二甲双胍通过parkin介导的线粒体自噬对糖尿病肾脏足细胞氧化损伤的影响及机制研究

The excessive release of reactive oxygen species triggered by aberrant induction of mitochondrial stress is the leading cause of podocytes oxidative injury under diabetic conditions, which is tightly associated with the genesis and development of diabetic nephropathy. Previous studies have proven the renoprotective effects of metformin, while it is still unclear whether metformin could alleviate mitochondrial stress in podocytes. Preliminary experiments from our group revealed that mitophagy, a process of selectively eliminating damaged mitochondria, is promoted by metformin so as to ameliorate mitochondrial stress in podocytes, possibly through the mechanisms of which metformin up-regulates the expression of parkin. However, the specific mechanisms underlying still need to be explored. Recent studies have proven that metformin could protect cardiac myocytes or hepatocytes from high glucose induced damage by activating PERK/eIF2α/ATF4 pathway; contemporaneous research illustrated that ATF4 directly facilitated the transcription of parkin. Thus, we hypothesize that metformin might mediate mitophagy via activating PERK/ATF4/parkin pathway, thereby ameliorating mitochondrial stress and reducing ROS-induced podocytes injury. Therefore, we intend to choose mouse podocytes and db/db mice as experimental subjects, following by utilizing the techniques of Crisper/Cas9 system combined with lentiviral vector transfection, immuno-electron microscope, immunofluorescent localization by confocal microscope, dual luciferase report gene assay system, chromatin immunoprecipitation, etc., so as to explore the effects and mechanisms of metformin-regulated mitophagy on podocyte injury through the perspective of molecules, cells and animals, which will shed light on novel strategy of preventing diabetic nephropathy.

足细胞损伤与糖尿病肾病的发生发展密切相关，而线粒体应激导致活性氧过度释放，是造成糖尿病足细胞氧化损伤的重要原因。二甲双胍对糖尿病肾脏病变具有保护作用，但其能否改善足细胞线粒体应激水平尚不明确。预实验结果提示，二甲双胍可上调足细胞中parkin表达，促进线粒体自噬发生，改善氧化损伤，但其机制仍需进一步探讨。研究证实，二甲双胍可激活PERK/eIF2α/ATF4通路改善高糖引起的心肌及肝细胞损伤；同时，ATF4可直接上调parkin表达。因此我们提出假设：二甲双胍可能通过激活PERK/ATF4/parkin介导线粒体自噬，调节氧化应激，改善足细胞损伤。为此，本研究拟选取小鼠足细胞、db/db小鼠为对象，采用Crisper/Cas9系统结合慢病毒转染、免疫电镜、共聚焦显微镜荧光定位、荧光素酶报告基因等技术，从细胞、组织及动物水平探讨二甲双胍调节线粒体自噬的具体机制，为糖尿病肾病的防治提供新思路。

足细胞损伤与糖尿病肾病的发生发展密切相关，而线粒体应激导致活性氧过度释放，是造成糖尿病足细胞氧化损伤的重要原因。二甲双胍对糖尿病肾脏病变具有保护作用，但其能否改善足细胞线粒体应激水平尚不明确，因此，本研究旨在揭示二甲双胍调节足细胞线粒体功能的具体机制。本研究首先构建正常糖、高糖及二甲双胍干预足细胞系，证实二甲双胍通过影响线粒体自噬改善足细胞功能，探讨二甲双胍对糖尿病肾脏足细胞内parkin介导的线粒体自噬的调节及机制；进而筛选二甲双胍干预后的足细胞lncRNA表达谱，确定lncRNA SNHG17为调控parkin的关键上游分子；进一步通过证实lncRNA SNHG17可通过影响MST1泛素化降解，抑制parkin介导的足细胞线粒体自噬过程；最后通过构建动物模型，体内论证二甲双胍、lncRNA SNHG17及parkin对足细胞线粒体自噬及线粒体应激的调节。基于以上研究内容，本研究结果如下：①高糖状态下足细胞形态异常，标记蛋白表达降低，线粒体功能受损，二甲双胍能够改善足细胞标记蛋白Nephrin、Podocin表达，维持足细胞线粒体膜电位水平、mtDNA及线粒体内ATP含量，降低足细胞内ROS水平；②高糖抑制parkin表达，从而抑制其介导的线粒体自噬过程，二甲双胍可以有效改善parkin介导的足细胞线粒体自噬；③采用Arraystar LncRNA-V4.0芯片构建正常糖、高糖及高糖+二甲双胍干预足细胞lncRNA表达谱，通过生物信息学手段筛选发现lncRNA SNHG17为parkin上游关键分子；④过表达lncRNA SNHG17抑制线粒体自噬，诱导足细胞凋亡; 干扰lncRNA SNHG17促进线粒体自噬，缓解高糖引起的足细胞凋亡；⑤lncRNA SNHG17通过结合Mst1并调控其蛋白泛素化水平，进而调控其下游parkin表达，影响线粒体自噬及细胞凋亡；⑥体内研究应用尾静脉注射重组腺相关病毒进行体内转染构建lncRNA SNHG17过表达小鼠，证实上述生物学调节过程。本课题研究运用多种先进技术，从细胞、组织及动物水平探讨二甲双胍通过lncRNA调节线粒体自噬的具体机制，可能为糖尿病肾病的防治提供新的思路和治疗靶点，具有一定的科学价值和应用前景。