Ethanol and high cholesterol diet causes severe steatohepatitis and early liver fibrosis in mice

Background and Aim: Because ethanol consumption is commonly associated with a high cholesterol diet, we examined whether combined consumption of ethanol and high cholesterol increases liver injury and fibrosis. Methods: Male C57BL/6J mice were fed diets containing: 1) 35% of calories from corn oil (CTR), 2) CTR plus 0.5% (w/v) cholesterol (Chol), 3) CTR plus ethanol (27% of calories) (EtOH), or 4) EtOH+Chol for 3 months. Results: In mice fed Chol or EtOH alone, ALT increased to ∼160 U/L, moderate hepatic steatosis occurred, and leukocyte infiltration, necrosis, and apoptosis increased modestly, but no observable fibrosis developed. By contrast in mice fed EtOH+Chol, ALT increased to ∼270 U/L, steatosis was more extensive and mostly macrovesicular, and expression of proinflammatory molecules (HMGB-1, TLR4, TNFα, ICAM-1) and leukocyte infiltration increased substantially. Necrosis and apoptosis also increased. Trichrome staining and second harmonic generation microscopy revealed hepatic fibrosis. Fibrosis was mostly sinusoidal and/or perivenular, but in some mice bridging fibrosis occurred. Expression of smooth muscle α-actin and TGF-β1 increased slightly by Chol, moderately by EtOH, and markedly by EtOH+Chol. TGF-β pseudoreceptor BAMBI increased slightly by Chol, remained unchanged by EtOH and decreased by EtOH+Chol. MicroRNA-33a, which enhances TGF-β fibrotic effects, and phospho-Smad2/3, the down-stream signal of TGF-β, also increased more greatly by EtOH+Chol than Chol or EtOH. Metalloproteinase-2 and -9 were decreased only by EtOH+Chol. Conclusion: High dietary cholesterol and chronic ethanol consumption synergistically increase liver injury, inflammation, and profibrotic responses and suppress antifibrotic responses, leading to severe steatohepatitis and early fibrosis in mice.