Alcohol, iNOS upregulation , leaky gut & liver disease

描述(由申请人提供)：临床上有意义的酒精性(A)肝损害(LD)继发于肝脏坏死炎性级联反应(HNIC)，仅发生在酒精者的一小部分中。因此，必须涉及乙醇(E)以外的其他因素。假设：ALD的关键辅助因素是由于长期服用E而导致的肠道屏障完整性的破坏(“渗漏的肠道”)，这使得肠道内毒素进入肝脏并引发HNIC；这种泄漏是由于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。我们发现：1)在人类中，酒精性肠梗阻与非酒精性肠梗阻相关；2)在大鼠中，E诱导的肠漏与LD有关；逆转肠漏可减弱LD；3)在肠道单层中，E诱导的iNOS表达上调导致细胞骨架和屏障的破坏。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，看看是否有肠漏：a)仅发生在酒精性酒精性肝病患者中，并先于肝硬变；b)在戒酒期间和ALD肝移植后持续存在，c)与酒精性肝病的严重程度定量相关，d)与没有生产过剩有关，以及HNIC，e)在女性中更为明显。我们预测肠道渗漏(口服糖负荷后尿乳果糖、甘露醇和三氯蔗糖水平过高)：i)仅见于酒精性酒精性肝病患者，出现在肝硬变之前；ii)与酒精性肝病的严重程度(临床参数、肝酶)相关；iii)与NO过量(肠道粘膜NO)、血清内毒素和HNIC(高新喋呤/细胞因子)有关。(2)看看在大鼠中，预防E诱导的肠漏是否也能阻止E诱导的LD&如果是过度活跃，则不涉及任何途径。我们预测，在E喂养的LD大鼠中：i)肠漏、内毒素血症、HNIC、肠道iNOS上调，不会发生肌动蛋白和微管蛋白的过度生产和氧化；ii)防止肠道渗漏(通过燕麦、iNOS抑制剂或精氨酸)可防止LD。(3)利用野生型(抑制物)和转基因细胞的单层细胞，观察E诱导的iNOS上调及其后果(以细胞骨架氧化/紊乱和屏障破坏来评估)是否由核因子-kappaB的激活所介导。我们预测：1)E通过降解IkappaBalpha来激活核因子-kappaB；ii)阻止核因子-kappaB的激活，阻止E诱导的iNOS上调及其后果。意义：显示ALD需要有渗漏的肠道，即不涉及细胞和细胞骨架途径，可以1)确定饮酒者有患LD(糖测试)的风险；2)导致那些无法戒酒的饮酒者预防LD的治疗。

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.