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Objectives and questions: Alcohol intoxication is common in polytrauma patients and influences post-traumatic inflammatory responses, yet the underlying mechanisms remain incompletely understood. Given the central role of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in trauma-induced inflammation and its negative regulation by A20 (TNFAIP3), this study aimed to investigate whether acute alcohol exposure modulates hepatic inflammatory responses following simulated polytrauma and to assess the role of A20 in this process.

Material and methods: An in vitro polytrauma model was established using human HepG2 hepatocytes, including wild-type (WT) cells and A20 knockout (A20 KO) cells. Cells were exposed to acute ethanol at low or high concentrations (50 or 200 mM) before stimulation with a defined polytrauma cocktail containing pro-inflammatory cytokines and complement factors to simulate polytrauma conditions. Inflammatory responses were assessed by quantitative PCR, multiplex cytokine analysis, flow cytometry of ICAM-1 expression, and functional neutrophil adhesion assays. Cell viability, apoptosis-related gene expression, and ethanol metabolism were additionally evaluated. Statistical analysis was performed using two-way ANOVA followed by Tukey’s post hoc test, with p < 0.05 considered statistically significant.

Results: Ethanol exposure and polytrauma stimulation did not affect hepatocyte viability or apoptosis-related gene expression. Polytrauma stimulation induced pronounced TNF-α and IL-6 expression and release, which were significantly amplified in A20 KO cells, indicating dysregulated NF-κB-dependent inflammation. Ethanol pre-exposure showed a tendency to attenuate polytrauma-induced inflammatory responses in WT hepatocytes but not in A20 KO cells. Chemokine expression, ICAM-1 surface expression, and neutrophil adhesion were increased following polytrauma stimulation in WT HepG2 cells, whereas these responses were reduced in the absence of A20.

Discussion and conclusions: Acute ethanol exposure modulates hepatocellular inflammatory responses following simulated polytrauma without inducing hepatocellular cytotoxicity. The anti-inflammatory effects of acute alcohol exposure appear to be mediated, at least partially, by A20, suggesting a functional interaction between alcohol and A20 in regulating NF-κB signaling and inflammatory responses. These findings provide mechanistic insight into the role of alcohol pre-exposure in modulating post-traumatic inflammation and identify A20 as a potential therapeutic target for hepatic and systemic inflammation following polytrauma.