The PI3K/Akt/FOXO1 signaling axis mediates acetaminophen hepatotoxicity by disrupting metabolic homeostasis and oxidative stress.

Acetaminophen (APAP) is a widely used antipyretic-analgesic drug. At high or cumulative doses, it can induce serious liver injury, though the full spectrum of its hepatotoxic mechanisms remains unclear. This study comprehensively explored its pathogenic mechanisms by integrating network toxicology, molecular docking, in vivo and in vitro validations. Multi-source database screening identified 27 cross-targets associated with APAP hepatotoxicity. Protein-protein interaction (PPI) network and topological analyses revealed core targets central to APAP-induced liver injury. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses indicated that APAP-triggered hepatotoxicity involves critical biological processes, including cell cycle dysregulation, genetic damage, and disrupted signal transduction, with significant correlations to lipid and glucose metabolism, oxidative stress, and the PI3K/Akt/FOXO1 axis. Molecular docking confirmed strong binding affinity between APAP and core targets. Systematic in vivo and in vitro validation demonstrated that APAP inhibits PI3K/Akt signaling, thereby activating FOXO1, leading to disorders in glycolipid metabolism and intensified oxidative stress, ultimately aggravating hepatocyte damage. This mechanism plays an important regulatory role in the late-stage of APAP-induced hepatotoxicity, operating in parallel with the existing JNK mechanism. These findings provide new insights into the integrated mechanistic model of APAP-induced liver injury and suggest potential therapeutic targets.