Probiotics and HIF signaling in Alcoholic Liver Disease

描述（由申请人提供）：酒精性肝病（ALD）的发病机制是多因素的。动物和人体研究表明，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菌的细胞壁，并且由于严格调节的肠屏障功能，通常仅以痕量渗透肠上皮。然而，在病理条件下，例如酒精滥用，内毒素渗漏增加。肠上皮的屏障功能由细胞旁顶端连接复合体和粘液层提供，所述细胞旁顶端连接复合体包括位于上皮细胞顶端的紧密连接和粘附连接。粘液层的保护涉及粘蛋白和许多调节肽，例如肠三叶因子（ITF），其在屏障的形成和稳定、快速肠修复（恢复）的增强、粘膜分化过程的调节和粘膜免疫应答的调节中起关键作用。屏障的形成和恢复也依赖于不间断的粘膜血流和持续的糖酵解。肠上皮细胞位于厌氧腔和高度代谢的固有层之间，形成陡峭的生理氧气梯度。生理性和病理性缺氧诱导缺氧诱导因子（HIF）-1和-2，其代表通过上调多种基因产物如肠ITF、粘蛋白和糖酵解酶的主要适应反应。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，最终导致肝损伤。补充益生菌菌株，鼠李糖乳杆菌GG（LGG），减弱这些有害作用和ALD。然而，其机制尚不清楚。我们的中心假设是，饮酒会导致有利于氧化应激、炎症和线粒体损伤的变化，从而减少HIF反应信号和靶蛋白，导致肠道屏障功能破坏、内毒素血症增加和随后的酒精性肝脏疾病，并且益生菌补充剂将减轻酒精通过增强HIF调节的反应来诱导肝损伤。为了验证这个假设，我们将进行我们的以下具体目标：具体目标1。确定乙醇和益生菌对ALD中肠道HIF信号传导和机制的影响。具体目标2。确定HIF信号传导是否是益生菌补充剂对以下疾病的保护作用所必需的： 酒精性肝损伤我们将使用精氨酸特异性HIF-1和HIF-2基因敲除小鼠来评估和比较HIF-1在ALD中的作用。然后，我们将使用脯氨酰-4羟化酶-1 PH. D. 1基因敲除小鼠的功能获得策略，以确定是否增加HIF减弱酒精诱导的ALD粘液层功能障碍。这项研究将对预防和治疗酒精性肝脏病的新治疗靶点的开发产生重大影响。

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.