TSPO/VDAC1-ROS-线粒体自噬在糖尿病视网膜病变中的作用与机制研究

Retinal vascular endothelial cell damage is a key factor in the progression of diabetic retinopathy (DR). ROS control imbalance is the crucial cause of mitochondrial DNA oxidation and endothelial cell injury. Whether the DR process can be delayed by changing ROS levels is unknown. Studies have shown that mitochondrial transmembrane complex TSPO/VDAC1 plays a vital role in regulating ROS content and therefore changes the autophagy of mitochondria. Our previous experiments showed that ROS and mitochondrial autophagy levels increased in high sugar, suggesting that ROS- mitochondrial autophagy may be involved in DR process. It is not clear whether TSPO/VDACI could regulate ROS content to affect mitochondrial autophagy and thus, we hypothesized that the rise of TSPO/VDACI could change the ROS content and mitochondrial autophagy level in retinal vascular endothelial cells, and promote DR development through the ROS- mitochondrial autophagy pathway. This project tested the activation status of TSPO/VDAC1-ROS-mitochondrial autophagy pathway in the pathogenesis of DR in vivo and in vitro. TSPO/VDAC1 was down-regulated in order to inhibit ROS and change the autophagy level of mitochondria, and then observed its protective effect on DR process. We will provide a theoretical foundation to understand the pathogenesis and find new treatments of diabetic retinopathy from the perspective of ROS- mitochondrial autophagy.

视网膜血管内皮细胞损伤是糖尿病视网膜病变（DR）发病的关键因素；ROS调控失衡是线粒体DNA氧化、内皮细胞损伤的重要原因；能否通过改变ROS水平延缓DR进程未知。研究表明线粒体跨膜复合物TSPO/VDAC1是调控ROS含量的关键，并因此改变线粒体自噬。我们的前期实验显示高糖下ROS及线粒体自噬水平增加，提示ROS-线粒体自噬可能参与DR进程；而TSPO/VDACI能否调节ROS含量影响线粒体自噬,对DR进行调控尚不清楚。为此，我们提出假设：TSPO/VDACI升高可以改变视网膜血管内皮细胞中ROS含量及线粒体自噬水平，通过ROS-线粒体自噬途径促进DR发展。本项目拟体内外实验检测TSPO/VDAC1-ROS-线粒体自噬在DR中的激活状态；靶向下调TSPO/VDAC1通过抑制ROS、改变线粒体自噬水平，以观察对DR的保护作用；从ROS-线粒体自噬角度探讨DR发病机制并为治疗靶点提供理论基础。

跨膜复合物TSPO/VDAC1是调控线粒体ROS含量的关键，而细胞内过量的ROS生成会阻碍细胞线粒体自噬。TSPO/VDACI能否调节ROS含量影响线粒体自噬，对糖尿病性视网膜病变进行调控尚不清楚。本研究中，我们在体内体外构建糖尿病视网膜病变模型，显示TSPO/VDAC1与线粒体自噬相关蛋白的表达变化几乎相反，显著抑制细胞线粒体自噬。在稳定低表达TSPO的细胞中线粒体自噬活性增高，加入自噬抑制剂3-MA后，能够抑制沉默TSPO后导致的自噬活性升高。另外高糖下TSPO过表达稳转株加入抗氧化剂MnTBAP清除大量ROS后，细胞线粒体自噬水平恢复。我们的研究证实在糖尿病视网膜病变中，TSPO/VDACI通过调节胞内大量ROS生成从而通过抑制PINK1/Parkin通路活性抑制细胞线粒体自噬，不能及时清除受损的线粒体，使细胞内炎症因子增加，促进细胞凋亡，从而促进糖尿病视网膜病变进展，为糖尿病性视网膜病变的发病机制研究及治疗奠定理论基础。目前关于TSPO/VDCA1 对DR 发病机制的研究还未见相关的报道，本课题探讨了糖尿病视网膜病变致病的一种新的线粒体自噬调控机制。