Dietary Fat in Ethanol-Induced Intestinal Barrier Dysfunction in ALD

描述（由申请人提供）：本申请将评价肠-肝和饮食-酒精相互作用，以确定肠屏障完整性/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。为什么一些酗酒者会患上酒精性肝病而另一些人则不会（我们假设饮食影响-见下文）。肠源性脂多糖（LPS）在ALD的发展中起着至关重要的作用。由于肠完整性的破坏而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发展的重要辅助因素。研究表明，富含饱和脂肪酸的饮食可以预防啮齿动物酒精诱导的肝病，而饮食中的多不饱和脂肪酸（如亚油酸，LA）会促进肝损伤。我们的工作假设是，饮食LA增强促氧化和促炎症作用的肠上皮细胞与随后的NF-κ B激活。这些事件通过MLCK依赖性机制导致肠紧密连接的改变，并导致乙醇相关的肠完整性破坏。我们假设LA和EtOH对肠屏障完整性的联合有害作用通过共同的NF-κ B-MLCK介导的机制发生。增加的肠道通透性导致血液内毒素血症升高和随后的肝损伤。因此，MLCK的抑制将减弱肠紧密连接破坏，减少肠渗漏和血液内毒素血症，并减弱乙醇/膳食亚油酸诱导的肝损伤。我们申请的具体目的是：1。确定：a）如果亚油酸诱导氧化应激，则在肠上皮屏障的体外模型Caco-2细胞中激活NF-kB并增加促炎细胞因子的产生; B）如果亚油酸通过NF-kB-MLCK介导的机制通过主要紧密连接蛋白的失调和重新分布来增强乙醇介导的肠屏障完整性的破坏，并且如果该作用是累加的或协同的。2.确定：a）在ALD的动物模型中，膳食亚油酸是否通过NF-κ B活化增强肠氧化应激并促进肠炎症; B）膳食亚油酸介导的肠炎症是否通过MLCK介导的体内机制促成乙醇相关的肠紧密连接功能障碍;和c）如果MLCK的药理学抑制/遗传缺失可以减弱紧密连接改变，降低肠通透性和血液内毒素血症，并因此减轻ALD小鼠模型中的肝损伤。饮食调节可能是ALD的一种新的预防/治疗方法，MLCK可能是一个特定的新的治疗靶点。

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.