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OBJECTIVE To investigate the mechanism of SIRT1/AMPK signaling pathway between hepatocytes and hepatic stellate cells (HSCs). METHODS Normal human Chang liver cells and human hepatic stellate cell line, LX-2 cells were treated with SRT1720 (10 μmol·L-1) and AICAR (500 μmol·L-1) prior to ethanol (50 mmol·L-1) for 24 and 48 h. Cell viability was analyzed by methyl thiazolyl tetrazolium assay. SIRT1, AMPK and p-AMPK mRNA levels for 24 h and 48 h were analyzed by RT-PCR, SIRT1, AMPK and p-AMPK protein expressions in the supernatant at 24 and 48 h was detected by Western blot. RESULTS SRT1720 and AICAR effectively decreased LX-2 cell viabilities and exhibited scarcely little toxicity in human Chang liver cells. SRT1720 and AICAR attenuated collagen-I, α-smooth muscle actin (α-SMA) levels, activated liver kinase B-1 (LKB1) and AMPK phosphorylation in ethanol treated LX-2 cells. Meanwhile, SRT1720 and AICAR enhanced SIRT1 expression mediated by ethanol both in Chang liver cells and LX-2 cells. Furthermore, SRT1720 and AICAR suppressed the expression of sterol regulatory element-binding protein-1 (SREBP-1) to regulate fatty acid synthesis. CONCLUSION SIRT1 agonist and AMPK agonist blocked the crosstalk between hepatocytes and HSCs via SIRT1/AMPK signaling pathway to modulate hepatocytes accumulation of lipid and HSCs activation.
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OBJECTIVE To investigate the hepato-protective mechanism of thymoquinone (TQ) onthe 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 12.5 μmol·L-1 TQ, 10 μmol·L-1 SP600125 and 500 μmol·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 gluta?thione (GSH) and glutathione peroxidase (GSH-PX) activities, while significantly inhibited interleukin-1β(IL-1β) 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 (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 cells, 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.