NR4A2和NF-κB的交互作用对氧化低密度脂蛋白致内皮细胞功能障碍的影响及其机制

The elevated level of oxidized low-density lipoprotein in serum caused by high-fat diet is an important risk factor to atherosclerosis and the endothelial cell dysfunction play an important role in the development of atherosclerosis. The nuclear receptor subfamily 4 group Amember 2 and NF-κB regulate the expression of genes responsible for atherosclerosis development, which the mechanism is still unclear. This project, which include cell culture experiment and animal experiment, plan to intervene NR4A2 and NF-κB in cell culture in vitro using gene silence and gene over-expression and study the transposing, expression as well as interaction between NR4A2 and NF-κB using confocal microscopy and immunoprecipitation in human umbilical vein endothelial cells induced by different concentrations of oxidized low-density lipoprotein with qualitative, quantitative and dynamic methods. This project will also analyze ICAM-1、VCAM-1、MCP-1、IL-1、IL-6、IL-8、eNOS、NO, which are regulate by NR4A2, NF-κB to unravel the interaction among NR4A2 with NF-κB on regulation of endothelial cell dysfunction molecular marks. Then those discovers in cell culture in vitro will investigated and determined further in mouse Atherosclerosis animal model established by high-fat diet, which will improve the understanding of mechanism of Atherosclerosis induced by oxidized low-density lipoprotein and provide scientific evidences for new control strategy.

高脂饮食引起的血中氧化低密度脂蛋白(oxidized low-density lipoprotein,Ox-LDL)升高是动脉粥样硬化（atherosclerosis，AS）的重要危险因素，其所致的内皮细胞功能障碍是AS早期病理变化的关键环节。孤儿核受体NR4A2（nuclear receptor subfamily 4 group Amember 2, NR4A2）和NF-κB是调节AS相关基因表达的重要转录因子，但两者对Ox-LDL致内皮细胞功能障碍的分子机理尚不明确。本项目采用细胞培养和动物模型研究NR4A2和NF-κB的交互作用对ICAM-1、VCAM-1、MCP-1、IL-1、IL-6、IL-8、eNOS的转录和翻译的影响，从而进一步阐明AS过程中Ox-LDL致内皮细胞功能障碍的分子机理。本研究将为进一步阐明动脉粥样硬化的机制和制订防治新策略提供科学依据。

动脉粥样硬化（AS）的发生、发展与营养膳食密切相关，由高脂饮食等因素引起的高脂蛋白血症，导致血浆低密度脂蛋白（LDL）升高，在体内被氧化修饰成Ox-LDL，进而引起血管内皮细胞功能障碍，促进早期AS的病变部位的形成。内皮细胞功能障碍主要表现为血管张力调节障碍，炎性和黏附分子表达异常。NF-κB是介导内皮功能障碍的重要信号通路，活化的NF-κB可促进血管内皮细胞受损后表达和分泌多种致AS效应分子，孤儿核受体NR4A2参与AS病变部位多种细胞表达炎症因子的调控，研究NR4A2、NF-κB通路与致AS效应因子的关系及相互作用机制，对发现动脉粥样硬化新的干预靶点显得非常重要。..本课题采用实时荧光定量PCR、酶联免疫吸附、免疫印迹等技术，发现ox-LDL损伤能剂量依赖型上调人脐静脉内皮细胞（HUVEC）多种白细胞介素、肿瘤坏死因子、粘附素、一氧化氮合成酶、NR4A2等基因转录与表达。Ox-LDL诱导HUVEC损伤后升高核内NR4A2浓度，损伤发生2h至浓度最大值。采用Ad-NR4A2腺病毒载体感染HUVEC过表达NR4A2，结果表明NR4A2过表达上调致炎细胞因子及粘附因子转录表达。NR4A2 RNAi转染HUVEC则下调Ox-LDL损伤HUVEC内NR4A2、细胞因子及粘附因子的表达。相对于单一转染，使用p65-RFP和NR4A2-EGFP共同转染HUVEC能够更加显著上调粘附及细胞因子表达，证明NR4A2和NF-κB信号通路间交互作用对内皮细胞功能障碍的影响。另外，本课题采用高脂饮食饲养C57BL/6J背景载脂蛋白E基因敲除（Apoe-/-）小鼠14周，建立了动脉粥样硬化动物模型，观察动脉斑块及血脂成分异常。..基于以上实验数据，说明ox-LDL能破坏HUVEC炎性和黏附分子的正常表达，从而引起内皮细胞功能障碍。NR4A2作为早期调控信号分子，与NF-κB信号通路相互作用，参与了ox-LDL诱导了内皮细胞功能障碍，进一步揭示孤儿核受体家族参与动脉粥样硬化的机制。