Dietary Fat in Ethanol-Induced Intestinal Barrier Dysfunction in ALD

描述（由申请人提供）：本申请将评估肠道-肝脏和饮食-酒精相互作用，以及肠道屏障完整性/功能在酒精性肝病（ALD）发生和进展中的关键作用。ALD仍然是肝脏疾病的主要原因之一，也是美国和世界范围内发病率和死亡率的主要原因。为什么一些重度饮酒者会患上ALD，而另一些则不会（我们假设饮食影响——见下文），目前还不清楚。肠源性脂多糖（LPS）在ALD的发展中起着至关重要的作用。肠道完整性破坏导致的肠通透性增加是酒精诱导的内毒素血症的主要原因。膳食脂肪是ALD发生的重要辅助因子。研究表明，富含饱和脂肪酸的饮食可以保护啮齿动物免受酒精引起的肝脏疾病，而饮食中的多不饱和脂肪酸（如亚油酸，LA）则会促进肝损伤。我们的工作假设是，膳食LA增强了肠上皮细胞的促氧化和促炎症作用，从而激活NF-kB。这些事件通过mlck依赖机制导致肠道紧密连接的改变，并导致乙醇相关的肠道完整性破坏。我们假设LA和EtOH对肠道屏障完整性的联合有害影响是通过nf - kb - mlck介导的共同机制发生的。肠道通透性增加导致血液内毒素血症升高，从而导致肝损伤。因此，抑制MLCK可以减轻肠道紧密连接破坏，减少肠道渗漏和血液内毒素血症，并减轻乙醇/亚油酸引起的肝损伤。我们的应用程序的具体目标是：1。为了确定：a)亚油酸诱导氧化应激是否激活NF-kB并增加Caco-2细胞（肠上皮屏障的体外模型）中促炎细胞因子的产生；b)亚油酸是否通过nf - kb - mlck介导的机制，通过解除主要紧密连接蛋白的管制和重新分配，增强乙醇介导的肠屏障完整性破坏，以及这种作用是加性的还是协同的。2. 为了确定：a)在ALD动物模型中，膳食亚油酸是否通过NF-kB激活增强肠道氧化应激并促进肠道炎症；b)在体内，膳食亚油酸介导的肠道炎症是否通过mlck介导的机制导致乙醇相关的肠道紧密连接功能障碍；c)在ALD小鼠模型中，MLCK的药理抑制/基因缺失是否可以减轻紧密连接改变，降低肠道通透性和血液内毒素血症，从而减轻肝损伤。饮食调节可能代表了一种新的预防/治疗ALD的方法，而MLCK可能是一种特殊的新的治疗靶点。

DESCRIPTION (provided by applicant): This application will evaluate Gut-Liver and Diet-Alcohol interactions with respect to the critical role of intestinal barrier integrity/function in he development and progression of alcoholic liver disease (ALD). ALD remains one of the leading causes of liver diseases and a major cause of morbidity and mortality in the United States and worldwide. Why some heavy drinkers develop ALD and other are spared is unclear (we postulate dietary effects-see below). Gut-derived lipopolysaccharide (LPS) plays a crucial role in the development of ALD. Increased intestinal permeability resulting from the disruption of intestinal integrity is a major cause of alcohol-induced endotoxemia. Dietary fat is an important cofactor for the development of ALD. It has been shown that diets enriched in saturated fatty acids protect against alcohol-induced liver disease in rodents, whereas dietary polyunsaturated fatty acids (e.g. linoleic acid, LA) promote liver damage. Our working hypothesis is that dietary LA potentiates pro-oxidative and pro-inflammatory effects on intestinal epithelial cells with consequent NF-kB activation. These events lead to alterations of intestinal tight junctions through an MLCK-dependent mechanism and contribute to the ethanol-associated disruption of intestinal integrity. We hypothesize that the combined deleterious effects of LA and EtOH on intestinal barrier integrity occur through a common NF-kB-MLCK-mediated mechanism. Increased gut permeability leads to elevated blood endotoxemia and consequent liver injury. Thus, inhibition of MLCK will attenuate intestinal tight junction disruption, decrease gut leakines and blood endotoxemia, and attenuate ethanol/dietary linoleic acid induced liver damage. The Specific Aims of our application are: 1. To determine: a) if linoleic acid induces oxidative stress activates NF-kB and increases pro-inflammatory cytokine production in Caco-2 cells, an in vitro model of the intestinal epithelial barrier; b) if linoleic acid enhances ethanol- mediated disruptin of intestinal barrier integrity by deregulation and redistribution of the major tight junctions proteins through an NF-kB-MLCK-mediated mechanism, and if this effect is additive or synergistic. 2. To determine: a) whether dietary linoleic acid potentiates intestinal oxidative stress and promotes intestinal inflammation through NF-kB activation in an animal model of ALD; b) whether dietary linoleic acid-mediated intestinal inflammation contributes to ethanol-associated intestinal tight junction dysfunction through the MLCK-mediated mechanism in vivo; and c) if pharmacological inhibition/genetic deletion of MLCK can attenuate tight junction alterations, decrease gut permeability and blood endotoxemia, and consequently attenuate liver injury in a mouse model of ALD. Dietary modulation may represent a novel preventive/therapeutic approach in ALD, and MLCK could be a specific novel therapeutic target.