Exosome/TLR4通路在细颗粒物诱发血压升高中的作用机制

Previous studies have demonstrated a strong link between the concentration of particulate matter (PM) in air and hypertension, in which endothelium dysfunction induced by inflammation and oxidative stress plays a pivotal role. Exosomes are bilipid membranous vesicles secreted from cells upon pathologic or stress stimulation and mediate immune response via activation of TRL4 pathways. However, whether exosome-TLR4 pathway is involved in the PM2.5-mediated elevation of blood pressure remains unknown. We hypothesize that PM2.5 inhalation will induce abnormal secretion of exosomes which activate TLR4 signaling pathway, leading to endothelium dysfunction and elevated blood pressure. Firstly, the effect of PM2.5 inhalation on blood pressure and vascular endothelium function will be examined. Secondly, Role of TLR4 in PM2.5-elevated blood pressure will be investigated with TLR4 antibody treatment or endothelium-specific TLR4-/- mice. Thirdly, effect of PM2.5 on concentration and characteristics of serum exosomes will be examined. Fourthly, Whether PM2.5-exposed mice-derived serum exosomes could induce endothelium dysfunction via TLR4 will be determined with isolated artery rings and cultured endothelial cells. Finally, the underlying mechanism of it will be explored at the integral level, tissue level and cellular level with focusing on inflammatory response and oxidative stress and by examining the TLR4 and eNOS signaling pathway. The present study will open new direction for the etiology and underlying mechanism of hypertension, and will provide important basis for the potential targets for its prevention and treatment.

高血压与细颗粒物（PM2.5）密切相关，血管内皮功能紊乱是其重要机制。Exosome是细胞受到刺激分泌的微小膜泡，通过激活TLR4调控免疫反应。但exosome-TLR4通路是否介导PM2.5调节的血压升高及机制尚无报道。我们假设PM2.5暴露引起exosome分泌异常，通过TLR4导致血管内皮功能受损从而引起血压升高。本项目将首先明确PM2.5对血压和血管内皮功能的影响；接下来借助TLR4中和抗体和内皮特异性TLR4-/-小鼠明确TLR4在这一过程中的作用；然后检测PM2.5吸入对exosome分泌的影响，并通过体外实验明确PM2.5暴露小鼠血清中exosome通过TLR4引起血管内皮功能损伤；最后在整体、组织和细胞水平就炎症和氧化应激深入探讨exosome调节PM2.5诱发血压升高的作用机制及信号通路。本项目将为高血压发病病因和机制提出新的研究方向，也为高血压防治新靶点的提出提供依据。

大气污染与高血压、糖尿病、非酒精性脂肪肝等代谢综合症密切相关。但是，其毒性作用及其机制远未阐清。本研究利用C57BL/6小鼠、AngII诱导的高血压模型鼠、CCR2-/-小鼠、TLR4-/-小鼠建立PM2.5全身暴露模型，深入探讨PM2.5诱发代谢综合症的机制。研究结果表明：1）杭州地区PM2.5吸入1个月并未改变基础血压，但引起AngII微渗泵处理小鼠血压升高、心率增快；并且微渗泵失效后PM组小鼠平均动脉压和收缩压显著高于FA组。PM2.5急性暴露引起小鼠主动脉内皮依赖性和非依赖性血管舒张作用显著受抑，肾动脉内皮非依赖性舒张作用受损；TLR4功能缺失或TLR4抑制剂处理部分阻断了这一损害效应。血浆外泌体CD63和CD81表达增强，蛋白组学分析显示原发性免疫反应信号通路异常。2）PM2.5吸入后小鼠出现胰岛素抵抗和肝脏脂质沉积，雌性小鼠更为敏感；脂质组学显示，PM2.5吸入引起肝脏中性脂肪、游离脂肪酸水平升高，甘油磷脂分布异常、肝脏醛缩磷脂水平增加；上述改变可能与长期PM2.5暴露抑制下丘脑-垂体-肾上腺皮质轴功能有关。3）PM2.5吸入后小鼠肝脏组织生物钟基因表达改变，并破坏转录因子和脂肪代谢酶的表达节律。表现为PM组小鼠肝脏时钟基因Bmal1、Cry1、Rev-erbα基因、肝脏脂肪功能相关调节因子Pparα、脂肪摄取和分解相关因子（Fabp2, Cpt1α, Vlcad）在不同ZT点表达增加，伴有节律异常。4）PM2.5吸入后小鼠白色脂肪和棕色脂肪组织昼夜节律关键基因表达模式和水平均出现显著改变，表现为生物钟基因、脂肪功能分子、脂质代谢和线粒体功能相关分子表达异常和节律紊乱。5）在正常饮食状态下，CCR2基因敲除虽然抑制了PM2.5吸入引起的小鼠内脏脂肪巨噬细胞浸润，但并不能改善PM2.5吸入引起的高血糖以及胰岛素敏感性的降低；CCR2基因敲除伴随有糖异生途径激活。本项目将为环境污染引发代谢性疾病提供新的治疗靶标，为脂类物质代谢异常提供代谢库和新的潜在研究靶点；从昼夜节律紊乱的角度揭示PM2.5暴露引发肝脏脂质代谢异常，为空气污染引发代谢综合症研究开辟新的视角。