NOX4-自噬途径介导apelin促单核细胞-血管内皮细胞粘附的研究

We reported for the first time that apelin-13 enhance monocyte-endothelial cells adhesion mediated by phosphoinositide 3 kinase (PI3K) signaling transduction pathway and G-protein coupled receptor APJ and endogenous ligand apelin system are reported to promote atherosclerosis,but it's molecular mechanism unclearly.Recently we find that apelin-13 induce autophagy、increases expression of NADPH oxdase 4(NOX4) and reactive oxygen species(ROS)in endothelial cells in pre-experiments.Hypothesis is that NOX4-autophagy pathway may involve in monocyte-endothelial cells adhesion induced by apelin.We will investigate that apelin effects on autophagy、monocyte-endothelial cell adhesion,chemotaxis in vitro;we will study that NOX4-autophagy pathway mediate monocyte-endothelial cell adhesion stimulated by apelin in siRNA and others;We will test that apelin effect on atherosclerosis and autophagy in apoE-/-atherosclerosis mice model by using high and low apelin expression technology.This hypothesis will show new mechenism that apelin/APJ system participate in progression of atherosclerosis in details.

申请者报道并提出磷酸肌醇激酶3（PI3K）信号通路介导apelin-13促进单核细胞-血管内皮细胞粘附参与APJ受体及其配体apelin促进动脉粥样硬化发展新观点，预实验发现apelin-13诱导血管内皮细胞自噬、促进NADPH oxidase 4(NOX4)表达和活性氧（ROS）生成，据此提出NOX4-自噬途径介导apelin促单核细胞-血管内皮细胞粘附的假说。课题拟明确apelin诱导血管内皮细胞自噬、自噬介导apelin促进单核细胞-血管内皮细胞粘附的影响；用RNA干扰等技术探讨NOX4-自噬途径是否介导apelin促单核细胞-血管内皮细胞粘附；分析体内高表达或沉默apelin对apoE-/-小鼠动脉粥样硬化斑块和自噬影响。阐明自噬参与apelin促单核细胞-血管内皮细胞粘附的新机理，为动脉粥样硬化防治提供实验依据。

本课题研究G蛋白偶联受体APJ的内源性配体apelin-13促单核细胞与血管内皮细胞粘附的NOX4源ROS和自噬机制。结果发现：1、Apelin-13诱导人脐静脉内皮细胞ROS的生成和NOX4表达；2、NOX抑制剂DPI和PI3K阻断剂LY294002抑制apelin-13诱导的人脐静脉内皮细胞ROS的生成；Class III PI3K抑制剂3-MA抑制apelin-13诱导人脐静脉内皮细胞ROS水平和NOX4表达的增加；3、Apelin-13诱导人脐静脉内皮细胞自噬标志蛋白beclin1和LC3-II/I的表达增加；mRFP-GFP-LC3融合蛋白标记追踪细胞内LC3，荧光显微镜观察细胞内自噬流，发现Apelin-13诱导人脐静脉内皮细胞自噬流增加，自噬溶酶体降解阻断剂羟基氯喹不能够抑制apelin-13诱导的自噬流增加；4、ROS清除剂NAC、过氧化氢酶类清除剂Catalase和NOX抑制剂DPI抑制了apelin-13诱导人脐静脉内皮细胞自噬标志蛋白beclin1和LC3-II/I表达的增加；NAC和Catalase抑制了apelin-13诱导人脐静脉内皮细胞自噬流增加；5、NAC、Catalase、DPI和3-MA抑制apelin-13促单核细胞-人脐静脉内皮细胞粘附作用； 6、Beclin1 siRNA和LC3 siRNA抑制apelin-13促单核细胞-人脐静脉内皮细胞粘附；自噬诱导剂Rapamycin促进单核细胞-人脐静脉内皮细胞粘附；7、NAC、Catalase、DPI和3-MA抑制apelin-13上调人脐静脉内皮细胞粘附蛋白ICAM-1表达；自噬诱导剂Rapamycin上调人脐静脉内皮细胞粘附蛋白ICAM-1表达。8、发现ApoE-/-（HFD）+Apelin组小鼠冠状动脉粥样硬化斑块明显加，血管内膜上单核细胞粘附明显增加，Apelin-13能够促进动脉粥样硬化斑块形成；结论：Class Ⅲ PI3K-NOX4-ROS-自噬途径介导apelin-13促单核细胞-人脐静脉内皮细胞粘附，实验结果对阐明As发生的apelin/APJ系统参与的自噬新机制有重要意义，apelin/APJ系统有望成为抗As药物新的筛选靶点。