Alcohol, iNOS upregulation, leaky gut & liver disease

描述（由申请人提供）：继发于肝坏死炎症级联（HNIC）的临床显著性酒精性(A)肝损伤（LD）仅发生在酗酒者的一个亚群中。因此，除乙醇(E)以外的其他因素必须参与其中。假设：ALD的关键辅助因素是由于慢性E的使用导致肠道屏障完整性的破坏（“漏肠”），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}在男性中，酒精性LD患者存在肠道渗漏，而非酒精性LD患者和非酒精性LD患者没有肠道渗漏；2}在大鼠中，e诱导的肠漏与LD有关；肠漏的逆转可减轻LD；在肠单分子层中，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转化方法（单层细胞，老鼠和人）来测试我们目前的假设。目的：(1)观察在更大的样本中，肠漏是否：a)只发生在酗酒的LD患者和肝硬化之前；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量）：i)仅见于LD的酗酒者，发生于肝硬化之前；ii)与LD的严重程度相关（临床参数、肝酶）；iii)与NO过量产生（肠黏膜NO）、血清内毒素和HNIC（高neopterin/cytokines）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通路。我们预测，在e -饲大鼠LD: 1)漏肠，内毒素血症，HNIC，肠道iNOS上调，NO过量生产和肌动蛋白和微管蛋白氧化发生；ii)防止肠道渗漏（通过燕麦、iNOS抑制剂或精氨酸）防止LD。(3)观察，使用野生型（抑制剂）和转染细胞单层，e诱导的iNOS上调及其后果（通过细胞骨架氧化/紊乱和屏障破坏评估）是否由NF-kappaB激活介导。我们预测i) E通过降解IkappaBalpha激活NF-kappaB；ii)阻止NF-kappaB激活可阻止e诱导的iNOS上调及其后果。意义：表明ALD需要一个渗漏的肠道，并且NO和细胞骨架通路参与其中，可以1)识别有LD风险的饮酒者（糖测试）；2)导致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.