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

描述（由申请人提供）：本提案的目的是检验新假设，即直接通过酒精或通过环境或遗传操纵破坏脑-肠昼夜节律之间的同步性是导致EtOH诱导的肠通透性过高的不同易感性的“脆弱性”因素，这解释了为什么只有一部分酗酒者会发生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）肠漏是内毒素血症的主要原因，肠源性内毒素是ASH所必需的;（2）虽然乙醇普遍破坏肠上皮细胞单层通透性，但肠漏仅发生在一部分酗酒者中，这表明可能涉及其他因素-肠漏的变异性;（3）核心生物钟分子机制存在于所有器官中，包括下丘脑视交叉上核（SCN）中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间，可能包括所谓的“脑-肠轴”（BGA）的脑-肠相互作用：（4）BGA可以调节肠通透性，并且病理性刺激如生理和心理应激可以引起肠漏;（5）脑肠通讯的昼夜节律调节可能通过昼夜节律基因调节顶端连接复合体（AJC）而影响肠通透性这些蛋白质基因直接参与调节肠道通透性。我们最近的体内小鼠数据表明，昼夜节律的破坏使肠道容易受伤。此外，我们在Caco-2肠单层中的试验数据显示，酒精刺激时钟基因Clock和Per 2的表达，并且这些基因的siRNA敲低防止酒精诱导的单层通透性过高。我们还表明，Clock和Per 2蛋白在酒精喂养的肠漏大鼠的肠道中增加。为了验证我们的假设，我们将采取两种不同的方法。首先（在目标1中），我们将使用环境[夹带LD循环中的恒定相移]和遗传[Clock突变体和Per 1/Per 2 KO小鼠]方法来破坏小鼠的整体昼夜节律组织，以确定这种破坏是否会导致乙醇诱导的肠道内毒素泄漏的易感性增加（8周慢性模型）。我们预测，这些昼夜节律的操作将阐明昼夜节律的脑-肠同步性和肠细胞时钟基因在调节肠顶端紧密连接蛋白和肠道通透性响应慢性酒精喂养的作用。其次（目标2），我们将评估EtOH诱导的中枢和外周生物钟功能和基因表达的变化如何影响内毒素的渗透性（3天急性模型和8周慢性模型）。我们预测，酒精介导的中枢和/或肠道昼夜节律紊乱将导致对心尖连接复合体（AJC）的损伤增加，从而导致肠道渗漏。证明昼夜节律介导的脑-肠通信中断是酒精诱导的内毒素血症的一个关键“易感性”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：叙述性大多数人都知道，我们的身体根据生物节律运作。这些生物节律似乎调节着我们健康的许多方面，但对所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酒精性肝病和其他酒精相关疾病的新疗法。

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.