FGF21通过Sirt1-FoxO1通路抑制高糖诱导的自噬延缓血管内皮细胞衰老的机制研究

Vascular endothelial cell senescence plays a critical role in diabetic vascular complications, and the autophagy induced by high glucose mediates the celluar senescence. In the previous study, we demonstrated both in type I diabetic mice and high glucose stimulated endothelial cells that FGF21 could alleviate celluar senescence through its role in inhibiting autophagy, and the inhibitory effect was achieved by promoting the association between Sirt1 and FoxO1, which could lead to the deacetylation of FoxO1 and its dissociation from Atg7. However, the mechanism remains to be further explored. Thus, we will firstly confirm that FGF21 mediated FoxO1 deacetylation constitutes the main molecular basis for the alleviation of high glucose induced endothelial cell senescence by Sirt1 gene silencing. Secondly, the FoxO1 deacetylation sites modulated by FGF21 will be revealed via targeted proteomics. Finally, the identified deacetylation sites will be replaced with glutamine for mimicking continuous acetylation. Thereby, the underlying mechanism could be elucidated by examining the alteration of FGF21 mediated protective effects against high glucose stimulation after different site mutation. This project is expected to provide new targets for the prevention and treatment of diabetic vascular complications.

血管内皮细胞衰老在糖尿病血管并发症中扮演着重要角色，且高糖诱导的内皮细胞过度自噬是造成其衰老的关键环节。前期在小鼠I型糖尿病模型及高糖损伤的内皮细胞中我们已证实FGF21可通过抑制高糖诱导的细胞自噬延缓细胞衰老，并且FGF21是通过促进Sirt1与FoxO1的结合，调节FoxO1的去乙酰化修饰及其与Atg7的解离，实现对自噬的抑制作用，但其中的分子机制有待更为详实的阐明。因此，我们将利用Sirt1基因沉默等手段证实FGF21调控的FoxO1去乙酰化是其抑制高糖诱导的细胞自噬乃至改善细胞衰老的分子基础；随后通过靶向蛋白质组学技术，进一步揭示FGF21调控下FoxO1发生去乙酰化修饰的潜在位点；最后将鉴定出的靶位点替换为谷氨酰胺以模拟不受Sirt1调控的持续乙酰化修饰，通过检测位点突变后FGF21逆转高糖损伤效应的改变，从而详细阐明相关分子机制。本项目有望为糖尿病血管并发症的防治提供新靶点。

内皮细胞衰老在糖尿病血管并发症中扮演重要的角色，且高糖诱导的内皮细胞过度自噬是造成其衰老的关键环节。既往研究表明，FGF21在糖尿病血管并发症中起着积极的作用，但其具体机制并不明确。本课题旨在探究FGF21在延缓血管内皮细胞衰老中的作用并探讨其内在机制。.我们分别在I型糖尿病小鼠的主动脉和高糖损伤的HUVEC中证实: FGF21在体内实验中能明显改善高糖引起的主动脉内皮衰老；FGF21在体外实验中能显著改善高糖损伤后HUVEC的成管抑制及衰老。同时，在糖尿病小鼠主动脉及高糖损伤的HUVEC中发现，LC3蛋白水平明显上调，FGF21可明显降低其自噬水平。在HUVEC中亦发现，干扰自噬相关基因ATG7的表达，FGF21缓解高糖诱导自噬的效果减弱。随后，我们发现由高糖刺激而明显上调的乙酰化FoxO1主要聚集于细胞质内，而合并FGF21给药后则促进去乙酰化的 FoxO1 入核。在高糖损伤情况下，FoxO1的乙酰化修饰程度明显升高，而合并FGF21给药后高糖诱导的FoxO1乙酰化修饰程度下调，该过程与FGF21促进FoxO1与Sirt1的结合增多相关。最后，我们在HUVEC中发现，在干扰Sirt1的情况下，FGF21的抗衰老及抑制自噬作用下降。.综上所述，本实验结果显示，在高糖情况下，自噬与衰老呈正相关趋势，FGF21依赖于Sirt1-FoxO1通路下调血管内皮细胞自噬水平，从而延缓高糖诱导的血管内皮细胞衰老。