粉尘超细颗粒致心血管疾病的下丘脑PVN机制

Pollution by particulates has been closely associated with increased cardiovascular morbidity and mortality. However, the mechanisms responsible for these effects are not well-elucidated. Our previous results indicate that ultrafine particles (PM2.5) cause decreased heart rate, blood pressure, increased inducible arrhythmias, myocardial mitochondrial injury and myocardial inflammation. In addition, results from ELISA show that TNF-α is increased in lung, heart and hypothalamus. Interestingly, results from whole-cell patch clamp and calcium imagine show that PM2.5 and IL-1 directly affect neurons activity. These findings led to a central hypothesis that paraventricular nucleus of hypothalamus (PVN) play an important role in PM2.5-induced cardiovascular dysfunction, via direct effect of local PM2.5 and/or the effect of increased inflammatory mediators on PVN GABAergic/glutamatergic neurons. The following 3 specific aims will be pursued in this proposal. The Aim 2 and 3 will be served to test our central hypothesis. Specific Aim 1: To study the effects of inhaled PM2.5 on cardiovascular, hypothalamus, pituitary, adrenal, and kidney in mice. Specific Aim2: To determine whether PVN GABAergic/glutamatergic neurons are required for PM2.5-induced cardiovascular dysfunction. Specific Aim3: To determine whether TNF-α and PM2.5 directly act on PVN GABAergic/glutamatergic neurons, which contributes to PM2.5-induced cardiovascular dysfunction. We have observed obvious cardiovascular injury in PM2.5 intratracheal instillation model rats. In this study, a PM2.5 (DQ12 and FWNT) inhalation mouse model will be served. Stereotaxic surgery, neuroanatomy, comprehensive cardiovascular function measurements and cutting-edge techniques including whole-cell patch clamp, long-term telemetric measurement, Cre-LoxP and optogenetic will be engaged for our 3 aims. The current proposal will definitely determine the contribution of PVN GABAergic/glutamatergic neurons to PM2.5-induced cardiovascular dysfunction (Aim 1 and 2), and to explore the underlying cellular mechanisms how inhaled PM2.5 affects PVN GABAergic/glutamatergic neurons activity (Aim 3). To our knowledge, the proposed experiments are the first study that will use the powerful genetic mouse models, whole-cell patch clamp, and optogenetic to determine how inhaled PM2.5 causes cardiovascular dysfunction and the contribution of PVN GABAergic/glutamatergic neurons. Findings from this proposal may not only reveal the fundamental mechanisms underlying PM2.5-induced cardiovascular diseases, but also may provide a rationale for the development of preventing and therapies for patients with air pollution.

粉尘超细颗粒物（PM2.5）浓度每增加10μg/m3，总死亡率、心肺死亡率分别增加13%和17%。前期工作表明PM2.5致心血管系统损伤，心、肺、下丘脑炎症反应。但粉尘致心血管损伤的机制尚不明了。我们提出粉尘致心血管疾病的下丘脑心血管中枢PVN假说，即进入脑组织的粉尘直接作用和/或通过增加的炎症介质，作用于PVN神经元，参与心血管疾病。本课题中，我们将建立PM2.5染毒小鼠模型和光遗传学实验模型，应用形态学，机能学，脑片膜片钳，Cre-loxP等，研究PM2.5对小鼠心、下丘脑、垂体、肾上腺、肾等器官的影响；PVN 的GABA或Glutamate神经元是否在PM2.5致心血管功能紊乱中起重要作用以及关键离子通道；炎症介质和PM2.5是否直接作用于PVN神经元，影响心血管功能。本课题的完成将论证粉尘致心血管疾病的PVN假说，并填补这一研究领域的空白。同时，为有效减轻大气污染致人类健康危害提供具有新的解决。

过去十年间，大量临床和流行病学资料揭示粉尘损害人类健康，和心血管疾病以及猝死之间存在关联。这些环境污染物与心血管疾病所致住院率和致死率增加之间有密切关联，尤其是原先患有心衰和/心律失常的病人对大气颗粒物致损伤作用更加易感。然而，粉尘如何导致心血管疾病的机制目前尚不明了，有待研究。我们提出粉尘致心血管疾病的下丘脑中枢PVN假说，即进入脑组织的粉尘直接和/或通过增加的炎症介质，影响PVN神经元的兴奋性，参与心血管疾病的发生发展。为论证我们的假说，本课题将进行下述四方面实验：1）利用气管滴注法建立长期粉尘染毒小鼠模型，检测长期染毒低剂量粉尘是否对小鼠心肺等器官造成损伤；2）利用脑立体定位PVN注射AAV-ChR2-EGFP病毒于小鼠，制备ChR2-EGFP特异性表达于PVN神经元的光遗传学实验模型，运用脑片膜片钳，验证粉尘是否改变光遗传学小鼠PVN神经元电生理功能；3）运用脑片膜片钳，检测TNF－alpha和粉尘是否对小鼠PVN神经元电生理活性有直接影响；4）利用体外培养的乳鼠心肌细胞，检测粉尘是否对心肌细胞有直接作用。通过以碳纳米管为主要研究对象，我们发现碳纳米管经气管滴注染毒小鼠10周，可致小鼠肺部肺泡融合及局部炎性病灶发生以及心肌局部炎性病灶发生及肌纤维排列方向紊乱。运用我们制备的光遗传学小鼠，我们发现光刺激（10Hz）可引起PVN神经元GABAergicIPSC，并增加神经元动作电位频率。孵育碳纳米管可减低同等强度光刺激引起的PVN神经元GABAergic IPSC幅度。运用脑片膜片钳，我们发现碳纳米管和TNF－alpha直接给药直接增加PVN神经元兴奋性。粉尘超细颗粒孵育离体心肌细胞，可造成心肌细胞一定的损伤。本课题首次结合光遗传学小鼠模型，脑片膜片钳，形态学和生化检测等实验方法研究长期暴露碳纳米管对心血管的损伤及中枢PVN机制。本研究为深入了解粉尘对心血管的损伤及其发生发展过程，并为制定粉尘的生物安全性评价标准以及防护粉尘对人类的卫生健康提供理论依据。