A20的O-GlcNAc糖基化修饰介导的血管损伤后保护及分子机制

Accumulating evidence has shown that inflammation activation is a critical pathological mechanism of vascular remodeling post-injury. However, the specific and effective therapeutic medicine is still unavailable so far. More recent studies have demonstrated that glucosamine treatment potently attenuated inflammation activation and neointima formation after vascular injury via up-regulating local O-linked N-acetylglucosamine modification (O-GlcNAcylation) level. However, the molecular mechanism remains unclear. A20 functions as an endogenous inhibitor for NF-kB pathway activation. The present proposal aims to test the hypothesis that increasing A20 O-GlcNAcylation level in vascular smooth muscle cells elicits anti-inflammatory and vasoprotective effects via a negative feedback regulation on NF-kB signaling activation. Therefore, we will utilize various modern molecular biological methods and techniques, including co-immunoprecipitation, in vitro O-GlcNAcylation assay and RNAi etc., to demonstrate the effect and mechanism of A20 O-GlcNAcylation on its biological activity and NF-kB pathway activation; to clarify the role of A20 O-GlcNAcylation on the modulation of inflammatory reaction response in vascular smooth muscle cells and vascular remodeling post-injury. Taken together, these proposed studies will contribute to demonstrate the anti-inflammatory and vasoprotective mechanism of A20 O-GlcNAcylation and provide new promising molecular target and yield a novel therapeutic strategy for the prevention and treatment of various vascular diseases.

炎症激活是血管损伤后血管壁重构的一个重要病理机制，目前尚无特异性的治疗药物。我们近来研究显示，葡糖胺可通过增加局部O连接的N已酰葡糖胺（O-GlcNAc）修饰水平有效减轻损伤血管炎症激活及内膜增生，但其分子机制不清楚。A20作为内源性NF-kB通路活化的关键负性调控分子，本项目研究旨在测试假说：增加血管平滑肌细胞内A20的O-GlcNAc修饰，通过反馈抑制转录因子NF-kB的激活而发挥抗炎及血管保护效应。为此，本研究将通过免疫共沉淀、体外糖基化修饰分析、基因沉默等多种方法共同阐明A20的O-GlcNAc修饰对其生物活性、NF-kB通路活化的影响及机制，揭示A20 O-GlcNAc修饰在调控血管平滑肌细胞炎症反应应答以及血管损伤后血管重构中的作用，从而在分子、细胞及整体水平剖析A20 O-GlcNAc修饰发挥抗炎及血管保护的作用机制，为研发新型血管保护药物提供新的思路和分子药物干预靶标。

炎症激活是血管损伤后血管壁重构的一个重要病理机制，目前尚无特异性的治疗药物。我们近来研究显示，葡萄糖胺可通过增加局部O连接的N已酰葡糖胺（O-GlcNAc）修饰水平有效减轻损伤血管炎症激活及内膜增生，但其分子机制不清楚。近年来大量的研究证实，血管损伤局部的炎症反应在血管重构过程中发挥重要的作用，抑制NF-kB信号通路的过度激活能有效抑制血管损伤后新生内膜的形成。本项目研究通过利用大鼠血管平滑肌细胞培养，免疫共沉淀、基因沉默等多种方法探讨葡萄糖胺是否可通过促进A20的O-GlcNAc修饰来增强其对NF-kB通路活化的抑制，从而介导葡萄糖胺的血管保护效应。我们发现GlcN或OGT抑制剂thiamet G处理培养的血管平滑肌细胞可促进A20的O-GlcNAc修饰，同时增强A20与TAXBP1的结合来增强其活性。过表达A20可进一步增强thiamet G对TNF-a诱导的IkBa的降解，p65激活以及DNA结合的抑制，以及促炎细胞因子表达，细胞迁移及增殖；相反，沉默A20的表达可显著减弱上述ThiametG介导的效应。在球囊拉伤的大鼠颈动脉损伤模型中，我们观察到GlcN治疗可显著抑制抑制损伤血管局部p65激活和新生内膜形成，局部注射腺病毒编码的A20-shRNA可有效抑制血管损伤局部的A20表达和p65激活，从而废除GlcN介导的血管保护作用。这些研究结果提示，GlcN主要通过促进A20的O-GlcNAc修饰来增强其生物学效应，从而抑制NF-kB信号通路介导的血管炎症反应及重构，而A20是GlcN发挥血管保护的主要作用分子靶标。本项目研究对开发新型的血管损伤保护剂以及预防血管成形术后再狭窄治疗具有重要的科学意义。