哮喘宁对心理应激哮喘大鼠肺内神经肽影响的实验研究

【作者】 杨保林；

【导师】 武维屏；

【作者基本信息】 北京中医药大学 ， 中医内科学， 2002， 博士

【摘要】 支气管哮喘是当前世界范围内严重威胁公众健康的重要慢性疾病之一。其病因繁多、发病机制复杂，治疗困难。主要以气道非特异性炎症和气道高反应为特征，其中神经机制在其发病中起有重要作用，非胆碱能非肾上腺能神经分泌的神经肽P物质和VIP对气道有调节作用，能使气道收缩，并且使气道粘膜水肿，腺体分泌增加，微血管渗漏，引起神经原性气道炎症。另外哮喘过程中，气道上皮损伤，神经末梢裸露，易受外来刺激使气道反应性升高。由于哮喘是典型的心身疾病，与情绪J心理因素关系密切。目前尚无特效西药。中医学认为情绪心理与肝关系密切，武维屏教授创制的哮喘宁从肝论治哮喘临床效果显著，为探讨其作用机制，本实验尝试建立心理应激哮喘大鼠模型，模拟临床情志因素加重哮喘，评价其可行性；并通过对心理应激模型进行哮喘宁药物干预，观察其对呼吸阻抗、肺通气功能，以及肺组织病理变化，血和肺组织中SP、VIP、IL-4以及NK₁受体基因表达等影响的研究，从功能、组织形态学、以及分子蛋白、基因等几个方面探讨情绪心理因素诱发和加重哮喘的病理机制及哮喘宁的作用途径，为临床应用提供实验依据。 1 心理应激哮喘大鼠模型的建立 按文献法采用卵蛋白加佐剂腹腔注射致敏，卵蛋白气雾吸入引喘法，复制哮喘大鼠模型，利用束缚和夹尾复合因素使其情志不遂，大怒伤肝，从哮喘表现，呼吸阻抗、肺顺应性、肺活量、0.3秒率、用力呼气流量、呼气中期流速，肺组织炎性细胞浸润，血清和支气管肺泡灌洗液中IL-4等改变，评价心理应激哮喘大鼠模型的成功。评价方法：a．体质：大鼠好动易怒，易激惹，属肝郁体质。B应激因素：束缚可制大鼠好动于不能，使其情志不遂，夹尾可使大鼠激怒而伤肝，肝与气机条畅密切相关。c哮喘表现：应激后大鼠除具有较哮喘模型加剧的表现外，还有拒捕、抓咬、不安、狂躁和惊恐、依从两种不同的行为特点。d．肺功能：应激可使哮喘大鼠呼气阻力有明显增高，肺顺应性、0.3秒率以及呼气中期流速等降低。e．组织形态学：正常组支气管、肺组织结构正常，气管上皮完整，肺泡间隔正常，炎性细胞侵润少见。哮喘组肺泡扩张，肺泡间质增宽，炎性细胞浸润，小气道周围炎性细胞浸润，支气管壁有上皮细胞脱落，炎性细胞浸润。心理应激组肺泡扩张较著，甚则断裂融合成肺大泡，肺间质大量炎性细胞浸润，气道及血管周围炎性细胞明显增多，可见大量嗜酸性细胞，支气管管腔狭窄。以上符合哮喘特有的气道炎症、呼吸阻抗升高以及哮喘表现，符合公认的哮喘标准，具有体质、病因、证候特征符合中医证候诊断标准。因此评价模型成功。 2 哮喘宁对心理应激哮喘大鼠模型肺功能和病理变化的影响 本实验运用哮喘宁干预，观察其对心理应激哮喘大鼠呼吸阻抗、肺通气功能及肺组织形态学的改变。结果干预后呼气阻抗明显下降，与应激大鼠有显著差异（P＜0.05）；肺顺应性显著升高，0.3秒率、用力肺活量、呼气高峰流量、呼气中期流速等都有明显升高趋势。肺组织病理变化：哮喘宁干预大鼠肺支气管、肺血管周围未发现嗜酸细胞浸润，其它炎性细胞较应激大鼠和哮喘模型有明显减少，肺间隔增厚较少，肺泡壁断裂少见，气道粘膜较少增厚，卑 哮喘宁对。七理应过哮喘大鼠肺内神经肽影响的试验研究管腔有较少粘液栓．由上可见，哮喘宁能够眺心理应激哮喘大鼠气道阻力，开高其肺顺应性、0．3秒率、用力肺活量、呼气高峰流量、呼气中期流速等肺通气功能．减少肺组织和气道炎性细胞浸润。3哮喘宁对。c理应激哮喘大鼠摸型肺内神经肽及M；受体InRNA表达的影响 按试剂盒要求制备肺组织和血浆SP、VIP检测样品，采用放免法检测各组大鼠肺组织和血浆中SP、VIP的含量。结果哮喘时大鼠血浆和肺组织中SP M均高于正常对照组。束缚加 ’夹尾应激组肺组织SP较哮喘组又有增加（P＜0．05）。Vr的含量，哮喘组较正常组明显俐氏，尤以应激组阐氏趋势较大．哮喘宁干预后其含量升高，而补肾防喘组次之。说明哮喘宁可以陇心理应激哮喘大鼠血浆和肺组织中SP的含量，提高VIP水平。 实验采用放免法检测了哮喘宁对支气管肺泡灌洗液和血清IL－4的含量，结果显示，哮喘模型和。心理应激哮喘大鼠IL－4水平明显高于正常大鼠，哮喘宁干预的大鼠支气管肺泡灌洗液和血清IL－4均明显陶氏。 实验迈三豆用逆转录PCR技术检测肺组织中NK；受体的表达水平，应激大鼠和哮喘模型大鼠水平较高，哮喘宁干预大鼠的水平与正常大鼠无差异，说明哮喘宁可以下调NK；受体的表达水平． 以冶踉明：哮喘宁具有抗气道炎症、提高VIP、阐氏IL叫、SP及其受体的基因表达等作用而防治哮喘．其分子机制可能与调节神经肽，抑制炎性因子的作用有关。提示调节神经－内分泌－免疫网络的调节可能是哮喘宁治疗哮喘的作用途径之一。更多还原

