Alcohol, iNOS upregulation, leaky gut & liver disease

描述（由申请方提供）：继发于肝坏死性炎症级联反应（HNIC）的临床显著酒精性（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的关键辅因子是由于长期使用E导致的肠屏障完整性的破坏（“肠漏”），其允许肠内毒素到达肝脏并引发HNIC;这种泄漏是由于细胞骨架蛋白的氧化引起的细胞骨架不稳定性，所述细胞骨架蛋白的氧化由E诱导的肠iNOS上调和一氧化氮（NO）过量产生引起。 我们发现：1}在人类中，LD酗酒者的肠漏，但在那些没有LD或LD非酗酒者中没有; 2}在大鼠中，E诱导的肠漏与LD相关;肠漏的逆转减弱LD; 3}在肠单层中，E诱导的iNOS上调导致细胞骨架和屏障破坏。我们将继续使用这种成功的翻译方法（单层，大鼠和人）来测试我们目前的假设。 目的：（1）在更大的样本中，观察肠漏是否：a）仅发生在患有LD的酗酒者和肝硬化之前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过度产生和HNIC相关，e）在女性中更明显。 我们预测肠道渗漏（口服糖负荷后过量的尿乳果糖、甘露醇和三氯蔗糖水平）：i）仅见于患有LD的酗酒者，在肝硬化之前; ii）与LD的严重程度（临床参数、肝酶）相关; iii）与NO过度产生（肠道粘膜NO）、血清内毒素和HNIC（高新蝶呤/细胞因子）相关。 (2)在大鼠中，观察预防E-诱导的肠漏是否也能预防E-诱导的LD &是否涉及过度活跃的NO通路。 我们预测，在患有LD的E-喂养大鼠中：i）肠漏、内毒素血症、HNIC、肠iNOS的上调、NO过度产生以及肌动蛋白和微管蛋白的氧化发生; ii）防止肠漏（通过燕麦、iNOS抑制剂或精氨酸）防止LD。 (3)要看到，使用野生型（抑制剂）和转染细胞的单层，如果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）是由NF-κ B激活介导的。我们预测i）E通过降解IkappaB α激活NF-κ B; ii）阻止NF-κ B激活阻止E诱导的iNOS上调及其后果。重要性：表明ALD需要肠道渗漏，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）导致治疗，以防止那些无法戒酒的饮酒者的LD。

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.