哮喘宁颗粒剂对哮喘大鼠糖皮质激素受体影响的实验研究
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摘要
上世纪60年代以来，支气管哮喘(简称哮喘)的发病率和死亡率均呈逐年上升趋势，加之哮喘病程绵长，迁延难愈，给社会带来了严重的经济负担。由此，哮喘已成为严重的公共卫生问题而引起了世界各国的极大关注。
哮喘是一种慢性气道炎症性疾患，气道高反应和可逆性气流受限与气道炎症密切相关，糖皮质激素是治疗哮喘慢性气道炎症的首选药物。近年来，大量研究发现，未经糖皮质激素治疗和经过糖皮质激素治疗的患者均存在糖皮质激素受体(glucocorticoid receptor GCR)的异常，包括数量的减少和功能的缺陷，而糖皮质激素受体异常的原因尚未明确。应用糖皮质激素必然导致其受体水平的进一步下降，可能导致哮喘加重。由此，近年来人们越来越关注应用糖皮质激素所导致的副作用。寻求从根本上治疗哮喘，而非单纯改善哮喘症状的药物成为当务之急。
导师武维屏教授认为哮喘病位虽在肺，但与肝关系密切，强调风、痰、气、瘀、虚为哮喘发作的主要病机，而肝肺失调、气血失和是其病机关键。治疗之时，从调肝理肺、和调气血入手。根据此法创制的中药复方制剂——哮喘宁颗粒剂，已在临床应用多年，疗效确切。
本课题即以此为基础，以卵蛋白为致敏原，氢氧化铝和百日咳杆菌为免疫佐剂，腹腔注射致敏SD大鼠，卵蛋白溶液雾化激发，制备大鼠过敏性哮喘模型，观察了哮喘宁颗粒剂对哮喘大鼠GCR的影响作用。并且在原大鼠哮喘模型基础上，通过腹腔注射地塞米松，制备经过激素干预后的哮喘大鼠模型，观察激素干预后哮喘大鼠的GCR水平以及哮喘宁颗粒剂对哮喘模型大鼠GCR的影响作用，探讨哮喘宁颗粒剂治疗哮喘的疗效机理。
1．对过敏性哮喘模型大鼠一般状态观察、小动物肺功能测定仪检测肺功能和对肺组织形态学进行光镜观察。结果表明：哮喘大鼠出现哮喘的典型症状(如呼吸急促、节律不整，呼吸幅度增大且不齐)，呼吸连续激发后出现呼吸频率加快；肺通气功能降低，表现为呼气阻力和吸气阻力升高，肺顺应性降低，最大通气量减小，0.3秒率，呼气峰流量和用力呼气中期流量下降；出现典型的肺组织病理形态学改变，支气管粘膜上皮细胞排列紊乱、部分粘膜上皮细胞脱落，淋巴细胞、嗜酸性粒细胞浸润。
哮喘宁颗粒剂可有效改善哮喘大鼠的哮喘症状，控制连续激发后哮喘大鼠呼吸频率的加快；改善哮喘大鼠的肺通气功能，降低呼吸阻力，增强肺顺应性，提高最大通气量，略增加0.3秒率、呼气峰流量和用力呼气中期流量；显著改善哮喘大鼠肺组织的病理形态学异常，减少淋巴细胞和嗜酸性粒细胞浸润。
2．检测大鼠血清和支气管肺泡灌洗液中的IL-4水平。结果表明，模型组大鼠血清和支气管肺泡灌洗液IL-4水平较正常组明显升高，支气管灌洗液IL-4水平的升高尤其显著；哮喘宁颗粒剂可显著降低哮喘大鼠血清和支气管肺泡灌洗液IL-4水平。
3．放射配基结合实验单点饱和分析法测定哮喘大鼠肺组织GCR的最大特异结合量，以RT-PCR半定量分析法检测GCR mRNA的表达情况，并检测了血清皮质醇浓度。结果表明哮喘大鼠肺组织GCR最大特异结合量显著下降，并且GCR mRNA表达
4 哮喘宁颗粒剂人哮喘大鼠糖皮质激素受体影响的实验研究
量明显减少，血清皮质醇浓度显著降低。哮喘宁颗粒剂可上调哮喘大鼠肺组织GCR
的最大特异结合量，且上调GCR fnR＊的表达，并且提高血清皮质醇浓度。
4．地塞米松干预后的哮喘大鼠激发后出现哮喘症状较普通模型组略迟，但是症
状迅速加重，连续激发后呼吸频率显著加快，肺通气功能降低，且出现肺组织病理
形态学改变，出现支气管粘膜上皮细胞脱落，大量淋巳细胞、嗜酸性粒细胞浸润。
哮喘宁颗粒剂可以改善激素干预后哮喘大鼠的症状，控制呼吸频率，改善肺通气功
能，降低气道阻力，提高肺顺应性，提高最大通气量和用力呼气中期流量，并且有
效改善肺组织病理形态学的异常，减少淋巴细胞、嗜酸性粒细胞浸润。
5．激素干预后哮喘大鼠模型肺组织 GCR最大特异结合量及 GCR ＂dNA显著下
降，且较普通哮喘模型有下降的趋势。哮喘宁颗粒剂可上调激素于预后哮喘大鼠肺
组织GCR的最大特异结合量和GCR lNA的表达。
本课题通过动物实验证实了哮喘宁颗粒剂治疗哮喘及经激素干预后哮喘的有效
性，其疗效机制可能与上调肺组织CCR水平有关。
According to the survey of epidemiology the incidence rate and the death rate of asthma is increasing since 1960s. Together with the long course and the difficulty of cure, asthma brings heavy economic burden to society. So it attracts much attentions of countries around the world.
