Background & Aims
Binge alcohol 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 adherent 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 ZO-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.
Layman’s Summary
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 binge alcohol-induced gut leakiness and endotoxemia, contributing to inflammatory liver disease.
Keywords : Cytochrome P450-2E1 (CYP2E1); ethanol; gut leakiness; nitroxidative stress; apoptosis; protein nitration; ubiquitin-dependent proteolysis; tight and adherent
junction proteins.