OBJECTIVE To investigate the hepato-protective mechanism of thymoquinone(TQ) on the development of acetaminophen(APAP)-induced liver injury.METHODS In vivo,male kunming mice were injected with a single dose of 300 mg·kg~(-1) APAP.Some mice were pretreated with TQ(5 or 20 mg·kg~(-1))and N-acetylcysteine(NAC,300 mg·kg~(-1))2 h before APAP injection.Mice were euthanized at 2 h,6 h,12 h after APAP treatment.In vitro,human Chang liver cells were incubated with 3.125,6.25 or 12.5μmol·L~(-1) TQ,10μmol·L~(-1) SP600125 and 500μmol·L~(-1) AICAR in the presence of APAP for 24 h.Cell viability were analyzed by MTT assay,protein expressions were assessed by Western blot.RESULTS TQ pretreatment significantly reduced serum aminotransferase and increased hepatic glutathione(GSH)and glutathione peroxidase(GSH-PX)activities,while significantly inhibited interleukin-1β(IL~(-1)β)levels.TQ significantly inhibited c-Jun N-terminal kinase(JNK),extracellular signal regulated kinase(ERK)and P38 phosphorylation induced by APAP.Moreover,TQ inhibited phosphatidylinositol 3-kinase(PI3K)/mammalian target of rapamycin(m TOR)signaling activation and activated AMPK phosphorylation induced by APAP.In addition,TQ inhibited signal transducer and activator of transcription 3(STAT3)phosphorylation on APAP-induced liver injury.In vitro,APAP enhanced JNK phosphorylation and attenuated AMPK phosphorylation in Chang liver cel s,and these effects were blocked by pretreatment with TQ,SP600125(JNK inhibitor)and AICAR(AMPK activator).CONCLUSION Our findings suggest that TQ may actively prevent APAP-induced liver injury,and this effect may be mediated by JNK and AMPK signaling pathways. OBJECTIVE To investigate the hepato-protective mechanism of thymoquinone (TQ) on the development of acetaminophen (APAP) -induced liver injury. METHODS In vivo, male kunming mice were injected with a single dose of 300 mg · kg -1 APAP .Some mice were pretreated with TQ (5 or 20 mg · kg -1) and N-acetylcysteine ​​(NAC, 300 mg · kg -1) for 2 h before APAP injection.Mice were euthanized at 2 h, 6 h and 12 h after APAP treatment. In vitro, human Chang liver cells were incubated with 3.125, 6.25 or 12.5 μmol·L -1 TQ, 10 μmol·L -1 SP600125 and 500 μmol·L -1, 1) AICAR in the presence of APAP for 24 h. Cell viability were analyzed by MTT assay, protein expressions were assessed by Western blot .RESULTS TQ pretreatment significantly reduced serum aminotransferase and increased hepatic glutathione (GSH) and glutathione peroxidase (GSH-PX) activities, while significantly reduced interleukin-1β (IL-1β) levels.TQ-induced c-Jun N-terminal kinase (JNK), extracellular signal regulated kinase (ERK) Phorylation induced by APAP. Moreover, TQ inhibited phosphatidylinositol 3-kinase (PI3K) / mammalian target of rapamycin (mTOR) signaling activation and activated AMPK phosphorylation induced by APAP. In addition, TQ inhibited signal transducer and activator of transcription 3 (STAT3) phosphorylation on APAP-induced liver injury. In vitro, APAP enhanced JNK phosphorylation and attenuated AMPK phosphorylation in Chang liver cel s, and these effects were blocked by pretreatment with TQ, SP600125 (JNK inhibitor) and AICAR (AMPK activator) .CONCLUSION Our findings suggest that TQ may actively prevent APAP-induced liver injury, and this effect may be mediated by JNK and AMPK signaling pathways.