Itaconate alleviates thioacetamide-induced acute liver injury by suppressing pyroptosis-associated mitochondrial dysfunction and activating the Nrf2/HO-1 pathway

Abstract

Acute liver injury is closely associated with inflammatory responses, oxidative stress, hepatocyte death, and mitochondrial dysfunction, whereas effective pharmacological strategies remain limited. This study aimed to investigate the protective effect of 4-octyl itaconate (4-OI) against thioacetamide (TAA)-induced acute liver injury and to further explore the underlying mechanisms. A TAA-induced acute liver injury mouse model, an LPS-stimulated RAW264.7 macrophage inflammatory model, and an LPS/nigericin-stimulated bone marrow-derived macrophage (BMDM) pyroptosis model were established. Liver injury, inflammatory responses, oxidative stress, hepatocyte apoptosis, pyroptosis, and mitochondrial dysfunction were evaluated using histopathological staining, TUNEL staining, biochemical assays, ELISA, qPCR, Western blotting, LDH release assay, immunofluorescence staining, PI staining, MitoSOX staining, and JC-1 assay. ML385 was used to inhibit nuclear factor erythroid 2-related factor 2 (Nrf2) signaling. 4-OI markedly alleviated TAA-induced liver injury, as shown by improved liver histology, reduced hepatocyte apoptosis and macrophage infiltration, decreased serum transaminase levels, and suppressed inflammatory cytokine production. In parallel, 4-OI activated the Nrf2/heme oxygenase-1 (HO-1) pathway, whereas pharmacological inhibition of Nrf2 by ML385 partially reversed its hepatoprotective and anti-inflammatory effects. Increased hepatic GSDMD-N expression after TAA exposure was also reduced by 4-OI. In LPS/nigericin-stimulated BMDMs, 4-OI decreased NOD-like receptor family pyrin domain-containing 3 (NLRP3), cleaved caspase-1, and GSDMD-N expression, accompanied by reduced LDH release, IL-1β secretion, PI-positive cell death, and mitochondrial reactive oxygen species production, as well as restored mitochondrial membrane potential. Moreover, 4-OI reduced mitochondrial GSDMD-N accumulation and attenuated mtDNA-induced pyroptotic activation. These findings indicate that 4-OI alleviates TAA-induced acute liver injury, at least in part, through activation of the Nrf2/HO-1 signaling pathway and suppression of pyroptotic activation and mitochondrial dysfunction-associated inflammatory amplification.