Alpinetin pretreatment prevents lipopolysaccharide/D-galactosamine-induced acute liver injury in mice by inhibiting ferroptosis via the Nrf2/SLC7A11/GPX4 pathway

This study aims to investigate whether alpinetin prevents lipopolysaccharide/D-galactosamine (LPS/D-GalN)-induced acute liver injury (ALI) in mice by inhibiting ferroptosis, and to explore the potential molecular mechanisms involved. Thirty-six male BALB/c mice were randomly assigned to one of six groups (n = 6): the control group, the LPS/D-GalN group, the Ferrostatin-1 (Fer-1) + LPS/D-GalN group, and the alpinetin (12.5, 25, or 50 mg/kg) + LPS/D-GalN groups.The ferroptosis-specific inhibitor Fer-1 was used as a positive control to verify the involvement of ferroptosis in the injury. Following intraperitoneal (i.p.) administration of alpinetin (12.5, 25, or 50 mg/kg) for three consecutive days, acute liver injury was induced by an i.p. injection of LPS (30 µg/kg) and D-GalN (600 mg/kg). The effect of alpinetin on ALI was detected by serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and hematoxylin and eosin (H&E) staining of hepatic tissue. Ferroptosis levels were analyzed by malondialdehyde (MDA), reduced glutathione (GSH), and Fe²⁺. In addition, the expression levels of ferroptosis-related proteins were determined by western blot and quantitative real-time PCR (qRT-PCR). The results show that alpinetin inhibited the increase of serum ALT and AST levels and hepatic tissue pathological damage induced by LPS/D-GalN. Alpinetin also inhibited ferroptosis by reducing MDA and Fe²⁺ levels and enhancing GSH activity. Simultaneously, alpinetin regulates iron metabolism-related proteins by inhibiting the expression of transferrin receptor 1 (TFR1) and divalent metal transporter 1 (DMT1) while promoting the expression of ferritin heavy chain (FTH). Additionally, alpinetin increased the expression of the ferroptosis-related protein nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream target genes solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4). Alpinetin protects against LPS/D-GalN-induced ALI by inhibiting the ferroptosis pathway, and its protective effect may depend on the activation of the Nrf2/SLC7A11/GPX4 axis.