Oxidized Metabolites of Linoleic Acid in Alcohol-induced Liver Injury

描述(由申请人提供)：这是一项名为“亚油酸在酒精性肝损伤中的氧化代谢物”的壁外/壁内酒精研究合作(U01)申请的重新提交。酒精性肝病(ALD)仍然是慢性疾病和死亡的主要原因。尽管对ALD的发病机制进行了广泛的研究，但其发生发展的具体机制尚不完全清楚。氧化应激增加是ALD肝损伤的核心异常。基于我们广泛的初步发现，我们提出了一个中心假设，即通过12/15-LO介导的途径产生的氧化亚油酸(LA)代谢物(OXLAM)在酒精介导的肝和肠损伤的发生和发展中起关键作用。我们已经建立了一个拥有广泛专业知识和独特资源的协作团队，通过使用ALD的体内实验动物模型、体外细胞培养模型，并与正在进行的NIAAA临床中心关于减少饮食亚油酸(LA)的随机试验和NIAAA赞助的治疗酒精性肝炎(AH)的U01临床试验的独特人群合作，使我们能够实现我们的目标。这项提案将使用最先进的技术，包括脂质组学(LC/ESI/MS/MS)、海马(用于研究线粒体功能障碍)、细胞组学(用于细胞图像采集和分析的高通量技术)。首先，我们将检验OXLAM是ALD肝脏损伤和肠道屏障破坏的特异性介质的假设。我们将确定饮食中LA和OXLAMS在诱导ALD小鼠模型肝脏脂肪变性/损伤和肠道高通透性中的作用。其次，我们将在体外系统中评估OXLAM增强乙醇介导的肝损伤和破坏肠道屏障完整性的潜在机制。我们将确定OXLAM及其与酒精的相互作用对肝细胞线粒体功能和肠上皮紧密连接完整性的影响。第三，我们将评估受控饮食降低人类LA的能力，以减少循环OXLAM、内毒素/肠道通透性和肝脏脂肪变性/损伤(NIAAA内壁RCT)。我们将测试这一假设，与含有8%能量的对照饲料相比，将LA降低到能量的1%持续12周将导致显著减少：3T-MRI评估的肝脏脂肪变性；血浆OXLAM和LA含量，循环内毒素，以及血清CK-18(总和碎片)水平(肝细胞损伤的两个强有力的标志)。最后，我们将建立人类酒精性肝炎中OXLAM的水平及其与疾病严重程度和死亡率的关系(NIAAA U01计划)。本研究将有助于阐明ALD发病机制中的一条新的生化途径。这些发现可能为生物标记物和药物开发提供新的靶点，并可能确定改善肝病的潜在营养策略。

DESCRIPTION (provided by applicant): This is a resubmission of an Extramural/Intramural Alcohol Research Collaboration (U01) Application entitled "Oxidized Metabolites of Linoleic Acid in Alcohol-induced Liver Injury". Alcoholic liver disease (ALD) remains a major cause of chronic illness and death. Despite extensive investigation into ALD pathogenesis, the specific mechanisms responsible for development and progression are incompletely understood. Increased oxidative stress is a core abnormality responsible for liver injury in ALD. Based on our extensive preliminary findings we propose the CENTRAL HYPOTHESIS that oxidized linoleic acid (LA) metabolites (OXLAMs) generated via the 12/15-LO mediated pathway play a critical role in the development and progression of alcohol-mediated hepatic and intestinal injury. We have established a collaborative team with extensive expertise and unique resources that will allow us to accomplish our goals by using in vivo experimental animal models of ALD, in vitro cell culture models, and collaborating with the unique human populations of an ongoing NIAAA Clinical Center randomized trial on dietary linoleic acid (LA) reduction and an NIAAA sponsored U01 clinical trial on alcoholic hepatitis (AH). State-of-the-art technologies including Lipidomics (LC/ESI/MS/MS), Seahorse (to investigate mitochondria dysfunction), Cellomics (the high-throughput technique for cell image-acquisition and analysis) will be utilized in this proposal. FIRST, we will test the hypothesis that OXLAMs are specific mediators of liver damage and intestinal barrier disruption in ALD. We will establish the role of dietary LA and OXLAMs in the induction of hepatic steatosis/injury and intestinal hyper-permeability in murine models of ALD. SECOND, we will evaluate in in vitro systems potential mechanisms by which OXLAMs enhance ethanol-mediated liver damage and disruption of intestinal barrier integrity. We will determine the effect of OXLAMs and their interactions with alcohol on mitochondrial function in hepatocytes and integrity of tight junctions in intestinal epithelium. THIRD, we will evaluate the ability of controlled dietary lowering of LA in humans to reduce circulating OXLAMs, endotoxin/gut permeability, and liver steatosis/injury (NIAAA intramural RCT). We will test the hypothesis that, compared to a control diet containing 8 % of energy as LA, lowering LA to 1% of energy for 12 weeks will result in significant reductions in: liver steatosis assessed by 3T-MRI; OXLAM and LA content of plasma, circulating endotoxin, and serum CK-18 (total and fragmented) levels (two robust markers of hepatocyte injury). FINALLY, we will establish levels of OXLAMs in human Alcoholic Hepatitis and their relation to disease severity and mortality (NIAAA U01 program). This study will help to elucidate a novel biochemical pathway involved in the pathogenesis of ALD. The findings could provide novel targets for biomarkers and drug development, and could identify a potential nutritional strategy for ameliorating liver disease.