LAUSR

Acetaminophen (APAP) overdose‐induced acute liver injury (ALI) causes hepatocyte cell death, oxidative stress, and inflammation. Oridonin (Ori), a covalent NLRP3‐inflammasome inhibitor, ameliorates APAP‐induced ALI through an unclear molecular mechanism. This study found that Ori decreased hepatic cytochrome P450 2E1 level and increased glutathione content to prevent APAP metabolism, and then reduced the necrotic area, improved liver function, and inhibited APAP‐induced proinflammatory cytokines and oxidative stress. Ori also decreased activating transcription factor 4 (ATF4) protein levels and increased peroxisome proliferator‐activated receptor‐γ coactivator‐1α (PGC‐1α) to reduce APAP‐induced endoplasmic reticulum stress activation and mitochondrial dysfunction. Furthermore, western blot and luciferase assay found that ATF4 inhibited transcription in the PGC‐1α promoter −507 to −495 region to reduce PGC‐1α levels, while ATF4 knockdown neutralized the hepatoprotective effect of Ori. Molecular docking showed that Ori bound to ATF4's amino acid residue glutamate 302 through 6, 7, and 18 hydroxyl bands. Our findings demonstrated that Ori prevented metabolic activation of APAP and further inhibited the ATF4/PGC‐1α pathway to alleviate APAP overdose‐induced hepatic toxicity, which illuminated its potential therapeutic effects on ALI.