Alcohol, iNOS upregulation , leaky gut & liver disease

酒精、iNOS 上调、肠漏

• 批准号: 7057359
• 负责人: ALI KESHAVARZIAN
• 金额: $ 66.51万
• 依托单位: RUSH UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7057359
• 关键词: Lactobacillus; alcoholic liver cirrhosis; alcoholism /alcohol abuse; biopsy; clinical research; cytoskeletal proteins; disease /disorder etiology; drug /alcohol abstinence; ethanol; human subject; intestinal mucosa; laboratory rat; lipopolysaccharides; liver disorder; liver transplantation; membrane permeability; nitric oxide; nitric oxide synthase; nuclear factor kappa beta; oxidative stress; questionnaires; tissue /cell culture; transfection

DESCRIPTION (provided by applicant): Clinically significant alcoholic (A) liver damage (LD), secondary to a hepatic necroinflammatory cascade (HNIC), occurs only in a subset of alcoholics. Hence, factors other than ethanol (E) must be involved. Hypothesis: The key cofactor for ALD is a breakdown of gut barrier integrity ("leaky gut") due to chronic E use, which allows intestinal endotoxin to reach the liver & initiate a HNIC; this leakiness is due to cytoskeletal instability caused by oxidation of cytoskeletal proteins which is elicited by E-induced gut iNOS upregulation & nitric oxide (NO) overproduction. We found: 1} in man, gut leakiness in alcoholics with LD but not in those without LD or in nonalcoholics with LD; 2} in rats, E-induced leaky gut is associated with LD; reversal of gut leakiness attenuates LD; 3} in intestinal monolayers, E-induced iNOS upregulation causes cytoskeletal & barrier disruption. We will continue to use this successful translational approach (monolayers, rats & man) to test our current hypotheses. Aims: (1) To see if, in a larger sample, a leaky gut: a) occurs only in alcoholics with LD & precedes cirrhosis b) persists during abstinence & after liver transplant for ALD, c) correlates quantitatively with LD severity, d) is associated with NO overproduction & HNIC, e) is more pronounced in females. We predict that gut leakiness (excess urinary lactulose, mannitol & sucralose levels after oral sugar load): i) is seen only in alcoholics with LD, precedes cirrhosis; ii) correlates with severity of LD (clinical parameters, liver enzymes); iii) is associated with NO overproduction (gut mucosal NO), serum endotoxin & HNIC (high neopterin/cytokines). (2) To see if, in rats, prevention of E-induced leaky gut also prevents E-induced LD & if a hyperactive, NO pathway is involved. We predict that in E-fed rats with LD: i) leaky gut, endotoxemia, HNIC, upregulation of intestinal iNOS, NO overproduction & oxidation of actin & tubulin occurs; ii) preventing gut leakiness (by oats, iNOS inhibitors or Arginine) prevents LD. (3) To see, using monolayers of wild type ((inhibitors) & transfected cells, if E-induced iNOS upregulation & its consequences (assessed by cytoskeletal oxidation/disarray & barrier disruption) are mediated by NF-kappaB activation. We predict i) E activates NF-kappaB by degrading IkappaBalpha; ii) preventing NF-kappaB activation prevents E-induced iNOS upregulation & its consequences. Significance: Showing that ALD requires a leaky gut, & that NO & cytoskeletal pathways are involved, could 1) identify drinkers at risk for LD (sugar test); 2) lead to therapies to prevent LD in those drinkers unable to abstain.

描述（由申请人提供）：继发于肝脏坏死性炎症级联反应（HNIC）的临床显着的酒精性（A）肝损伤（LD）仅发生在一部分酗酒者中。 因此，必须涉及乙醇（E）以外的因素。假设：ALD 的关键辅助因子是由于长期使用 E 导致的肠道屏障完整性破坏（“肠漏”），这使得肠道内毒素到达肝脏并引发 HNIC；这种渗漏是由于 E 诱导的肠道 iNOS 上调和一氧化氮 (NO) 过量产生而引起的细胞骨架蛋白氧化引起的细胞骨架不稳定所致。 我们发现： 1} 在男性中，患有 LD 的酗酒者有肠道渗漏，但没有 LD 的人或患有 LD 的非酗酒者则没有； 2} 在大鼠中，E 诱导的肠漏与 LD 相关；逆转肠道渗漏可减弱 LD； 3} 在肠道单层细胞中，E 诱导的 iNOS 上调会导致细胞骨架和屏障破坏。我们将继续使用这种成功的转化方法（单层细胞、大鼠和人）来测试我们当前的假设。 目的：(1) 在更大的样本中观察肠漏是否：a) 仅发生在患有 LD 的酗酒者中且先于肝硬化 b) 在戒酒期间和 ALD 肝移植后持续存在，c) 与 LD 严重程度定量相关，d) 与 NO 过量产生和 HNIC 相关，e) 在女性中更为明显。 我们预测肠道渗漏（口服糖负荷后尿中乳果糖、甘露醇和三氯蔗糖水平过高）：i) 仅见于患有 LD 的酗酒者，发生在肝硬化之前； ii) 与 LD 的严重程度相关（临床参数、肝酶）； iii) 与 NO 过量产生（肠粘膜 NO）、血清内毒素和 HNIC（高新蝶呤/细​​胞因子）有关。 (2) 观察在大鼠中，预防 E 诱导的肠漏是否也能预防 E 诱导的 LD，以及是否涉及过度活跃的 NO 途径。 我们预测，在患有 LD 的 E 喂养大鼠中： i) 发生肠漏、内毒素血症、HNIC、肠道 iNOS 上调、NO 过量产生以及肌动蛋白和微管蛋白氧化； ii) 防止肠道渗漏（通过燕麦、iNOS 抑制剂或精氨酸）可预防 LD。 (3) 使用野生型（抑制剂）和转染细胞的单层，观察 E 诱导的 iNOS 上调及其后果（通过细胞骨架氧化/混乱和屏障破坏评估）是否是由 NF-κB 激活介导的。我们预测 i) E 通过降解 IkappaBalpha 来激活 NF-κB； ii) 防止 NF-kappaB 激活可防止 E 诱导的 iNOS 上调及其后果。意义：表明 ALD 需要肠漏，并且涉及 NO 和细胞骨架途径，可以 1) 识别有 LD 风险的饮酒者（糖测试）； 2) 为那些无法戒酒的饮酒者提供预防 LD 的治疗方法。