【Abstract】 Bronchial asthma is one of the important chronic diseases that seriously banned the healthy of public in the world at present Its etiological factor is various, pathogenesis i s complicated, and treatment is difficult Airway unspecific inflammation and high response arc the main character of this disease. Among the total, neural regulation play a chief role in die episode. Non-adrenergic nerves excreted substance P (SP) and vasoactive intestina porypeptide (VIP) can adjust die airway. In the detail, they can affect die relaxation and contraction, result in die mucosal edema, die increasing of glandular excretion, the leakage of microvessel, and the neurogenic airway inflammation In addition, in the process of asthma, airway epitheBa injured and nerve ending exposed, so die airway reacivity tend to increase on account of ecdemic stimulation. Asthma is a typical psychophysiological disorder, has close relation to die psychological factor. There is still no special effective western medicine.In die traditional Chinese medicine (TCM), emotion and psyche are closely related to the Liver. Professor Wuweiping created Xiaochuanning granules (XG) treated die astiima by way of treating the Liver, gained notable clinical effect For die reason of finding out die acted mechanism, we tried to create psychologically stressed asthma rat model, simulated clinical emotional agent added asthma, evaluated the feasibility.1 creation of psychologically stressed asthma rat modelAccording to die record of literature, we injected ovalbumin added adjuvant into abdominal cavity to create sensitization, and used ovalbumin aerosol evoked asthma method, reproduced asthma rat modeL At die same time, we adopted die compound method of tie and clip tail to cause stagnancy of the Qi of die liver. After that, we evaluated that die reproduction of psychological stress asthma rat model had been success according to the manifestation of asduna, breathing impedance, lung compliance, vital capacity, 0.3-econd rate, exerted expiratory flow, expiratory metaphase flow-rate, inflammatory cell infiltrate in lung constitution, IL-4 in blood serum and bronchoalveolar lavage fluid, etc.2 influence that XG to pathological alteration and pulmonary function on psychological stress asthma rat modelIn tiiis experiment, we used XG to intervene psychological stress asdima rat model The result manifested tiiat XG affected psychological stress asdima rat model The influence included depressing the airway resistance, and inflammatory eel infiltrate in lung constitution and airway, advancing pulmonary ventilation function. The latter included advancing lung compliance, 0.3-second rate, exerted expiratory flow, expiratory peak flow-rate expiratory metaphase flow-rate, etc.3 influence that XG to neuropeptide in lung and mRNA expression of NKi receptor onpsychological stress asthma rat modelWe used radioimmunoassay to detect the content of SP and VIP that derived from the lung constitution and plasm of each group, and IL-4 in blood semm and bronchoalveolar lavage fluid. The result manifested that all content of SP in the lung constitution and plasm at asthma episode were much more than the normal controlled group. The content of SP in the lung constitution of psychological stress group is more than the asthma group (P<0.05). In the respect of content of VIP, the asthma group obviously decreased than the normal controlled group, especially in the psychological stress group. After intervened by XG, the content of VIP i s more advanced than Bushenfangchuan tablet (BT) group. The result showed that XG can depress the content of SP and advanced the level of VIP in the lung constitution and plasm of psychological stress asthma rats. In the respect of the level of IL-4 in bronchoalveolar lavage fluid and blood serum, asthma model and psychological stress asthma rats is more than the normal controlled group, XG intervened rats are obviously decreased.In the experiment, we used RT-PCR technique to detect the expressed level of NK1 receptor, and discovered更多还原