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首页> 外文期刊>Alcohol >NF-kappaB activation as a key mechanism in ethanol-induced disruption of the F-actin cytoskeleton and monolayer barrier integrity in intestinal epithelium.
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NF-kappaB activation as a key mechanism in ethanol-induced disruption of the F-actin cytoskeleton and monolayer barrier integrity in intestinal epithelium.

机译：NF-κB活化是乙醇诱导的F-肌动蛋白细胞骨架破坏和肠上皮单层屏障完整性的关键机制。

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Intestinal barrier disruption has been implicated in several intestinal and systemic disorders including alcoholic liver disease (ALD). Using monolayers of intestinal (Caco-2) cells, we showed that ethanol (EtOH) disrupts the barrier integrity via destabilization of the cytoskeleton. Because proinflammatory conditions are associated with activation of NF-kappa B (NF-kappaB), we hypothesized that EtOH induces disruption of cytoskeletal assembly and barrier integrity by activating NF-kappaB. Parental cells were pretreated with pharmacological modulators of NF-kappaB. Other cells were stably transfected with a dominant negative mutant for the NF-kappaB inhibitor, I-kappaBalpha. Monolayers of each cell type were exposed to EtOH and we then monitored monolayer barrier integrity (permeability); cytoskeletal stability and molecular dynamics (confocal microscopy and immunoblotting); intracellular levels of the I-kappaBalpha (immunoblotting); subcellular distribution and activity of NF-kappaB (immunoblotting and sensitive ELISA); and intracellular alterations in the 43kDa protein of the actin cytoskeleton, polymerized F-actin, and monomeric G-actin (SDS-PAGE fractionation). EtOH caused destabilizing alterations, including I-kappaBalpha degradation, NF-kappaB nuclear translocation, NF-kappaB subunit (p50 and p65) activation, actin disassembly (upward arrow G-, downward arrow F-), actin cytoskeleton instability, and barrier disruption. Inhibitors of NF-kappaB and stabilizers of I-kappaBalpha (e.g., MG-132, lactacystin, etc) prevented NF-kappaB activation while protecting against EtOH-induced injury. In transfected I-kappaBalpha mutant clones, stabilization of I-kappaBalpha to inactivate NF-kappaB protected against all measures of EtOH-induced injury. Our data support several novel mechanisms where NF-kappaB can affect the molecular dynamics of the F-actin cytoskeleton and intestinal barrier integrity under conditions of EtOH injury. (1) EtOH induces disruption of the F-actin cytoskeleton and of intestinal barrier integrity, in part, through I-kappaBalpha degradation and NF-kappaB activation; (2) The mechanism underlying this pathophysiological effect of the NF-kappaB appears to involve instability of the assembly of the subunit components of actin network.
机译：肠道屏障的破坏与包括酒精性肝病（ALD）在内的几种肠道和全身性疾病有关。使用单层肠（Caco-2）细胞，我们显示乙醇（EtOH）通过细胞骨架的不稳定破坏了屏障的完整性。因为促炎性疾病与NF-κB（NF-κB）的激活有关，我们假设EtOH通过激活NF-κB诱导细胞骨架组装和屏障完整性的破坏。亲代细胞用NF-κB的药理调节剂预处理。其他细胞用NF-kappaB抑制剂I-kappaBalpha的显性负突变体稳定转染。每种细胞类型的单层细胞都暴露于EtOH中，然后我们监测单层细胞屏障的完整性（通透性）。细胞骨架稳定性和分子动力学（锥镜和免疫印迹）; I-κBalpha（免疫印迹）的细胞内水平; NF-κB的亚细胞分布和活性（免疫印迹和灵敏ELISA）;肌动蛋白细胞骨架，聚合的F-肌动蛋白和单体G-肌动蛋白的43kDa蛋白的胞内变化（SDS-PAGE分级分离）。 EtOH导致不稳定的变化，包括I-κB降解，NF-κB核易位，NF-κB亚基（p50和p65）活化，肌动蛋白拆卸（向上箭头G-，向下箭头F-），肌动蛋白细胞骨架不稳定和屏障破坏。 NF-κB抑制剂和I-kappaBalpha的稳定剂（例如MG-132，lacticacystin等）可防止NF-kappaB活化，同时防止EtOH诱导的损伤。在转染的I-kappaBalpha突变体克隆中，使I-kappaBalpha稳定以灭活NF-kappaB可以抵抗EtOH诱导的所有损伤。我们的数据支持了几种新机制，其中NF-κB可以在EtOH损伤的情况下影响F-肌动蛋白细胞骨架的分子动力学和肠屏障完整性。 （1）EtOH部分地通过I-κB降解和NF-κB活化而诱导F-肌动蛋白细胞骨架的破坏和肠屏障完整性的破坏; （2）NF-κB的这种病理生理作用的机制似乎涉及肌动蛋白网络亚基组件装配的不稳定性。

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