Objective: To study the expression changes of mitochondrial (mt) DNA encoding ATP6, 8 of hepatocytes in rat liver transplantation with cold preservation-reperfusion and explore the relationship between the expression of F(0)F(1)-ATPase induced by changes of ATP 6, 8 genes.
Methods: Orthotopic liver transplantation was performed in Wistar rats using the cuff technique as described by Kamada with modifications. Rats were randomly divided into four groups: group A (30 min cold preservation), group B (6 h cold preservation), group C (12 h cold preservation) and group D (sham operation). Samples were collected at 12 h, 24 h and on Day 3, 5 and 7 post-operation. The levels of mtDNA encoding ATPase 6, ATPase 8 mRNA were determined by RT-PCR. The expression changes of F(0)F(1)-ATPase protein were examined by Western blot. The ATP level of liver was observed in each group.
Results: The expression of ATPase 6, ATPase8 mRNA, F(0)F(1)-ATPase protein expression (ng/mg protein) and the level of ATP decreased (U/L) in groups A, B and C at 12 h post-operation. The lowest was in group C (0.81 +/- 0.08, 0.71 +/- 0.07, 0.47 +/- 0.07, 0.44 +/- 0.05) when compared with groups A (1.11 +/- 0.04, 1.07 +/- 0.07, 0.86 +/- 0.15, 0.70 +/- 0.11) and B (1.02 +/- 0.07, 0.90 +/- 0.10, 0.65 +/- 0.07, 0.55 +/- 0.08) (P < 0.05). The expression of ATPase 6, ATPase 8 mRNA increased after 24 h in groups A, B and C. The changes of F(0)F(1)-ATPase protein expression and ATP level were consistent with the expression of ATPase 6, ATPase 8 mRNA. Both ATPase 6 mRNA, ATPase 8 mRNA and F(0)F(1)-ATPase protein in group C increased slowly compared with groups A and B after 24 h. The mtDNA encoding ATPase 6, ATPase 8 changes of hepatocytes in rats liver transplantation with cold preservation-reperfusion suggested that mitochondrial function disorders are related to the changes of mitochondrial DNA encoding ATPase6, ATPase8. mtDNA encoding ATPase 6, ATPase 8 genes of hepatocytes decreased significantly and F(0)F(1)-ATPase protein expression decreased remarkably and resulted in the decrease of ATP levels. The energy deficiency led to liver injury during cold preservation-reperfusion in rats liver transplantation. The injury of liver was more obvious with the prolongation of cold preservation period.
Conclusion: The abnormal expressions of mtDNA ATPase 6, ATPase 8 genes are important causes of the changes of F(0)F(1)-ATPase protein expression resulting in a disorder of mitochondrial energy metabolism.