A novel in vitro model for joint effects of alcohol and free fatty acids on hepatocellular steatosis and inflammation

Alcoholic steatohepatitis (ASH) and nonalcoholic steatohepatitis (NASH) are the most frequent conditions leading to elevated liver enzymes and liver cirrhosis, respectively, in the Western world. However, despite strong epidemiological evidence for combined effects on the progression of liver injury, the mutual interaction of the pathophysiological mechanisms is incompletely understood.

The aim of this study was to establish an in vitro model for joint effects of alcohol and lipids on hepatic steatosis and inflammation.

Methods and Results: Initially, we established the dose range in which neither alcohol nor incubation with the free fatty acid oleat affected viability or mitochondrial activity in HepG2 hepatoma cells and primary human hepatocytes (PHH). Subsequently, we assessed the combined effect of alcohol (1–2%) and oleat (0.2mM) on hepatocellular lipid accumulation in HepG2 cells and PHH. Under these conditions, alcohol significantly enhanced oleat induced cellular triglyceride content and free fatty acid (FFA) levels, while alcohol alone had only a minimal effect on hepatocellular lipid content. Analysis of heme oxygenase-1 (HMOX-1) expression and malondialdehyde levels revealed that the combination of alcohol and oleat caused significantly higher oxidative stress and lipid peroxidation than either of the two substances alone. Further, we observed a synergistic effect of alcohol and FFA on ERK-activation, while ERK-inhibition ameliorated the effect of this combination on pro-inflammatory (IL-8 and ICAM-1) gene expression.

Conclusion: In summary, this new model allows the investigation of isolated or joint effects of alcohol and FFA on hepatocellular lipid metabolisms and inflammatory signaling. Our present findings indicate ERK-activation as critical mediator of a synergistic effect of alcohol and FFA on hepatic inflammation.