NOX1及mtROS介导的eNOS解偶联在PM2.5致心血管毒性中的作用及机制研究

Recently, the relationship of air particulate with public health has aroused great attention. Mounting epidemiological and experimental data revealed that PM2.5 exposure is closely related to the onset and progression of cardiovascular disease. Additionally, inflammatory reaction mediated by ROS is the independent risk factor of cardiovascular lesion in response to PM2.5, in which multiple signaling pathways are involved. However, with regard to the cardiovascular toxicity of PM2.5, the activation of inflammasome and the pyroptosis of vascular endothelial cells have not yet been reported. And the molecular mechanism of vascular endothelial pyroptosis is still not clearly defined. Based on the results of epidemiological data, preliminary transcriptome sequencing and molecular biology experiments, we choose the in vitro model of vascular endothelial cells, in vivo model and blood samples of population in order to explore the sources of ROS production after PM2.5 exposure, which can be attributed to NADPH oxidase and mitochondrial reactive oxygen species. Meanwhile, the role of ROS burst, eNOS uncoupling, NLRP3 inflammasome and inflammatory cascade reaction in PM2.5-induced cardiovascular lesion will be investigated. Combined with in vitro, in vivo and epidemiological research, the inflammatory mechanism of PM2.5-induced cardiovascular lesion will be clarified. Meanwhile, models with a particular gene knock down or/and knock in would be used to comprehensively analyze the ROS/eNOS/NLRP3 signaling pathway so as to look for sensitive biomarkers, and provide a theoretical reference for the public health protection.

近年来，大气污染与人群健康引起了高度关注。大量流行病学和体内、外实验资料显示，PM2.5与心血管疾病的发生、发展呈相关性，而ROS介导的炎性反应是PM2.5诱导心血管损伤的独立危险因素，涉及多条信号通路，其中PM2.5致血管内皮细胞炎性小体的激活尚未见报道，细胞焦亡分子机制尚不明确。基于人群流行病学资料及前期转录组测序和分子生物学实验结果，我们选择发挥重要屏障作用的血管内皮细胞、小鼠模型及人群血液样本，拟从ROS的来源途径--NADPH氧化酶和线粒体活性氧簇出发，探讨PM2.5诱导的ROS爆发和eNOS解偶联，进而激活NLRP3炎性小体和炎性级联反应在心血管毒性中的作用；并通过目的基因沉默和/或过表达的细胞和动物验证ROS/eNOS/NLRP3信号通路在PM2.5致心血管毒性中的作用；寻找PM2.5致心血管损伤的生物标志，为人群心血管疾病的防护提供理论参考。

近年来，城市大气颗粒物引起人群的心血管损伤受到广泛关注。本项目以城市大气颗粒物的标准参考材料1648a（PM SRM1648a）暴露的血管内皮屏障毒效应为切入点，通过BALB/c小鼠的动物实验，结合体外内皮细胞暴露模型，进一步深入探讨PM SRM1648a暴露对心血管系统的毒性效应。以人脐静脉血管内皮细胞系EA.hy926和HUVECs进行体外实验，首先评估了血管内皮细胞在PM SRM1648a刺激下的氧化应激状态，并明确PM SRM1648a暴露导致EA.hy926和HUVEC细胞中产生氧化应激的来源和eNOS解偶联的发生，以及引起的内皮功能障碍和炎症反应；通过NOX1/4抑制剂GKT137831的预处理反向验证，证明了PM SRM1648a诱导的NOX1和NOX4上调是内皮细胞中ROS的来源，并通过引起血管内皮细胞eNOS解偶联，激活AKT/eNOS/NO信号反应，导致细胞内皮功能障碍和炎症损伤。其次，以线粒体动力学探讨颗粒物暴露扰乱血管内皮细胞线粒体功能，包括mtROS增高、MMP减少和ATP消耗，损害线粒体生物发生。用流式细胞仪检测细胞焦亡率和caspase1活性，PM SRM1648a激活caspase1酶活性和炎症级联反应，caspase1抑制剂Z-YVAD-FMK可以显著降低颗粒物引起的caspase1激活及IL-1β炎性因子的释放；以及运用siRNA慢病毒转染敲降技术反向讨论PM SRM1648a暴露经DRP1/caspase1/IL-1β信号通路扰乱血管内皮细胞线粒体分裂-融合平衡，进而触发炎症反应，细胞焦亡不是PM SRM1648a引起血管内皮细胞程序性死亡的主要作用模式。BALB/c小鼠在急性和亚急性暴露后，出现局部和全身的氧化应激以及炎性反应，亚急性组出现主动脉壁增宽以及主动脉根部胶原蛋白增多趋势，心肌组织线粒体结构层面分子表达出现改变，高剂量组（20 μg/cm2）触发了心脏组织mTOR/AKT/GSK-3β通路激活，导致心肌细胞的肥大。通过一系列实验，我们首次证明了城市大气颗粒物（PM SRM1648a）可以通过损害线粒体功能进而导致血管内皮细胞产生炎症反应和心肌肥大。