pink1-prkn PARK2通路与线粒体自噬和肝脏急性缺血再灌注损伤的关联研究

Association of pink1-prkn PARK2 pathway with mitochondrial autophagy and acute hepatic ischemia-reperfusion injury

目的探讨pink1-prkn PARK2通路与线粒体自噬和肝脏急性缺血再灌注损伤的关联。方法选择C57BL/6小鼠,将Pink1-KO小鼠与Park2-KO小鼠杂交以产生Pink1Park2double-KO小鼠,制作肝脏急性缺血再灌注损伤模型。取肝脏组织行SP3的免疫组织化学染色。用质粒DNA或RNAi瞬时转染HK-2细胞;HK-2细胞在含20mM CCCP的无糖RKRB缓冲液诱导ATP耗竭,模拟再灌注;进行细胞凋亡分析;分离线粒体,测量线粒体DNA(mtDNA)的相对含量;测量HK-2细胞中的线粒体ROS;免疫印迹测定蛋白质浓度。结果缺血性急性肝脏损伤体外和体内模型的肝脏近端肝脏细胞中可诱导线粒体自噬。Pink1和Park2的缺失可消除这种条件下的线粒体自噬,证明PINK1-PARK2途径在肝脏细胞线粒体自噬中的关键作用。pink1和park2单基因或双基因敲除的小鼠中,缺血性急性肝脏损伤均加剧。Pink1和Park2缺失会增加线粒体损伤,活性氧产生和炎症反应。结论 PINK1-PARK2介导的线粒体自噬对急性肝脏损伤期间的线粒体质量控制,肝脏细胞存活和肝脏功能起重要作用。

Objective To assess the association of pink1-prkn PARK2 pathway with mitochondrial autophagy and acute hepatic ischemia-reperfusion injury.Methods C57 BL/6 mice were selected and the Pink1-KO mice were hybridized with Park2-KO mice to produce Pink1 Park2 double-KO mice,and the acute ischemia-reperfusion injury of the liver was made.Liver tissues were taken for immunohistochemical staining of SP3.HK-2 cells were transfected instantaneously with plasmid DNA or RNAi;HK-2 cells induced the depletion of ATP with 20 mM CCCP free RKRB buffer,simulated reperfusion,performed apoptosis analysis,isolated mitochondria,measured the relative content of mitochondrial DNA（mtDNA）,measured the mitochondrial ROS in HK-2 cells,and measured protein concentration by Western blot.Results In vitro and in vivo models of ischemic acute liver injury can induce autophagy in the liver proximal liver cells.The deletion of Pink1 and Park2 can eliminate mitochondrial autophagy under this condition,demonstrating the key role of PINK1-PARK2 pathway in mitochondrial autophagy of liver cells.In addition,ischemic and acute liver injury was aggravated in PINK1 and PARK2 monogenic or double knockout mice.From the point of view of mechanism,Pink1 and Park2 deficiency increase mitochondrial damage,reactive oxygen species production and inflammation.ConclusionPINK1-PARK2 mediated mitochondrial autophagy plays an important role in mitochondrial quality control,liver cell survival and liver function during acute liver injury.