自噬调控线粒体介导NLRP3炎症体信号轴在单核/巨噬细胞极化及动脉粥样硬化中的作用及机制

Inflammation plays a primordial role in all phases of atherosclerosis, from the initial recruitment of innate and acquired immune cells to the arterial wall to later atherothrombotic events. Monocyte/Macrophages compose the majority of the inflammatory burden in plaques and incite many of the deleterious responses that exacerbate disease. In response to stimulation, they demonstrate the extent of heterogeneity and exert their different function as well. Pro-inflammatory monocytes, and classically activated (M1) macrophages could produce a wide variety of pro-inflammatory cytokines; while surveillance or resident monocytes and alternatively activated（M2）macrophages participate in anti-inflammatory responses and promote inflammatory resolution and tissue repair. The polarization of monocyte/macrophage, referred as the switch between the different phenotypes, could mediate the inflammatory response that operates in atherosclerosis. It is important to explore the key mediators in the regulation of the phenotype and role of monocyte/macrophage in inflammation. Autophage is an evolutionary conserved process involved in the delivery of long-lived proteins and excess or dysfunctional organelles to the lysosomal machinery for ultimate degradation and recycling. The results showed autophage becomes dysfunctional in atherosclerosis and its deficiency promotes vascular inflammation, oxidative stress, and plaque rupture. However, how autophage is induced in atherosclerotic lesions and influence plaque stability is little known. In our previous study, we found some key factors protect against inflammation by the inhibition of pro-inflammatory COX-2 pathway and reduce oxLDL-induced intracellular lipid loading by inhibiting lipid influx and enhancing lipid efflux in activated macrophage; activation of autophage could inhibit pro-inflammatory cytokines secretion. Our preliminary data also showed that in mouse model for vulnerable atherosclerotic plaque, atorvastatin improves plaque stability by regulating chemokines and chemokine receptors. Moving forward, in this project, we will explore the role and mechanism of autophage in the polarization of monocyte/macrophage, the stages of inflammation and eventually the progression of atherosclerosis; whether NLRP3 inflammasome mediated by mitochondrial damage is involved in the process. Thus we explore the feasibility and effectiveness therapeutic targets for atherosclerosis through the switch between the different phenotypes of monocyte/macrophages by selective autophage.

动脉粥样硬化是危险因素及免疫机制共同参与的慢性炎症过程，激活的炎症是斑块破裂的主要因素。单核/巨噬细胞是血管炎症反应的主要效应细胞，其极化状态调控着炎症反应转归，动脉粥样硬化的进程。明确调控免疫细胞极化状态的因素及机制是斑块稳定性的关键。自噬是将衰老细胞器运输到溶酶体，降解或再循坏的细胞代谢过程。研究报道，动脉粥样硬化斑块内显示自噬功能紊乱，而自噬基因缺陷更易形成动脉粥样硬化。但自噬是如何调控炎症反应及动脉粥样硬化知之甚少。我们前期研究，发现了激活巨噬细胞中脂质负荷、炎症反应的一些调控因素及信号通路；激活自噬抑制了炎症因子的分泌；在动脉粥样硬化动物模型中，调控炎症因子及受体可以改善斑块的稳定性。结合前期工作，此次课题，拟探讨自噬是否通过线粒体介导炎症体信号轴调控单核/巨噬细胞极化状态，炎症反应转归及动脉粥样硬化斑块稳定性，以探索通过选择性自噬介导免疫细胞极化状态来防治动脉粥样硬化的可行性。

巨噬细胞是血管炎症反应的主要效应细胞，其极化状态调控着炎症反应转归，动脉粥样硬化（AS）的进程。明确调控巨噬细胞极化状态的因素及机制是斑块稳定性的关键。AS斑块内显示自噬功能紊乱，而自噬基因缺陷更易形成AS。该项目中，我们探讨了自噬是否通过线粒体介导NLRP3炎症体信号轴调控巨噬细胞极化，炎症反应转归，进而AS斑块的稳定性。我们发现，自噬参与了巨噬细胞炎症反应的调控，阿托伐他汀（at）呈剂量及浓度依赖性诱导巨噬细胞自噬，而抑制LPS诱导的巨噬细胞炎症因子IL-1β和TNFα分泌，给予自噬抑制剂3MA或LY294002抑制自噬及小干扰RNA (ATG5siRNA)后，at对LPS诱导的炎症因子分泌无明显抑制作用，机制表明at通过AKT/mTOR信号通路诱导巨噬细胞自噬，抑制炎症因子分泌，发挥抗炎作用；我们建立了易损斑块的动物模型，斑块内可见大量巨噬细胞浸润，自噬相关蛋白（LC3-Ⅱ、p62/S QSTM1）的表达及NLRP3炎症小体的激活,而at明显诱导巨噬细胞自噬，且通过减轻脂质沉积，减少巨噬细胞的浸润及增加胶原的含量，减少炎症因子IL-1β、TNFα和IL-6的分泌，促进AS的稳定性；在AS易损斑块模型中，自噬激动剂雷帕霉素(Rap)通过减少巨噬细胞凋亡，抑制斑块坏死核心的形成，而3-MA具有相反作用，机制研究表明，激活自噬减轻了线粒体功能紊乱，线粒体结构受损、细胞色素c释放、线粒体膜电位降低、线粒体裂变、ROS的产生、Caspase-9和Caspase-3的激活等，自噬抑制剂则加重了线粒体功能失调，MAPK‐NF‐κB通路参与了调控过程。课题通过自噬对炎症型巨噬细胞生物学功能的调控及AS不稳定斑块的研究，发现激活自噬通过促进线粒体稳态，调节活化型巨噬细胞向替代型巨噬细胞转换，抑制炎症反应、细胞凋亡，而稳定斑块的新机制，为AS的防治提供了新靶点及理论依据。