活性氧介导内毒素诱导的人真皮内皮细胞NF-κB激活。

Reactive oxygen species mediate endotoxin-induced human dermal endothelial NF-kappaB activation.

作者信息

Chan Emily L, Murphy Joseph T

机构信息

Department of Surgery, Division of Burns, Trauma, Critical Care, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA.

出版信息

J Surg Res. 2003 May 1;111(1):120-6. doi: 10.1016/s0022-4804(03)00050-7.

DOI:10.1016/s0022-4804(03)00050-7

PMID:12842456

Abstract

BACKGROUND

Microvascular endothelial cell "activation" by endotoxin is an early and critical phenomenon underlying organ dysfunction related to sepsis. Dermal endothelial cells contribute to many of the manifestations of septic shock, such as cutaneous interstitial edema and loss of peripheral vasomotor regulation. Human dermal endothelial cell activation by endotoxin (lipopolysaccharide [LPS]) is characterized by the generation of reactive oxygen species (ROS) that enhance nuclear translocation of the transcription factor kappa-B (NF-kappaB).

METHODS

We tested our hypothesis by stimulating human dermal microvascular endothelial cells (HMEC.1) with endotoxin and assaying for endothelial generation of ROS and nuclear translocation of NF-kappaB subunits. HMEC.1 cultures were treated individually with LPS, hydrogen peroxide, or xanthine, xanthine oxidase, and ferrous sulfate (xanthine/XO/Fe(2+)). Nuclear proteins were isolated and consensus sequence binding was assessed by electrophoretic mobility shift assay (EMSA). 2',7'-Dichlorofluorescin diacetate and confocal microscopy were used to examine ROS production in LPS-stimulated HMEC.1.

RESULTS

Nuclear translocation of the p65/p50 NF-kappaB heterodimer was detectable 30 min after stimulation with LPS alone or the xanthine/XO/Fe(2+) combination, but not with hydrogen peroxide. Antioxidant N-acetylcysteine (NAC) inhibited LPS-stimulated ROS production in HMEC.1. Antioxidant prior to or simultaneously with LPS exposure, but not following LPS, also prevented NF-kappaB activation. NAC was ineffective at inhibiting NF-kappaB translocation at increased LPS concentrations.

CONCLUSIONS

Dermal endothelial cells, a microvascular cell type that may contribute to the systemic response to blood-borne endotoxemia, generate ROS in the absence of other inflammatory cells. These LPS-activated endothelial cells, in turn, rapidly translocate transcription factor NF-kappaB to cell nuclei, a process regulated in part by intracellular ROS.

摘要

背景

内毒素引起的微血管内皮细胞“激活”是脓毒症相关器官功能障碍的早期关键现象。皮肤内皮细胞在脓毒性休克的许多表现中起作用，如皮肤间质水肿和外周血管舒缩调节功能丧失。内毒素（脂多糖 [LPS]）激活人皮肤内皮细胞的特征是产生活性氧（ROS），ROS 可增强转录因子κB（NF-κB）的核转位。

方法

我们通过用内毒素刺激人皮肤微血管内皮细胞（HMEC.1）并检测内皮细胞 ROS 的产生和 NF-κB 亚基的核转位来验证我们的假设。HMEC.1 培养物分别用 LPS、过氧化氢或黄嘌呤、黄嘌呤氧化酶和硫酸亚铁（黄嘌呤/黄嘌呤氧化酶/Fe(2+)）处理。分离核蛋白并通过电泳迁移率变动分析（EMSA）评估共有序列结合情况。用 2',7'-二氯荧光素二乙酸酯和共聚焦显微镜检查 LPS 刺激的 HMEC.1 中 ROS 的产生。

结果

单独用 LPS 或黄嘌呤/黄嘌呤氧化酶/Fe(2+)组合刺激 30 分钟后可检测到 p65/p50 NF-κB 异二聚体的核转位，但用过氧化氢刺激则未检测到。抗氧化剂 N-乙酰半胱氨酸（NAC）抑制 HMEC.1 中 LPS 刺激的 ROS 产生。在 LPS 暴露之前或同时给予抗氧化剂，但不是在 LPS 暴露之后，也可防止 NF-κB 激活。在 LPS 浓度增加时，NAC 抑制 NF-κB 转位无效。