotease activity. We further conclude that endogenous proteases play a role in the biology of constitutive neutrophil apoptosis.

摘要

背景

二异丙基氟磷酸酯（DFP）是一种丝氨酸蛋白酶抑制剂，广泛用于体外中性粒细胞研究中内源性蛋白酶的抑制剂。其对中性粒细胞功能的影响尚不清楚。我们试图确定 DFP 对人中性粒细胞凋亡和氧化爆发的生物学影响。

方法

我们从健康志愿者中分离中性粒细胞，用 DFP（2.5mM）孵育它们，并评估中性粒细胞弹性蛋白酶（NE）活性、中性粒细胞脱颗粒、凋亡（反映在低二倍体 DNA 形成和磷脂酰丝氨酸（PS）外化）、半胱天冬酶-3 和 -8 的加工和活性、氧化爆发活性和过氧化氢释放。

结果

与作为丝氨酸蛋白酶抑制剂的活性一致，DFP 显著抑制 NE 活性，但不抑制嗜中性粒细胞的脱颗粒作用。DFP 抑制了固有中性粒细胞凋亡，表现为 DNA 片段化，以及半胱天冬酶-3 和 -8 的加工和活性。DFP 还抑制了中性粒细胞氧化爆发活性和过氧化氢释放的启动。然而，DFP 以剂量依赖的方式增强 PS 的外化。