FoxO1介导的线粒体自噬在糖尿病肾脏足细胞氧化应激损伤中的作用

Oxidative stress is the leading cause of podocytes injury of diabetic nephropathy, which is tightly associated with excessive production of reactive oxygen species (ROS) induced by damaged mitochondria. Mitochondrial autophagy (mitophagy), a type of specific autophagy, plays a vital role in selectively removing damaged mitochondria. Therefore, it is important that regulating the level of mitophagy in podocytes under diabetic conditions may contribute to alleviating oxidative injury in podocytes. Previous studies from our group demonstrated that Forkhead box class O1 (FoxO1), a transcriptional regulator of oxidative stress, is implicated in ameliorating renal oxidative stress by up-regulating the expression of target genes involving in anti-oxidative effects. However, whether FoxO1 could alleviate podocyte injury through restraining inordinate release of ROS is still unknown. Current researches have confirmed the direct binding of FoxO1 on promoter of PTEN-induced putative kinase 1 (PINK1)-a kinase capable of inducing mitophagy, which facilitates the transcription of PINK1. Moreover, Preliminary experiments from our group have proved the decrease of FoxO1 bioactivity in podocytes under high glucose conditions, which was accompanied with significant attenuation of PINK1 expression. Thus, we hypothesize that FoxO1 might regulate mitophagy of podocytes in diabetic kidney via up-regulating PINK1 expression, thereby reducing ROS-induced podocytes injury. Therefore, by utilizing the technique of lentiviral vector transfection, constitutive active mutation and RNA interference, we are intended to explore the effects and mechanisms of FoxO1-induced mitophagy on podocyte injury through the perspective of molecules, cells and animals, which will shed light on novel strategy of protecting from diabetic nephropathy.

受损线粒体过度释放活性氧（ROS）所致氧化应激，是糖尿病足细胞损伤的中心环节。线粒体自噬是一种选择性的自噬，是特异清除损伤线粒体的过程。FoxO1是调节氧化应激的关键转录因子，我们前期研究证实FoxO1可促进下游抗氧化靶基因表达，增加ROS清除，改善肾脏氧化应激。但FoxO1能否抑制线粒体ROS过度释放，改善足细胞氧化应激损伤，目前尚无报道。近期研究发现，FoxO1可激活线粒体自噬关键启动因子PINK1表达。我们预实验也表明，糖尿病足细胞中FoxO1活性下降，伴随有PINK1表达降低，ROS产生增多。据此提出假设：FoxO1可能通过PINK1调节线粒体自噬，减少ROS过度释放，改善糖尿病足细胞氧化应激损伤。为此，本研究采用慢病毒转染、组成型活性突变、RNAi等手段，从细胞及动物整体水平，探讨FoxO1介导的线粒体自噬在糖尿病足细胞氧化应激损伤中的作用及机制，为糖尿病肾病的防治提供新的思路。

线粒体自噬是一种选择性的自噬，是特异清除损伤线粒体的过程。受损线粒体过度释放活性氧（ROS）所致氧化应激，是糖尿病足细胞损伤的中心环节。FoxO1是调节氧化应激的关键转录因子，我们前期研究证实FoxO1可促进下游抗氧化靶基因表达，增加ROS清除，改善肾脏氧化应激。PINK1是线粒体自噬关键启动因子，研究表明，FoxO1可通过与PINK1启动子结合，促进其表达。但FoxO1能否抑制线粒体ROS过度释放，改善足细胞氧化应激损伤，目前尚无报道。在该研究中，我们发现糖尿病足细胞中FoxO1活性下降，伴随有PINK1表达降低，线粒体损伤增多，ROS产生增多。为了进一步验证我们的假设：FoxO1通过对糖尿病小鼠足细胞内线粒体自噬的调节，减少受损线粒体ROS的过度释放，改善足细胞氧化应激损伤。我们开展了以下研究：①利用慢病毒转染技术，调节高糖培养的小鼠足细胞内FoxO1表达及活性，观察FoxO1对足细胞线粒体自噬及氧化应激的影响；②在FoxO1过表达基础上下调PINK1，进一步明确了FoxO1调节线粒体自噬及在对抗氧化应激损伤中的具体机制；③通过肾脏靶向性——肾皮质多部位注射组成型活性FoxO1慢病毒，体内验证FoxO1介导的线粒体自噬在糖尿病小鼠足细胞氧化应激损伤中的效应。研究结果表明：①过表达FoxO1可促进PINK表达，激活足细胞线粒体自噬，减少氧化应激和ROS生成；②通过肾皮质靶向注射组成型活性FoxO1过表达慢病毒，体内进一步证明FoxO1通过线粒体自噬对糖尿病肾脏足细胞内氧化应激的调节。③通过慢病毒转染、组成型活性突变、RNAi等手段明确了FoxO1再糖尿病肾病足细胞损伤中的具体作用机制。本课题研究有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意义。