Asthma is a kind disease of chronic airway inflammation, and airway hyperreactivity (AHR) and reversible airstream restriction are related to airway inflammation closely, and glucocorticoid is the principle choice for asthma treatment. However, many recent researches suggest that there is abnormal GCR (glucocorticoid receptor) level including quantity reduction and functional defect in asthma patients with and without glucocorticoid treated, and reason of abnormal GCR standard is still confirmed. It is truism that glucocorticoid will lead to further descent of GCR standard. So, it is urgent to seek theraputic drugs for asthma in essence not simply improving asthmatic symptom.
In the opinions of my advisor professor Wu Weiping, the position of asthma is the lung, it is related to the liver closely. "Wind", "phlegm", "Qi", "stasis", "asthenia" are the main pathogenesis of asthma, and disharmony of the liver and lung, disturbance of Qi and blood is the key point of pathogenesis. So regulate the function the liver and the lung, adjust Qi and blood is in common use when treat asthma, and it's effective in clinic. The complex prescription of traditional Chinese madicine梄iao Chuan King granule created on the basis of the theory and has been used in clinic for many years shows definite therapeutic effects. This prescription gained good effects not only in mild and moderate asthma but also in severe glucocorticoid dependend asthma by reducing the oral dosage of glucocorticoid.
The purpose of this study is finding out the pharmacological mechanism of Xiao Chuan King granule treating ashtma. During the experiment, we repeat animal models of allergic asthmatic rats by ovalbumin mixture intraperitoneal injection, ovalbumin acting as allergen, aluminium hydroxide and bordetella pertussis acting as immune adjuvent. Using Bu Shen Fang Chuan pill as the control medicament, search possible pharmacological effect of Xiao Chuan King granule on GCR.
1 observing asthmatic rats' common state, inspecting pulmonary function of asthmatic rats, observing morphology of pulmonary tissues under microscope suggest, that the asthmatic rats appeared typical symptoms of asthma including fast respiratory frequency > lower pulmonary ventilation function and typical pathologic changes of lung tissues. Xiao Chuan King granule can (1) effectively improve rats' asthmatic symptoms. (2) lower asthmatic rats' respiratory frequency. (3) improve pulmonary ventilation function by reducing resistance of expiration and resistance of inspiration, rising pulmonary compliance, rising maximum ventilatory volume, appreciably rising 0. 3FEV% , appreciably rising peak expiratory flow and appreciably rising forced expiratory flow 25% -75%. (4) improve asthmatic rats morphological abnormal by decreasing lymphocytic cells and eosinophilic granulocytes soakage.
2 Examining asthmatic rats' IL-4 level in serum and BALF suggest, that asthmatic rats' IL-4 level is increasing in serum, especially in BALF, and Xiao Chuan Ning granule can reduce the IL-4 level in serum and in BALF.
3 Examining GCR maximum specific binding in asthmatic rats' lung tissues, GCR mRNA expression and cortisol concentration in serum suggest, that GCR maximum specific binding reduce obviously as well as GCRmRNA expression, while cortisol concentration in serum reduce obviously. Xiao Chuan Ning granule can increase GCR maximum specific binding reduce obviously, GCRmRNA expression, and cortisol concentration in serum.
