中枢抗利尿激素过度激活气道迷走神经的突触机制与哮喘发病

Excessive activation of the airway vagal nerves followed by antidiuretic hormone-neurons excited plays a critical role in the pathogenesis of psychosocial-stress-induced asthma.The airway vagal nerves derive from airway vagal preganglionic neurons (AVPNs) located in the external of nuleus ambiguus. Previous studies indicated that AVPNs receive projections from paraventricular vasopressin-containing neurons and express antidiuretic hormone receptors. The modulation of AVPNs by ADH maybe involved in asthma attack.However, how ADH modulates AVPNs at synaptic levels remains unkonwn. And what's more, the hypothesis that the synaptic control of AVPNs by ADH involved in the pathogenesis of psychosocial stress-induced asthma is lack of experimental evidence.Therefore, in a murine model of asthma caused by psychological stress, radioimmunoassay method is used to show more ADH release and immunohistochemistry technique is used to demonstrate ADH receptors upregulate when asthma attacks.In addition, in medullary slices, AVPNs are labelled using retrogradely fluorescent labeling techniques.Then, we use patch clamp techniques to study how ADH and its receptor-antagnoists as well as PKA/PKC antagnoists regulate the activity of AVPNs at synaptic levels. We will clarify central mechanisms of asthma caused by psychosocial stress.

中枢抗利尿激素（ADH）神经元活性增加，导致气道迷走神经过度激活，是心理社会应激诱发哮喘发作的关键机制。气道迷走神经主要起源于延髓疑核外侧部的气道迷走节前神经元（AVPNs）。AVPNs兴奋程度决定了气道迷走神经活性的大小。研究提示：AVPNs表面广泛表达ADH受体，并接受ADH的调控，可能与心理社会应激诱导哮喘发病密切相关。然而，在突触水平，ADH调控AVPNs的生理机制未经证实，参与心理社会应激诱发哮喘发病的学说更缺乏实验依据。因此，本项目拟：（1）分别在野生型及ADH受体基因敲除型心理应激哮喘模型小鼠，应用放射免疫、免疫组织细胞化学等方法，证实ADH在呼吸中枢释放增加、ADH受体表达上调参与心理应激诱发哮喘发病；（2）在小鼠离体延髓脑片，应用逆行荧光标记及膜片钳技术，研究ADH及受体拮抗剂、PKA/PKC拮抗剂对AVPNs的调控及机制，阐明心理社会应激诱发哮喘发病的中枢机制。

气道迷走神经功能亢进导致气道阻力增加，为哮喘加重的主要呼吸动力学机制。气道迷走神经的活性主要取决于位于延髓腹外侧的气道迷走节前神经元（AVPNs）。心理应激影响“神经-免疫-内分泌”网络，使中枢相关内分泌激素分泌失调，进而激活AVPNs，为心理应激时哮喘恶化的重要原因。最新文献报道，心理应激时中枢组胺结合H1型受体，导致哮喘恶化。研究发现，下丘脑室旁核（PVN）内的抗利尿激素（ADH）纤维接受情绪中枢-杏仁核的支配，投射至AVPNs，应激时ADH释放增加，参与呼吸调控。本课题以前期研究为基础，制备哮喘、心理应激诱发哮喘加重（PS）的小鼠模型，应用逆行荧光标记、real-time PCR、western-blot、酶联免疫吸附测定、免疫组织化学等方法，验证心理应激时，中枢ADH释放增加，结合于AVPNs表面ADH受体，进而激活PKA/PKC信号转导通路，参与心理应激所致的哮喘恶化的假说。本研究结果如下：与对照组及哮喘组相比，PS小鼠脑脊液中ADH含量明显升高、AVPNs表面ADH受体表达明显增加；PS小鼠肺组织中EPO、MPO活性增强、IgE及组胺含量明显增加；肺泡灌洗液中炎症细胞及炎症因子含量显著增高。ADH受体拮抗剂及PKA、PKC拮抗剂明显抑制PS小鼠PKAα、PKCα蛋白含量的增加，降低EPO、MPO活性，抑制IgE、组胺的含量增加，明显抑制炎症细胞及细胞因子的释放。这些结果表明，心理应激时，中枢ADH释放增多，通过AVPNs表面的ADH受体激活PKA/PKC途径，诱发哮喘加重，揭示了难治性哮喘或哮喘持续状态发病的重要中枢机制。此研究成果已投稿Cellular physiology and biochemistry杂志并接受。