OBJECTIVE: To study the role of tumor necrosis factor-alpha (TNFalpha) and caspase-3 expression on hepatocyte apoptosis in experimental model of fulminant hepatic failure (FHF). METHODS: Mouse experimental model of FHF was induced by lipopolysaccharide (LPS) and D-galactosamine (D-GalN). Serum TNFalpha level and TNFalpha mRNA expression in liver were tested by ELISA and reverse transcriptase PCR (RT-PCR) method, respectively. The expression of caspase-3 in liver tissue was determined by in situ hybridization. Hepatocyte apoptosis was examined by DNA agarose gel electrophoresis and TUNEL method. TNFalpha, caspase-3 and hepatocyte apoptosis were observed in different stages after drug administration. In addition, we observed the changes of above variables after pretreatment with anti-TNFalpha IgG1. RESULTS: TNFalpha mRNA expression in liver increased significantly (0.91 +/- 0.75, that of normal control was 0.32 +/- 0.10) 2 to 4 hours after administration of LPS and D-GalN. The level of serum TNFalpha increased [(320.50 +/- 86.57) ng/L; that of normal control was (16.66 +/- 7.01) ng/L] and caspase-3 expressed to a slight extent, too. Typical manifestation of hepatocyte apoptosis appeared 8h after drug administration. 8 hours after drug administration the level of serum ALT and total bilirubin (TBil) remarkably increased [(560.66 +/- 60.20) U/L and (163.66 +/- 34.51) micro mol/L, respectively, that of normal control was (23.56 +/- 8.03) U/L and (14.90 +/- 4.80) micro mol/L, respectively] and the expression of caspase-3 reached the peak. 12 hours after drug administration, hepatocyte apoptosis and necrosis coexisted, and the level of serum ALT and TBil reached a peak [(668.30 +/- 78.69) U/L and (203.17 +/- 19.92) micro mol/L, respectively] whereas the expression of caspase-3 already decreased. Hepatocyte apoptosis and liver injury and the expression of caspase-3 could be blocked by antagonizing TNFalpha. CONCLUSIONS: TNFalpha played an important role in hepatocyte apoptosis and liver injury in fulminant hepatic failure. The hepatocyte apoptosis induced by TNFalpha is correlated with the activation of caspase-3. Hepatocyte apoptosis occurs earlier than necrosis.
OBJECTIVE: To study the role of tumor necrosis factor-alpha (TNFalpha) and caspase-3 expression on hepatocyte apoptosis in experimental model of fulminant hepatic failure (FHF). METHODS:Mouse experimental model of FHF was induced by lipopolysaccharide (LPS) and D-galactosamine (D-GalN). Serum TNFalpha level and TNFalpha mRNA expression in liver were tested by ELISA and reverse transcriptase PCR (RT-PCR) method, respectively. The expression of caspase-3 in liver tissue was determined by in situ hybridization. Hepatocyte apoptosis was examined by DNA agarose gel electrophoresis and TUNEL method. TNFalpha, caspase-3 and hepatocyte apoptosis were observed in different stages after drug administration. In addition, we observed the changes of above variables after pretreatment with anti-TNFalpha IgG1. RESULTS:TNFalpha mRNA expression in liver increased significantly (0.91 +/- 0.75, that of normal control was 0.32 +/- 0.10) 2 to 4 hours after administration of LPS and D-GalN. The level of serum TNFalpha increased [(320.50 +/- 86.57) ng/L; that of normal control was (16.66 +/- 7.01) ng/L] and caspase-3 expressed to a slight extent, too. Typical manifestation of hepatocyte apoptosis appeared 8h after drug administration. 8 hours after drug administration the level of serum ALT and total bilirubin (TBil) remarkably increased [(560.66 +/- 60.20) U/L and (163.66 +/- 34.51) micro mol/L, respectively, that of normal control was (23.56 +/- 8.03) U/L and (14.90 +/- 4.80) micro mol/L, respectively] and the expression of caspase-3 reached the peak. 12 hours after drug administration, hepatocyte apoptosis and necrosis coexisted, and the level of serum ALT and TBil reached a peak [(668.30 +/- 78.69) U/L and (203.17 +/- 19.92) micro mol/L, respectively] whereas the expression of caspase-3 already decreased. Hepatocyte apoptosis and liver injury and the expression of caspase-3 could be blocked by antagonizing TNFalpha. CONCLUSIONS:TNFalpha played an important role in hepatocyte apoptosis and liver injury in fulminant hepatic failure. The hepatocyte apoptosis induced by TNFalpha is correlated with the activation of caspase-3. Hepatocyte apoptosis occurs earlier than necrosis.