Dietary Fat in Ethanol-Induced Intestinal Barrier Dysfunction in ALD

描述(申请人提供)：这项申请将评估肠道-肝脏和饮食-酒精的相互作用，关于肠道屏障完整性/功能在酒精性肝病(ALD)的发展和进展中的关键作用。ALD仍然是导致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ALD，而另一些人则幸免于难，这一点尚不清楚(我们假设饮食影响--见下文)。肠源性脂多糖(LPS)在ALD的发生发展中起着重要作用。由于肠道完整性的破坏而导致的肠道通透性增加是酒精引起的内毒素血症的主要原因。膳食脂肪是ALD发生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酒精引起的啮齿动物肝病，而饮食中的多不饱和脂肪酸(例如亚油酸，LA)则会促进肝脏损伤。我们的工作假设是，饮食中的LA增强了肠道上皮细胞的氧化和促炎作用，从而激活了核因子-kB。这些事件通过MLCK依赖的机制导致肠道紧密连接的改变，并有助于乙醇相关的肠道完整性破坏。我们假设LA和EtoH对肠屏障完整性的联合有害作用是通过共同的NF-kB-MLCK介导的机制发生的。肠道通透性增加会导致血液内毒素血症升高，进而导致肝脏损伤。因此，抑制MLCK将减轻肠道紧密连接的破坏，降低肠道细胞因子和血液内毒素血症，减轻乙醇/饮食亚油酸所致的肝损伤。我们应用的具体目的是：1.确定：a)亚油酸是否诱导氧化应激激活Caco-2细胞中的核因子-kB并增加促炎细胞因子的产生，这是一个肠上皮屏障的体外模型；b)亚油酸是否通过放松调控并通过核因子-kB-MLCK介导的机制重新分配主要的紧密连接蛋白来增强乙醇介导的肠屏障完整性的破坏，以及这种作用是相加的还是协同的。2.确定：a)饮食亚油酸是否通过激活核因子-kB增强ALD动物模型的肠道氧化应激并促进肠道炎症；b)饮食亚油酸介导的肠炎症是否通过体内MLCK介导的机制促进乙醇相关性肠紧密连接功能障碍；以及c)MLCK的药理抑制/基因缺失是否可以减轻ALD小鼠的紧密连接改变，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ALD的一种新的预防/治疗方法，而MLCK可能是一种特异的新的治疗靶点。

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.