BACKGROUND & AIMS Binge alcohol exposure causes gut leakiness, contributing to increased endotoxemia and inflammatory liver injury, although the molecular mechanisms are still elusive. This study was aimed at investigating the roles of apoptosis of enterocytes and nitration followed by degradation of intestinal tight junction (TJ) and adherens junction (AJ) proteins in binge alcohol-induced gut leakiness. METHODS The levels of intestinal (ileum) junctional complex proteins, oxidative stress markers and apoptosis-related proteins in rodents, T84 colonic cells and autopsied human ileums were determined by immunoblot, immunoprecipitation, immunofluorescence, and mass-spectral analyses. RESULTS Binge alcohol exposure caused apoptosis of gut enterocytes with elevated serum endotoxin and liver injury. The levels of intestinal CYP2E1, iNOS, nitrated proteins and apoptosis-related marker proteins were significantly elevated in binge alcohol-exposed rodents. Differential, quantitative mass-spectral analyses of the TJ-enriched fractions of intestinal epithelial layers revealed that several TJ, AJ and desmosome proteins were decreased in binge alcohol-exposed rats compared to controls. Consistently, the levels of TJ proteins (claudin-1, claudin-4, occludin and zonula occludens-1), AJ proteins (β-catenin and E-cadherin) and desmosome plakoglobin were very low in binge alcohol-exposed rats, wild-type mice, and autopsied human ileums but not in Cyp2e1-null mice. Additionally, pretreatment with specific inhibitors of CYP2E1 and iNOS prevented disorganization and/or degradation of TJ proteins in alcohol-exposed T84 colonic cells. Furthermore, immunoprecipitation followed by immunoblot confirmed that intestinal TJ and AJ proteins were nitrated and degraded via ubiquitin-dependent proteolysis, resulting in their decreased levels. CONCLUSIONS These results demonstrated for the first time the critical roles of CYP2E1, apoptosis of enterocytes, and nitration followed by ubiquitin-dependent proteolytic degradation of the junctional complex proteins, in promoting binge alcohol-induced gut leakiness and endotoxemia, contributing to inflammatory liver disease. LAY SUMMARY Binge alcohol exposure causes gut leakiness, contributing to increased endotoxemia and inflammatory liver injury. Our results demonstrated for the first time the critical roles of apoptosis of enterocytes and nitration followed by ubiquitin-dependent proteolytic degradation of the junctional complex proteins in promoting this gut leakiness and endotoxemia. These results provide insight into the molecular mechanisms of alcohol-induced inflammatory liver disease.

中文翻译：

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肠细胞的凋亡和连接复合蛋白的硝化促进酒精诱导的肠道渗漏和肝损伤

背景与目的 暴饮暴食会导致肠道渗漏，导致内毒素血症和炎症性肝损伤增加，尽管分子机制仍然难以捉摸。本研究旨在研究肠细胞凋亡和硝化作用以及肠道紧密连接 (TJ) 和粘附连接 (AJ) 蛋白降解在酗酒引起的肠道渗漏中的作用。方法通过免疫印迹、免疫沉淀、免疫荧光和质谱分析测定啮齿动物、T84结肠细胞和尸体解剖的人回肠中肠（回肠）连接复合蛋白、氧化应激标志物和凋亡相关蛋白的水平。结果 酗酒导致肠道肠细胞凋亡，血清内毒素升高和肝损伤。肠道 CYP2E1、iNOS、在暴饮暴食的啮齿动物中，硝化蛋白和凋亡相关标记蛋白显着升高。肠道上皮层富含 TJ 的部分的差异、定量质谱分析显示，与对照组相比，暴露于狂饮酒精的大鼠中的几种 TJ、AJ 和桥粒蛋白减少。一致地，TJ 蛋白（claudin-1、claudin-4、occludin 和 zonula occludens-1）、AJ 蛋白（β-catenin 和 E-cadherin）和桥粒 plakoglobin 在暴饮暴食的大鼠、野生-型小鼠，并尸检人回肠，但不在 Cyp2e1 缺失小鼠中。此外，用 CYP2E1 和 iNOS 的特异性抑制剂进行预处理可防止暴露于酒精的 T84 结肠细胞中 TJ 蛋白的解体和/或降解。此外，免疫沉淀和免疫印迹证实肠道 TJ 和 AJ 蛋白通过泛素依赖性蛋白水解作用被硝化和降解，导致它们的水平降低。结论 这些结果首次证明了 CYP2E1、肠细胞凋亡和硝化作用，随后连接复合蛋白的泛素依赖性蛋白水解降解，在促进酗酒引起的肠道渗漏和内毒素血症中的关键作用，从而导致炎症性肝病。概述 暴饮暴食会导致肠道渗漏，导致内毒素血症和炎症性肝损伤增加。我们的研究结果首次证明了肠细胞凋亡和硝化作用以及连接复合蛋白泛素依赖性蛋白水解降解在促进肠道渗漏和内毒素血症中的关键作用。这些结果提供了对酒精诱导的炎症性肝病的分子机制的深入了解。