Brain-gut circadian rhythm interactions in alcohol-induced gut leakiness

描述(申请人提供)：这项建议的目标是测试这一新的假设，即酒精直接或环境或遗传操作扰乱大脑-肠道昼夜节律之间的同步性，是导致乙醇诱导的肠道高通透性差异易感性的“脆弱”因素，这解释了为什么只有部分酗酒者会出现内毒素和脂肪性肝炎[ASH]的肠道泄漏。我们的假设得到以下支持：(1)肠漏是导致内毒素血症的主要因素，而肠源性内毒素是ASH所必需的；(2)虽然乙醇普遍能扰乱肠上皮细胞的单层通透性，但只有少数酗酒者会发生肠漏，这表明可能还涉及其他因素--肠漏的变异性；(3)核心生物钟分子机制存在于所有器官，包括下丘脑视交叉上核(SCN)的中央生物钟和肠上皮细胞。SCN调节和协调多种外周昼夜分子节律的表达和时序，可能包括所谓的“脑-肠轴”(BGA)的脑-肠相互作用；(4)BGA可以调节肠道通透性，而生理和心理应激等病理刺激可导致肠道渗漏；(5)大脑肠道通讯的昼夜调节可以影响肠道通透性，因为昼夜节律基因调控与肠道通透性调节直接相关的顶端连接复合体(AJC)蛋白基因。我们最近的活体小鼠数据显示，昼夜节律的扰乱使肠道容易受到伤害。此外，我们在Caco-2肠道单层中的试点数据表明，酒精刺激时钟基因Clock和PER2的表达，而这些基因的siRNA敲除可防止酒精诱导的单层高通透性。我们还发现，在酒精喂养的肠漏大鼠的肠道中，时钟和PER2蛋白增加。为了检验我们的假设，我们将采取两种不同的方法。首先(在目标1)，我们将使用环境[夹带LD周期中的恒定相移]和遗传[时钟突变和PER1/PER2 KO小鼠]的方法来扰乱小鼠的整体昼夜节律组织，以确定这种干扰是否导致酒精喂养小鼠(8wk慢性模型)乙醇诱导的肠道内毒素渗漏的易感性增加。我们预测，这些昼夜节律操作将阐明昼夜脑-肠道同步性和肠道细胞时钟基因在调节肠道顶端紧密连接蛋白和肠道通透性方面的作用，以响应长期酒精喂养。其次(目标2)，我们将评估乙醇诱导的中枢和外周时钟功能和基因表达的变化如何影响内毒素的通透性(3d急性和8wk慢性模型)。我们预测，酒精介导的中枢和/或肠道昼夜节律失同步性将导致心尖连接复合体(AJC)损伤增加，从而导致肠道渗漏。证明昼夜节律调节的脑-肠道通讯障碍是酒精引起的内毒素血症的一个关键的易感因素，将为ASH的预防和治疗干预提供新的靶点。 公共卫生相关性：叙述大多数人都知道，我们的身体是按照生物节律运作的。这些生物节律似乎调节着我们健康的许多方面，但人们对其中涉及的细胞机制知之甚少。这项研究将测试酒精如何影响肠道以及大脑和肝脏的生物节律，看看这是否是酒精导致酒精性肝病等疾病的一种方式。确定酒精如何影响这些器官的生物节律，可能有助于我们设计治疗酒精性肝病和其他与酒精相关的疾病的新方法。

DESCRIPTION (provided by applicant): The goal of this proposal is to test the novel hypothesis that disruption of synchrony between brain-gut circadian rhythms either directly by alcohol, or by environmental or genetic manipulations, is the "vulnerability" factor responsible for the differential susceptibility for EtOH-induced intestinal hyperpermeability that explains why only subset of alcoholics develop gut leakiness to endotoxins and steatohepatitis [ASH]. Our hypothesis is supported by: (1) Gut leakiness is a major contributor to endotoxemia and gut-derived endotoxin is required for ASH; (2) while EtOH universally disrupts intestinal epithelial monolayer permeability, gut leakiness occurs in only a subset of alcoholics, suggesting other factors might be involved-variability in gut leakiness; (3) The core circadian clock molecular machinery is within all organs including the central circadian clock in the hypothalamic suprachiasmatic nucleus (SCN), and intestinal epithelial cells. The SCN regulates and coordinates the expression and timing of multiple peripheral circadian molecular rhythms, possibly including brain-gut interactions of the so called "brain-gut axis," (BGA); (4) The BGA can regulate intestinal permeability, and pathological stimuli like physical and psychological stress can cause gut leakiness; (5) The circadian modulation of the brain gut communication could affect intestinal permeability since circadian genes regulate apical junctional complex (AJC) protein genes that are directly involved in regulation of intestinal permeability. Our recent in vivo mice data showed that disruption of circadian rhythms makes the intestine susceptible to injury. Also, our pilot data in Caco-2 intestinal monolayers show that alcohol stimulates expression of the clock genes Clock and Per2 and that siRNA knockdown of these genes prevents alcohol-induced monolayer hyperpermeability. We also show that Clock and Per2 proteins are increased in the intestines of alcohol fed rats with leaky gut. To test our hypothesis, we will take two different approaches. First (in Aim 1), we will use both environmental [constant phase shifts in the entraining LD cycle] & genetic [Clock mutant and Per1/Per2 KO mice] approaches to disrupt the overall circadian organization of mice to determine if such disruption leads to increased vulnerability for EtOH-induced gut leakage to endotoxins in alcohol-fed mice (8 wk chronic model). We predict that these circadian manipulations will elucidate the roles of circadian brain-gut synchrony and intestinal cell clock genes in regulating intestinal apical tight junctional proteins and gut permeability in response to chronic alcohol feeding. Second (in Aim 2), we will assess how EtOH-induced changes in central and peripheral clock function and gene expression impacts permeability to endotoxin (3 day acute and 8 wk chronic models). We predict that alcohol-mediated central and/or intestinal circadian desynchrony will result in increased injury to apical junctional complex (AJC) leading to gut leakiness. Demonstrating that circadian- mediated disrupted brain-gut communication is one critical contributing "susceptibility" factor for alcohol- induced endotoxemia would provide new targets for preventive and therapeutic interventions in ASH. PUBLIC HEALTH RELEVANCE: NARRATIVE Most people are aware that our bodies operate according to biological rhythms. These biological rhythms appear to regulate many aspects of our health but little is known about the cellular mechanisms involved. This study will test how alcohol affects the biological rhythms of the intestine as well as the brain and liver to see if this is one way alcohol can cause diseases such as alcoholic liver disease. Identifying how alcohol affects these biological rhythms in these organs may help us design new treatments for alcoholic liver disease and other alcohol related diseases.