Probiotics and HIF signaling in Alcoholic Liver Disease

描述(申请人提供)：酒精性肝病(ALD)的发病机制是多因素的。动物和人类研究表明，肠源性内毒素是导致ALD的关键因素。内毒素来源于革兰氏阴性细菌的细胞壁，由于严格调控的肠道屏障功能，通常只有微量的内毒素穿透肠道上皮。然而，在病理条件下，如酗酒，内毒素泄漏会增加。肠上皮的屏障功能是由细胞旁的顶端连接复合体提供的，包括位于上皮细胞顶端的紧密连接和粘连连接以及粘液层。粘液层的保护涉及粘蛋白和许多调节多肽，如肠道三叶因子(ITF)，它们在屏障的形成和稳定、促进肠道快速修复(‘修复)、调节粘膜分化过程和调节粘膜免疫反应中发挥关键作用。屏障的形成和重建也依赖于不间断的粘膜血流和正在进行的糖酵解。肠道上皮细胞位于厌氧腔和高度代谢的固有层之间，形成了陡峭的生理氧气梯度。生理和病理低氧诱导低氧诱导因子(HIF)-1和-2，它们通过上调肠道ITF、粘蛋白和糖酵解酶等多种基因产物而成为一种主要的适应性反应。我们的初步研究表明，慢性酒精喂养降低了肠道HIF-2、ITF和紧密连接的表达，导致内毒素血症增加，最终导致肝脏损伤。添加益生菌菌株鼠李糖乳杆菌(LGG)可以减轻这些有害影响和ALD。然而，其机制尚不清楚。我们的中心假设是，酒精喂养引起有利于氧化应激、炎症和线粒体损伤的改变，从而减少HIF反应信号和靶蛋白，导致肠道屏障功能障碍，增加内毒素血症和随后的ALD，而益生菌补充剂将通过增强HIF调节反应来减轻酒精诱导的肝损伤。为了验证这一假说，我们将进行以下具体目标：具体目标1.确定乙醇和益生菌对肠道HIF信号的影响及其在ALD中的作用机制。特定目的2.确定益生菌补充剂的保护作用(S)是否需要低氧诱导因子信号转导 酒精性肝损伤。我们将使用肠道特异的HIF-1和HIF-2基因敲除小鼠来评估和比较HIF-1和HIF-2在ALD中的作用。然后，我们将使用Prolyl-4羟基酶-1 PH.D.1基因敲除小鼠进行功能获得策略，以确定增加的HIF是否可以减轻酒精诱导的ALD粘液层功能障碍。这项研究将对开发预防和治疗ALD的新的治疗靶点产生重大影响。

DESCRIPTION (provided by applicant): The pathogenesis of alcoholic liver disease (ALD) is multi-factorial. Animal and human studies suggest that gut-derived endotoxin is a critical factor contributing to ALD. Endotoxins are derived from the cell wall of Gram-negative bacteria, and normally penetrate the gut epithelium in only trace amounts due to tightly regulated intestinal barrier function. However, endotoxin leakiness is increased under pathological conditions, such as alcohol abuse. The barrier function of the intestinal epithelium is provided by paracellular apical junction complexes, including tight junctions and adherens junctions located at the apical end of epithelial cells, and by mucus layers. Protection of the mucus layer involves mucins and a number of regulatory peptides, such as intestinal trefoil factors (ITFs), which play critical rols in formation and stabilization of the barrier, enhancement of rapid intestinal repair ('restitution), modulation of mucosal differentiation processes, and modulation of the mucosal immune response. Formation and restitution of the barrier is also dependent on uninterrupted mucosal blood flow and ongoing glycolysis. Intestinal epithelial cells are positioned between an anaerobic lumen and a highly metabolic lamina propria forming a steep physiologic oxygen gradient. Physiological and pathological hypoxia induces hypoxia-inducible factor (HIF) -1 and -2, which represent a major adaptation response by upregulating a variety of gene products such as intestinal ITF, mucins and glycolytic enzymes. Our preliminary studies showed that chronic alcohol feeding decreased intestinal HIF-2, ITF and tight junction expression leading to increased endotoxemia and ultimately liver injury. Supplementation of a probiotic strain, lactobacillus rhamnosus GG (LGG), attenuates those deleterious effects and ALD. However, the mechanisms are unknown. Our central hypothesis is that alcohol feeding causes a change favoring oxidative stress, inflammation and mitochondrial damage, which reduces HIF responsive signaling and the targeted proteins, leading to disruption of gut barrier function, increased endotoxemia and subsequent ALD, and that probiotic supplementation will attenuate alcohol-induced liver injury by potentiating HIF regulated responses. To test this hypothesis, we will carry our following specific aims: Specific Aim 1. Determine the effects of ethanol and probiotics on intestinal HIF signaling and mechanisms in ALD. Specific Aim 2. Determine whether HIF signaling is required for the protective effect(s) of probiotic supplementation against alcohol-induced liver injury. We will use intestine-specific HIF-1¿ and HIF-2¿ knockout mice to evaluate and compare the role of HIF-1¿ and HIF-2¿ in ALD. We will then use prolyl-4 hydroxylase-1 PH.D.1 knockout mice for a gain-of-function strategy to determine whether an increase HIF attenuates alcohol-induced mucus layer dysfunction in ALD. This study will have a major impact on the development of new therapeutic targets for the prevention and treatment of ALD.