基于AMPK-TFEB通路研究高车前苷调控自噬改善糖尿病血管病变的作用机制

Autophagy protects endothelial cells during the development of diabetic vascular complications. The transcription factor EB (TFEB) is one of the important regulatory proteins associated with autophagy and its activity is controlled by the energy receptor AMPK. Our earlier studies have shown that homoplantaginin (Hom), the main active component of Salvia plebeia, significantly ameliorates arterial lesions in diabetic mice induced by high-sugar plus high-fat diet combined with streptozotocin. In vitro cell culture studies demonstrated that Hom increases the endothelial cell viability and enhances autophagy function induced by high glucose. Therefore, in the present study, we firstly study the protective effect of Hom on high glucose-induced endothelial cell inflammation, mitochondrial oxidative stress, apoptosis and endothelial dysfunction. Secondly, gene overexpression/interference, immunofluorescence and other techniques will be utilized. We will manipulate about autophagy, TFEB, AMPK/mTOR pathways and examine how these manipulations affect the protective actions of Hom on vascular endothelial cells. Finally, it is verified in the animal model that Hom activates AMPK, down-regulates mTOR and up-regulates TFEB transcriptional activity, repairs lysosomes and enhances autophagy, then inhibits mitochondrial oxidative stress and inflammatory responses, and improves diabetes mellitus with major vascular disease. This multi-link effect mechanism of Hom provides a theoretical basis for the treatment of diabetic vascular complications, and strengthens a better understanding of the mechanism that autophagy regulated by AMPK-TFEB ameliorates diabetic vascular disease.

在糖尿病血管并发症的发生过程中，自噬对内皮细胞起到保护作用。转录因子TFEB是细胞自噬的重要调控蛋白之一，接受能量感受器AMPK的调控。我们前期研究发现，荔枝草主要活性成分高车前苷（Hom）可显著改善高糖高脂饲料联合链脲佐菌素诱导的糖尿病小鼠动脉病变，提高高糖损伤的内皮细胞存活率，增强自噬功能。因此，本课题首先研究Hom对高糖诱导内皮细胞炎症、线粒体氧化应激、凋亡和内皮功能失调的保护作用。再采用基因过表达/干扰、免疫荧光等技术分别干预自噬、TFEB、AMPK/mTOR不同环节，探索Hom保护血管内皮细胞的机制。最后，在模型动物验证Hom通过激活AMPK，下调mTOR，上调TFEB转录活性，修复溶酶体而增强细胞自噬，进而抑制线粒体氧化应激和炎症反应，改善糖尿病大血管病变的多环节效应机制。这为其治疗糖尿病血管并发症提供理论依据，并加强对AMPK-TFEB调控的自噬改善糖尿病血管病变机制的认识。

糖尿病血管并发症是糖尿病患者高致残率、高死亡率的主要原因。血管内皮细胞损伤是糖尿病血管并发症发生发展的始动环节。荔枝草为唇形科鼠尾草属植物荔枝草（Salvia plebeia R. Br.）的全草，高车前苷是荔枝草的主要黄酮类成分。本研究以高糖致血管内皮细胞损伤和2型糖尿病db/db小鼠为模型，结合RT-qPCR、Western blot、免疫荧光、基因干扰等方法证实了高车前苷具有：①多环节保护血管内皮细胞：抑制高糖所致内皮细胞凋亡、线粒体氧化应激和炎症反应；促进内皮细胞NO生成。②增强自噬水平：促进高糖所致内皮细胞Atg5、Beclin1和LC3-Ⅱ蛋白表达，抑制P62蛋白表达，上调自噬小体数量；采用早期自噬抑制剂 3-methyladenine、晚期抑制剂氯喹和巴佛洛霉素A1干预证实高车前苷通过增强自噬而保护内皮细胞。③经TFEB调节自噬：促进高糖所致内皮细胞LAMP1、CTSB mRNA 和蛋白表达；改善溶酶体膜通透性；促进TFEB mRNA表达, 促进TFEB核转位，激活TFEB。si-TFEB条件下，高车前苷显著上调高糖所致内皮细胞自噬，促进LAMP1和CTSB蛋白表达，抑制ROS生成和细胞凋亡的作用被逆转。④经AMPK/mTOR/TFEB通路调节自噬水平：分子对接研究发现高车前苷和AMPK蛋白有良好的相互作用；高车前苷显著升高高糖所致的内皮细胞p-AMPK蛋白水平，降低p-mTOR、p-p70s6K和p-TFEB蛋白表达。使用AMPK抑制剂Compound C后, 高车前苷上调内皮细胞p-AMPK蛋白表达，降低p-mTOR和p-TFEB蛋白表达的作用被逆转。⑤保护db/db小鼠动脉：改善db/db小鼠口服糖耐量、胰岛素耐量和胰岛素抵抗；改善糖代谢和脂质代谢；降低ICAM-1、IL-1β、MDA水平，升高SOD水平；上调db/db小鼠主动脉内皮细胞p-AMPK和TFEB蛋白表达，促进自噬体形成，抑制内皮细胞凋亡，减轻动脉组织病理损伤，改善胸主动脉内皮依赖性舒张功能。本研究明确了高车前苷通过AMPK/mTOR/TFEB通路，促进TFEB核转位进而修复溶酶体功能，增强自噬水平，抑制高糖所致血管内皮损伤，继而改善糖尿病大血管病变。本研究为将高车前苷开发为治疗糖尿病血管并发症的药物提供理论依据，并加强对 AMPK-TFEB调控的自噬改善糖尿病血管病变机制的认识。