Banan A, Keshavarzian A, Zhang L, Shaikh M, Forsyth C B, Tang Y, Fields J Z
Department of Internal Medicine, Section of Gastroenterology & Nutrition, Rush University Medical Center, Chicago, IL 60612, USA.
Alcohol. 2007 Sep;41(6):447-60. doi: 10.1016/j.alcohol.2007.07.003. Epub 2007 Sep 14.
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.
肠道屏障破坏与包括酒精性肝病（ALD）在内的多种肠道和全身性疾病有关。利用肠道（Caco-2）细胞单层，我们发现乙醇（EtOH）通过破坏细胞骨架的稳定性来破坏屏障完整性。由于促炎状态与核因子κB（NF-κB）的激活有关，我们推测EtOH通过激活NF-κB诱导细胞骨架组装破坏和屏障完整性受损。亲代细胞用NF-κB的药理学调节剂进行预处理。其他细胞用NF-κB抑制剂I-κBα的显性负突变体进行稳定转染。将每种细胞类型的单层暴露于EtOH，然后我们监测单层屏障完整性（通透性）；细胞骨架稳定性和分子动力学（共聚焦显微镜和免疫印迹）；I-κBα的细胞内水平（免疫印迹）；NF-κB的亚细胞分布和活性（免疫印迹和灵敏酶联免疫吸附测定）；以及肌动蛋白细胞骨架43kDa蛋白、聚合的F-肌动蛋白和单体G-肌动蛋白的细胞内变化（十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分级分离）。EtOH引起了包括I-κBα降解、NF-κB核转位、NF-κB亚基（p50和p65）激活、肌动蛋白解聚（G-上升、F-下降）、肌动蛋白细胞骨架不稳定和屏障破坏在内的不稳定变化。NF-κB抑制剂和I-κBα稳定剂（如MG-132、乳胞素等）可防止NF-κB激活，同时预防EtOH诱导的损伤。在转染的I-κBα突变体克隆中，I-κBα稳定化以失活NF-κB可预防所有EtOH诱导损伤的指标。我们的数据支持了几种新机制，即在EtOH损伤条件下，NF-κB可影响F-肌动蛋白细胞骨架的分子动力学和肠道屏障完整性。（1）EtOH部分通过I-κBα降解和NF-κB激活诱导F-肌动蛋白细胞骨架破坏和肠道屏障完整性受损；（2）NF-κB这种病理生理效应的潜在机制似乎涉及肌动蛋白网络亚基组件组装的不稳定性。
