gms | German Medical Science

Objectives: Ethanol (EtOH) and its primary metabolite acetaldehyde (MeCHO) are directly impairing the physiological functions of the liver, inducing mutagenesis and leading to the tissue injury due to their high cellular toxicity. Ethanol intoxication is frequently a common background for accidental physical trauma injuries, which prevents the proper medical treatment and the trauma patient management, profounding the systemic inflammation and physical harm even further, with organ and notably liver malfunctions. Hepatic inflammation roots itself from the upregulated activity of the transcription factor nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NF-kappaB), which responses to the both trauma-related and alcohol-related stimuli. A20 protein (TNFAIP3) inhibits NF-kappaB activity by sequestrating its components inactive in the cytoplasm. This study aimed to characterize the impact of A20 on alcohol-induced NF-kappaB-driven inflammatory response in the liver.

Methods: Liver tissue was taken from a mouse double-hit model (hemorrhagic shock, femoral fracture) with previous alcohol intoxication at 24 hours post-trauma. Immortalized hepatic cells (HepG2) were treated with either EtOH or MeCHO in a time- and dose-dependent manner. Inflammatory changes, apoptosis, secretory capacity for pro-inflammatory cytokines, DAMP-receptor expression by flow cytometry as well as adhesion of isolated neutrophils from healthy volunteers to pretreated HepG2 were elaborated. A20 protein expression, localization and NF-kappaB regulatory activity were further elaborated by immunocytochemistry/immunofluorescence (ICC/IF) and western blot (WB).

Results and Conclusion: A20 protein expression decreased significantly after 100 mM EtOH treatment for 24 hours and even more decreased expression was observed after 250 mM EtOH treatment for 24 hours. The MeCHO study (5 mM and 15 mM, respectively) for the same incubation period yielded almost no expression of A20 in neither concentration in hepatic cells. DAMP-receptor Toll-like receptor (TLR)4 and intercellular adhesion molecule (ICAM)1, which both act as the response indicators to the inflammation, increased significantly upon EtOH treatment, going along with the enhanced NF-kappaB activation in the liver. In addition, specified protein analyses uncovered three distinguished populations of A20 - full length protein, 90 kDa, and two cleaved polypeptides, 50 and 37 kDa, which corresponds to A20 both catalytic and structural domains.

In conclusion, an evaluation of the molecular correlation between the alcohol-based liver damage, along with the on-going tissue inflammation and the activity levels of ubiquitin-editing A20 enzyme, which moderates the NF-kappaB pro-inflammatory pathway was shown.