Study On Changs Of Mitochondrial DNA ATP6, 8 Gene Expression And Function Of Hepatocytes With Cold Preservation-refusion In Rats Liver Transplantation

Backgroud:Liver transplantation is the only effective therapy for end-stage liver diseases,and has been carried out successfully all over the world.However,cold preservation-reperfusion remains one of the most important factors in determining the effect of liver transplantation. Cold preservation-reperfusion is closely related to primary nonfunction(PNF) and delayed function recovery of the graft.Our experiments aimed to study the changes and regulatory mechanisms of mt DNA encoding ATP6,8 of hepatocytes after liver transplantation in rats.Objective:(1) To study the changes o f the expression of mt DNA encoding ATP6,8 of hepatocytes in rat liver transplantation with cold preservation-refusion,and explore the relationship between the expression of F₀F₁-ATPase and function induced by changes of ATP6,8 genes. (2)To investigate the mechanism of the energy metabolism disorder of mitochondria and the cellular damages induced by cold preservation-refusion in hepatocytes.(3) To observe the effects of NRF-1 and mtTFA on the expression of ATP6,8 genes and study the molecular mechanism of NRF-1 and mtTFA on repair of mitochondria DNA injury.Methods:Orthotopic liver transplantations were performed in wistar rats using the cuff technique as described by Kamada with modifications.Rats were randomly divided into four groups namely:group A(cold preservation of 30 min),group B(cold preservation of 6 hours),group C(cold preservation of 12 hours)-and group D(sham operation).Samples were collected at 12 h,24 h and on 3^st,5^th,7^th days after operation.The pathological changes of liver tissue,liver function,SOD,ATPase,ATP of liver and mitochondrial morphology were observed in each group.The changes of the expression of NFR-1,mtTFA and mtDNA encoding ATPase6,ATPase8 mRNA were determined by RT-PCR.The changes of the expression of F₀F₁-ATPase protein was examined by Western-blot. Results:(1)The pathological changes of liver tissue and liver function were more severe in group C than in groups A and B.SOD,ATPase,ATP of liver and mitochondrial morphology in group C were significantly lower than in groups A and B(p＜0.05).The liver function gradually recovered in groups B and C,but SOD,ATPase and ATP of liver in group C were increased gradually on day 7 after operation.(2) The expression of ATPase6,ATPase8 mRNA were decreased in groups A,B and C on 12h after operation.ATPase6,ATPase8 mRNA were the lowest in group C when compared with groups A and B(p＜0.05).The expression of ATPase6,ATPase8 mRNA increased after 24h in groups A,B and C.The changes of F₀F₁-ATPase protein expression were consistent with the expression of ATPase6,ATPase8 mRNA.Both ATPase6,8 mRNA and F₀F₁-ATPase protein in group C increased slowly compared with groups A and B after 24h.(3) The expression of mtTFA and NRF-1mRNA were decreased in groups A,B and C at 12h after operation,and mtTFA and NRF-1 mRNA were the lowest in group C(p＜0.05).The expression of mtTFA and NRF-1 mRNA were consistent with the expression of ATPase6,ATPase8 mRNA after 12h.Conclusions:(1) Our studies showed that there were mtDNA encoding ATPase6,ATPase8 changes of hepatocytes in rats liver transplantation with cold preservation-refusion,suggesting that mitochondrial function disorders are related to changdes of mitochondrial DNA encoding ATPase6,ATPase8.(2)The RT-PCR and Western-blot results showed that the expression of mtDNA encoding ATPase6,ATPase8 genes of hepatocytes decreased significantly,and F₀F₁-ATPase protein expression was decreased remarkably,which lead to the ATP levels decreased.The energy deficiency lead to liver injury druing cold preservation-refusion in rats liver transplantation.The injury of liver is more obvious with the prolongation of the cold preservation period.The results suggested that the abnormal expression of mtDNA ATPase6,ATPase8 genes were important causes of disorder of mitochondrial energy metabolism.(3) mtTFA and NRF-1 increased the expression of ATPase6,ATPase8 mRNA, suggest which that of mtTFA and NRF-1 may be important factors in controling the expression of ATPase6,ATPase8 mRNA transcription.