Chronic aniline exposure causes prominent hepatotoxicity accompanied by hepatic lipid metabolic disorders and cellular senescence, yet the molecular cascade underlying aniline-triggered liver injury remains unclear. Here, a 90-day chronic aniline intoxication mouse model and an AML12 murine hepatocyte model were established to systematically dissect toxic mechanisms and identify candidate hepatoprotective agents. Adverse outcome pathway analysis revealed lipid metabolic disturbance and cellular senescence as core toxic events, with lipid metabolic disturbance acting as an upstream driver of aniline-induced hepatocyte senescence and injury. Integrated multi-omics analysis showed that acyl-CoA thioesterase 2 (ACOT2), a key rate-limiting enzyme in fatty acid β-oxidation, was markedly downregulated in aniline-damaged livers. Hepatic ACOT2 overexpression restored mitochondrial fatty acid oxidation capacity, alleviated lipid overload, excessive ROS accumulation and hepatic dysfunction, and reduced bis(monoacylglycero)phosphate (BMP) accumulation by suppressing neuronal ceroid lipofuscinosis protein 5 (CLN5) expression. Mechanistically, ACOT2 deficiency caused ROS overproduction and activated JAK2/STAT3 signaling. Phosphorylated STAT3 translocated into the nucleus and directly bound the Cln5 promoter, enhancing its transcription. Elevated CLN5 disrupted lysosomal structural integrity and acidification, thereby blocking lipid degradation during lipophagy. Conversely, Cln5 silencing restored lysosomal homeostasis and autophagic flux and reduced hepatic lipid deposition. Virtual screening identified dioscin as a natural small molecule that directly interacts with ACOT2, as confirmed by molecular docking, molecular dynamics simulation and surface plasmon resonance assays. PPARα-dependent transcriptional regulation contributed to ACOT2 suppression across multiple liver injury models, whereas dioscin restored ACOT2-dependent fatty acid oxidation and conferred broad hepatoprotection against aniline-, CCl4-, and APAP-induced liver injury. Collectively, this study reveals, in male mice and murine hepatocyte models, a previously unreported ACOT2-regulated ROS-JAK2/STAT3-CLN5 axis linking impaired mitochondrial fatty acid oxidation, lysosomal dysfunction and defective lipid clearance during aniline hepatotoxicity. These findings further support dioscin as a promising hepatoprotective candidate drug that directly targets ACOT2.
