MYDGF对糖尿病动脉粥样硬化性血管病变保护作用及机制研究

Atherosclerotic vascular diseases are the main causes of mortality in diabetes. However, the underlying mechanisms for Atherosclerotic vascular diseases are not fully clear, moreover, the effective treatment strategies for diabetic atherosclerotic vascular diseases are scare. It was reported that myeloid-derived growth factor (MYDGF) treatment reduces scar size and contractile dysfunction after myocardial infarction. However, the data are not available on the protective roles of MYDGF in atherosclerotic vascular diseases under diabetic condition. Our preliminary in vitro experiments revealed that MYDGF could attenuate the endothelial cell damage induced by high glucose. Therefore, we hypothized that MYDGF can protect endothelial damage and ameliorate atherosclerosis induced by high glucose. In this study, ① In vitro experiments, endothelial cells will be treated with high glucose and MYDGF, and the protective effects of MYDGF on endothelial cell apoptosis will be detected. ② The artery endothelial damage and atherosclerosis in MYDGF deficiency condition will be investigated in apoE(-/-) diabetic mice by gene knockout assay. ③ The protective roles of MYDGF on artery endothelial damage and atherosclerosis will be investigated in apoE(-/-) diabetic mice by adeno-associated viruses and recombinant MYDGF assays. ④ In both in vitro and in vivo experiments, STAT3, eNOS and autophagy pathways will be detected using gene siRNA silencing assay to test whether or not these signal pathways are involved in the protective roles of MYDGF on atherosclerotic vascular diseases induced by high glucose. We aim to reveal the new mechanisms of diabetic atherosclerotic vascular diseases, and provide a new therapeutic target and a new drug for the treatment of diabetic atherosclerotic vascular complications.

糖尿病（DM）动脉粥样硬化性血管病变发生机制未完全阐明，且缺乏有效防治方法。文献报道骨髓源性生长因子（MYDGF）对心肌梗死后心脏有保护作用。但MYDGF是否具有抗DM动脉粥样硬化作用未见报道。申请者预实验显示，MYDGF有潜在抗高糖内皮损伤作用。据此，申请者提出如下假说：MYDGF有抗DM动脉粥样硬化作用。本项目拟探讨：①体外实验探讨MYDGF对高糖动脉内皮损伤的保护作用；②采用apoE(-/-)/MYDGF（-/-）双敲除鼠探讨MYDGF缺乏对apoE(-/-)糖尿病鼠动脉粥样硬化的影响；③采用MYDGF注射及腺相关病毒载体转染法探讨升高体内MYDGF对DM鼠动脉粥样硬化保护作用；④采用siRNA腺病毒载体基因沉默法，从体外和体内探讨STAT3、eNOS和自噬通路是否介导MYDGF的内皮和动脉保护作用。旨在探讨DM大血管病变新机制及防治靶点，并为防治DM血管病变提供新靶点及新药物。

动脉粥样硬化的发生率逐年升高，我们最近的研究表明，骨髓细胞（BMCs）分泌的骨髓源性生长因子（MYDGF）可改善胰岛素抵抗（IR）和葡萄糖/脂质代谢。然而，MYDGF对血管内皮功能及动脉粥样硬化是否有保护作用还不清楚。我们采用骨髓细胞特异性 MYDGF+/-小鼠和MYDGF-/-小鼠分别与ApoE-/-小鼠杂交繁殖而获得MYDGF+/+ApoE-/-小鼠（AKO）以及 MYDGF-/-/ ApoE-/-小鼠 (DKO)。自小鼠6-8周龄开始喂食西方饮食饲料，成功建立了动脉粥样硬化模型。本研究首先探讨了在ApoE基因敲除的基础上，MYDGF基因缺乏对动脉粥样硬化的影响。其次，通过小鼠的骨髓过表达MYDGF，探讨补充MYDGF对AKO和DKO小鼠动脉粥样硬化的影响。此外，在体外实验中，在棕榈酸 (PA) 刺激的条件下，采用重组MYDGF（rMYDGF）进行干预，观察对血管内皮细胞凋亡，炎症和通透性的影响。此外，从体内体外两个方面探索与MYDGF相关的信号通路。干预12周后，与AKO组相比，DKO组小鼠内皮依赖性血管舒张功能下降了40.02％；斑块的表面积增加了2.5倍；斑块的横截面积增加了2.1倍；小鼠主动脉炎性侵润面积、血清炎性因子、体重、三酰甘油、总胆固醇、低密度脂蛋白及 游离脂肪酸水平增加, 高密度脂蛋白水平降低。相反，补充MYDGF后，与AKO-GFP组和DKO-GPF组小鼠相比，AKO-MYDGF组和DKO-MYDGF组小鼠内皮依赖性血管舒张功能明显改善；斑块面积明显减少；炎性侵润及炎症因子水平显著降低，代谢紊乱得到改善。 rMYDGF促进了主动脉内皮细胞的增殖和迁移。MYDGF能拮抗PA诱导的下小鼠主动脉内皮细胞凋亡及炎症反应。MAP4K4 /NF-κB信号参与了MYDGF对动脉粥样硬化的保护作用。综上所诉, MYDGF可改善AopE-/-小鼠血管内皮功能，减少斑块面积，降低炎性侵润及炎症因子水平，改善机体代谢；MYDGF能拮抗PA诱导的下小鼠主动脉内皮细胞凋亡及炎症反应，提示MYDGF具有抗动脉粥样硬化作用。MAP4K4 /NF-κB信号参与了MYDGF对动脉粥样硬化的保护作用。MYDGF为防治动脉粥样硬化提供新靶点及新药物。