Itaconate facilitates methane-induced Nrf2 pathway activation for mitigating liver ischemia and reperfusion injury.

Background and aims: Methane has shown protective effects against ischemia and reperfusion injury (IRI) in the liver, but the mechanism underlying these beneficial effects is unclear. To investigate the hypothesis that itaconate facilitates in methane-induced Nrf2 pathway activation to mitigate liver IRI. Methods: An oxygen and glucose derivation (OGD) model using RAW 264.7 cells and a liver IRI model in mice were established. Methane's beneficial effects were assessed through hematoxylin and eosin (HE) staining, Suzuki's score, serum alanine transferase level, superoxide dismutase (SOD) level, malondialdehyde (MDA) level, and cell viability. The relative expression levels of Nrf2, its downstream molecules and some inflammatory factors were detected via western blotting. Itaconate levels were analyzed using liquid chromatography. RAW 264.7 cells were transfected with short hairpin RNA targeting mouse aconitate decarboxylase 1 (Acod1) mRNA for itaconate downregulation. Results: Methane significantly alleviated liver IRI, as shown by the significant reduction in Suzuki's scores and alanine transferase (ALT) levels in vivo. Methane treatment significantly increased MTT and SOD levels and decreased MDA levels in the OGD injury model in vitro. Methane also increased the total and nuclear Nrf2 expression levels, activated downstream molecules including heme oxygenase-1 (HO-1), NQO1 and affected the production of inflammatory cytokines such as IL-10, IL-1β, and IL-12. Itaconate levels were significantly elevated after methane treatment compared with the OGD injury group. The protective effects of methane were abolished after itaconate downregulation through Conclusions: Methane alleviates liver IRI through itaconate/Nrf2 pathway activation, with itaconate being critical for methane's beneficial effects.