Azithromycin induces liver injury in mice by targeting the AMPK/Nrf2 pathway

Azithromycin is an antibacterial and anti-inflammatory drug widely used for the treatment of various diseases, including those caused by atypical pathogens, bacterial or viral infections, chronic sinusitis, and bronchial asthma, particularly in pediatric patients. However, concerns have emerged regarding its hepatotoxicity and its precise mechanism of action remains unclear. To investigate the molecular mechanisms responsible for azithromycin-induced acute liver injury to advance our understanding of the progression and pathogenesis of antibiotic-induced liver damage, and to improve prevention and treatment strategies. C57BL/6 mice, Nrf2-/- mice, and primary hepatocytes were used. Primary hepatocytes from mice were isolated using a two-step perfusion method and cultured in vitro via the ‘sandwich’ culture model. The exposure to azithromycin resulted in increased apoptosis and reactive oxygen species (ROS) levels. In mouse models, intraperitoneal administration of azithromycin at varying concentrations and time points substantially induced hepatic disarray, swelling, and dysfunction. Azithromycin markedly upregulated the mRNA and protein levels of phosphorylated adenosine-activated protein kinase (AMPK) while downregulating nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), and NADPH: quinone oxidoreductase 1 (NQO-1). Moreover, HO-1 and NQO-1 protein levels remained largely unaffected in primary hepatocytes co-cultured with azithromycin in Nrf2-/- mice. Our findings suggest that azithromycin-induced acute liver injury is mediated by suppression of Nrf2 activation and ROS production. This sheds light on the potential mechanisms involved in azithromycin-induced liver damage, underscoring the importance of exploring targeted interventions to mitigate the hepatotoxic effects.