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

描述（由申请人提供）：本提案的目的是验证一个新的假设，即直接由酒精或环境或遗传操纵破坏脑-肠昼夜节律同步，是造成etoh诱导的肠道高通透性易感差异的“脆弱性”因素，这解释了为什么只有一小部分酗酒者会发生肠道内毒素泄漏和脂肪性肝炎[ASH]。我们的假设得到以下证据的支持：(1)肠道渗漏是内毒素血症的主要原因，而肠道内毒素是ASH所必需的；(2)虽然EtOH普遍破坏肠上皮单层通透性，但肠道渗漏仅发生在一小部分酗酒者中，这表明可能涉及其他因素——肠道渗漏的变异性；(3)核心的生物钟分子机制存在于包括下丘脑视交叉上核（SCN）和肠上皮细胞在内的所有器官中。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可调节肠道通透性，生理、心理应激等病理刺激可引起肠道渗漏；(5)脑肠通讯的昼夜节律调节可能影响肠通透性，因为昼夜节律基因调控了直接参与肠通透性调节的AJC蛋白基因。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到损伤。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，这些基因的siRNA敲低可防止酒精诱导的单层高通透性。我们还发现，在有漏肠的酒精喂养大鼠的肠道中，Clock和Per2蛋白增加。为了验证我们的假设，我们将采用两种不同的方法。首先（在目标1中），我们将使用环境[携带LD周期中的恒定相移]和遗传[时钟突变和Per1/Per2 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酒精喂养小鼠（8周慢性模型）中etoh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节律脑肠同步和肠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对慢性酒精喂养的反应中的作用。其次（在目标2中），我们将评估etoh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（3天急性和8周慢性模型）。我们预测，酒精介导的中枢和/或肠道昼夜节律不同步将导致顶端连接复合体（AJC）损伤增加，从而导致肠道渗漏。证明昼夜节律介导的脑肠通讯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。

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.