雷帕霉素对肝脏缺血再灌注中线粒体DNA损伤及修复机制的影响

Effect of rapamycin on mitochondrial DNA damage in rats after liver ischemiareperfusion injury and repair mechanisms

目的探讨大鼠肝缺血再灌注损伤中线粒体DNA的损伤和修复机制及雷帕霉素的干预作用。方法建立大鼠全肝缺血再灌注损伤模型,分为4组:A组:缺血30 min组;B组:缺血50 min组;C组:术前雷帕霉素灌胃,缺血50 min组;D组:假手术组。术后24 h,第3、5天分别处死各组动物,测定血清AST、ALT;取出肝脏作病理学检测;mtDNA D-loop区扩增测序,查找变异位点;RT-PCR检测肝组织内线粒体DNA聚合酶γmRNA的表达情况。结果术后早期(24 h),A、B、C 3组AST、ALT均升高,肝组织病理学变化较重,线粒体DNA聚合酶γmRNA表达量均升高,其中C组AST、ALT及肝组织病理学改变和线粒体DNA聚合酶γmRNA表达量明显低于B组(P<0.01);24 h后3组AST、ALT均降低,A、B 2组线粒体DNA聚合酶γmRNA表达量逐渐降低,C组则较A、B 2组为低(P<0.01),且术后一直维持在同一水平;C组mtDNA D-loop区DNA突变率明显低于B组(P<0.01)。结论肝脏缺血再灌注损伤存在mtDNA D-loop区突变,雷帕霉素可减轻大鼠肝脏缺血再灌注的病理损害,降低mtDNA的突变率,抑制线粒体DNA聚合酶γmRNA表达量的过度升高,从而对线粒体DNA的氧化损伤起保护作用。

Objective To investigate the injury and repair mechanism of mitochondrial DNA damage in rats after hepatic ischemia and reperfusion injury and to determine the intervention effect of rapamycin in the process. Methods Whole hepatic ischemia and reperfusion was performed in the rats according to the following ischemia period. The experimental rats were divided into 4 groups,group A（ ischemia 30 min）,group B（ ischemia 50 min）,group C [intra-gastric injection of 1. 5 mg /（ kg ·d） rapamycin for 3 d before surgery,then ischemia 50 min and rapamycin injection again],and group D（ sham operation）. The rats were sacrificed in 24 h,and 3 and 5 d respectively after reperfusion in each group,and their serum levels of AST and ALT were tested and hepatic pathology was observed after HE staining. The mtDNA D-loop region was them amplified and sequenced to find the mutations. The mRNA expression of the mitochondrial DNA polymerase γwas detected in hepatic tissues by RT-PCR. Results In the early stage after injury（ 24 h）,both serum levels of AST and ALT were increased in groups A,B and C,pathological injury was serious in hepatic tissues,mitochondrial DNA polymerase γ mRNA expression was increased. Among the 3 injured groups,the indexes were significantly lower in group C than group B（ P〈 0.01）. After 24 h,the serum levels of AST and ALT were decreased in the 3 groups,the expression level of mitochondrial DNA polymerase γ was gradually decreased in them,with those in the group C more lower（ P〈 0.01）,and maintaining at the same level after operation. The mutant rate of mtDNA D-loop region was significantly lower in group C than group B（ P〈 0.01）. Conclusion There are DNA mutations of mtDNA D-loop region in hepatic ischemia-reperfusion injury. Rapamycin attenuates the pathological damage induced by ischemia-reperfusion in rat liver,reduces the mtDNA mutation rate,suppresses the increased expression of mitochondrial DNA polymerase γ,and thus exerts a protective effect against oxidative damage.