脂联素N-糖链调控脂肪-血管轴抗冠状动脉粥样硬化的作用及机制研究

The previous studies showed that the atherosclerotic plaque was occurred in the coronary artery only, but not in the internal mammary artery and radial artery of coronary artery disease patients, in addition to the endocrine dysfunction in the epicardial adipose tissue around the lesion coronary artery, which indicates that the lesion of the coronary artery is affected by the local micro-environment of the epicardial adipose tissue.Although the human recombinant adiponectin was observed to delay the progress of atherosclerosis in the CAD model mice, ti is required to be administrated frequently due to the low activity and rapid elimination. It was found in the follow-up studies with the mass spectroscopy that 11 glycosylation sites existed in the normal human adiponectin; under the circumstance of endoplasmic reticulum stress, the glycosylation modification was dysfunctional; the polymeric adiponectin which secreted from the epicardial adipose tissue was reduced, and the glycosylation level decreased too, but the glycoform of adiponectin still remains unclear. Based on the previous work, we are planning to target the giycosylation from two aspects: first, on the molecular biology level, we will make use of mass spectroscopy to further explore the changes of N- linked oligosaccharide chains of adiponectin in structures, types and connections under the circumstance of atherosclerosis, to reveal the intrinsic relations between the changes of N- linked oligosaccharide chains of adiponectin and the atherosclerosis. Second, on the cultured cells and model animal level, we will make use of the site-directed mutagenesis to enrich the N-linked oligosaccharide chains by increasing the N-glycosylation sites in the adiponectin. Meanwhile, with the adipo-/- knockout mice, we will study the anti-atherosclerotic effects and its mechanism of the modified adiponectin. We expect that this project will theoretically clarify the functions of N-linked oligosaccharide chains, which will provide the theoretical guide for the precise and specific modulation of the proteins by dynamic modification of glycosylation, and practically present the new target and new method of the treatment of CAD.

本课题前期研究发现，冠心病者冠脉形成粥样斑块，而乳内动脉及桡动脉不易发生，且病变冠脉外周EAT分泌功能紊乱，提示EAT局部微环境影响冠脉病变的发生；在小鼠模型中应用人重组脂联素抗AS，可延缓AS的进程，因其活性低且消除快，需频繁给药；后续研究发现：正常人脂联素有11个糖基化位点；而在内质网应激下，EAT分泌的HMW减少，脂联素糖基化水平明显降低，但其糖型的变化尚不清楚。据此，本课题以糖基化的视角，从分子水平：用质谱技术进一步探讨AS状态下脂联素中糖链的结构、类型，组成及连接等变化，探究脂联素糖型的变化与AS的内在联系。从而改良脂联素，并用adipo-/-敲除小鼠研究改良后脂联素抗AS效果及机制。为阐明糖链的功能，通过动态的糖基化修饰以更精细、专一地调控蛋白质功能提供理论依据，为冠心病治疗提供新靶点和新手段。

本课题从糖基化的视角，在分子水平用质谱技术进一步探讨AS状态下脂联素中糖链的结构、类型，组成及连接等变化，探究脂联素糖型的变化与AS的内在联系。从而改良脂联素，并用adipo-/-敲除小鼠研究改良后脂联素抗AS效果及机制。本项目从临床角度阐明了冠心病、非冠心病患者心外膜脂肪细胞中脂质代谢的差异，证实了冠心病组心外膜脂肪细胞脂解受到抑制，冠心病患者心外膜脂肪细胞堆积。高浓度的ox-LDL和葡萄糖抑制3T3L1细胞的脂解，高浓度的LPS能促进3T3L1脂肪细胞的水解,主要表现为小脂滴增多、大脂滴减少,甘油含量增多、甘油三酯含量减少。阐明了病理状态下（冠状动脉粥样硬化）心外膜脂肪脂联素糖链的变化特点。在软脂酸、葡萄糖、TNF-α 模拟心外膜脂肪局部高脂、高糖及炎症环境对局部脂肪细胞N-糖基化程度及糖型变化的影响，并且还在体外构建突变的apM1质粒并转染HEK293T细胞，制备纯化的Mut-APN并分析Mut-APN三种聚体水平、N-糖基化位点及其糖型变化。另外在研究中还发现脂肪因子CTRP3具有与脂联素相似的结构和功能，可以减轻心肌细胞中脂滴积累，改善炎症、凋亡、氧化应激、细胞活性等。最后还验证了经糖工程改性后脂联素抗动脉粥样硬化的效果。并且积极验证了Mut-APN 对脂肪-血管轴调控的影响：Mut-APN 对脂肪细胞、巨噬细胞功能的影响。在本研究中，改性脂联素的作用和功能以及其血浆半衰期、清除率的变化仍需要积极的改进探索。本研究构建adipo-/-基因敲除鼠的动物模型，通过高脂饮食喂养研究心外膜脂肪细胞微环境的变化、冠状动脉粥样硬化的发生以及心肌功能的改变等，并且在研究中发现高脂饮食可以通过改变肠道菌群的环境。脱硫弧菌是影响肠道菌群的重要菌落之一，它可以损害肠粘膜屏障功能，减弱肠道屏障，为脱硫弧菌及其LPS入血提供可能，并通过激活TLR4/NF-κB通路，加重内皮细胞氧化应激，促进单核细胞粘附并分泌大量促炎因子，最终促进动脉粥样硬化。