Isoschaftoside protects against acetaminophen-induced acute liver injury by suppressing TLR4/NF-κB pathway activation

Acetaminophen (APAP) overdose is a leading cause of acute liver injury, yet effective protective therapies remain limited. Isoschaftoside (Iso) is a flavonoid phytochemical with reported antioxidant and anti-inflammatory activities, but its role in APAP-induced liver injury remains unclear. Here, we evaluated the hepatoprotective effect and mechanism of Iso in vivo and in vitro. BALB/c mice received APAP (300 mg/kg, i.g.) to induce acute liver injury, followed 1.5 h later by Iso (8 mg/kg, i.p.); samples were collected at 6 h. Liver pathology was examined by hematoxylin-eosin (H&E) staining; serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (AKP) were measured using commercial assay kits; and cytokines were quantified by enzyme-linked immunosorbent assay (ELISA). Signaling-related mRNA and protein levels were assessed by quantitative real-time PCR (RT-qPCR) and western blot. Network pharmacology and molecular docking suggested involvement of toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) signaling, which was further tested in AML12 hepatocytes. Iso markedly alleviated APAP-induced histological damage and reduced serum liver enzymes and pro-inflammatory cytokines. In AML12 cells, Iso suppressed APAP-triggered TLR4/NF-κB activation and apoptosis. These findings indicate that Iso mitigates APAP-induced acute liver injury largely by inhibiting TLR4/NF-κB-mediated inflammatory and apoptotic responses, supporting its potential as a therapeutic candidate.