4 By intervented with dexamethasone, asthmatic rats represent typically asthmatic appearance later but badly little by little, respiratory frequency becoming fast obviously, pulmonary ventilation function reducing and lung tissues appearing typical pathomorphologic changes. Xiao Chuan
哮喘是一种慢性气道炎症性疾患，气道高反应和可逆性气流受限与气道炎症密切相关，糖皮质激素是治疗哮喘慢性气道炎症的首选药物。近年来，大量研究发现，未经糖皮质激素治疗和经过糖皮质激素治疗的患者均存在糖皮质激素受体(glucocorticoid receptor GCR)的异常，包括数量的减少和功能的缺陷，而糖皮质激素受体异常的原因尚未明确。应用糖皮质激素必然导致其受体水平的进一步下降，可能导致哮喘加重。由此，近年来人们越来越关注应用糖皮质激素所导致的副作用。寻求从根本上治疗哮喘，而非单纯改善哮喘症状的药物成为当务之急。
导师武维屏教授认为哮喘病位虽在肺，但与肝关系密切，强调风、痰、气、瘀、虚为哮喘发作的主要病机，而肝肺失调、气血失和是其病机关键。治疗之时，从调肝理肺、和调气血入手。根据此法创制的中药复方制剂——哮喘宁颗粒剂，已在临床应用多年，疗效确切。
本课题即以此为基础，以卵蛋白为致敏原，氢氧化铝和百日咳杆菌为免疫佐剂，腹腔注射致敏SD大鼠，卵蛋白溶液雾化激发，制备大鼠过敏性哮喘模型，观察了哮喘宁颗粒剂对哮喘大鼠GCR的影响作用。并且在原大鼠哮喘模型基础上，通过腹腔注射地塞米松，制备经过激素干预后的哮喘大鼠模型，观察激素干预后哮喘大鼠的GCR水平以及哮喘宁颗粒剂对哮喘模型大鼠GCR的影响作用，探讨哮喘宁颗粒剂治疗哮喘的疗效机理。
1．对过敏性哮喘模型大鼠一般状态观察、小动物肺功能测定仪检测肺功能和对肺组织形态学进行光镜观察。结果表明：哮喘大鼠出现哮喘的典型症状(如呼吸急促、节律不整，呼吸幅度增大且不齐)，呼吸连续激发后出现呼吸频率加快；肺通气功能降低，表现为呼气阻力和吸气阻力升高，肺顺应性降低，最大通气量减小，0.3秒率，呼气峰流量和用力呼气中期流量下降；出现典型的肺组织病理形态学改变，支气管粘膜上皮细胞排列紊乱、部分粘膜上皮细胞脱落，淋巴细胞、嗜酸性粒细胞浸润。
哮喘宁颗粒剂可有效改善哮喘大鼠的哮喘症状，控制连续激发后哮喘大鼠呼吸频率的加快；改善哮喘大鼠的肺通气功能，降低呼吸阻力，增强肺顺应性，提高最大通气量，略增加0.3秒率、呼气峰流量和用力呼气中期流量；显著改善哮喘大鼠肺组织的病理形态学异常，减少淋巴细胞和嗜酸性粒细胞浸润。
2．检测大鼠血清和支气管肺泡灌洗液中的IL-4水平。结果表明，模型组大鼠血清和支气管肺泡灌洗液IL-4水平较正常组明显升高，支气管灌洗液IL-4水平的升高尤其显著；哮喘宁颗粒剂可显著降低哮喘大鼠血清和支气管肺泡灌洗液IL-4水平。
3．放射配基结合实验单点饱和分析法测定哮喘大鼠肺组织GCR的最大特异结合量，以RT-PCR半定量分析法检测GCR mRNA的表达情况，并检测了血清皮质醇浓度。结果表明哮喘大鼠肺组织GCR最大特异结合量显著下降，并且GCR mRNA表达
4 哮喘宁颗粒剂人哮喘大鼠糖皮质激素受体影响的实验研究
量明显减少，血清皮质醇浓度显著降低。哮喘宁颗粒剂可上调哮喘大鼠肺组织GCR
的最大特异结合量，且上调GCR fnR＊的表达，并且提高血清皮质醇浓度。
4．地塞米松干预后的哮喘大鼠激发后出现哮喘症状较普通模型组略迟，但是症
状迅速加重，连续激发后呼吸频率显著加快，肺通气功能降低，且出现肺组织病理
形态学改变，出现支气管粘膜上皮细胞脱落，大量淋巳细胞、嗜酸性粒细胞浸润。
哮喘宁颗粒剂可以改善激素干预后哮喘大鼠的症状，控制呼吸频率，改善肺通气功
能，降低气道阻力，提高肺顺应性，提高最大通气量和用力呼气中期流量，并且有
效改善肺组织病理形态学的异常，减少淋巴细胞、嗜酸性粒细胞浸润。
5．激素干预后哮喘大鼠模型肺组织 GCR最大特异结合量及 GCR ＂dNA显著下
降，且较普通哮喘模型有下降的趋势。哮喘宁颗粒剂可上调激素于预后哮喘大鼠肺
组织GCR的最大特异结合量和GCR lNA的表达。
本课题通过动物实验证实了哮喘宁颗粒剂治疗哮喘及经激素干预后哮喘的有效
性，其疗效机制可能与上调肺组织CCR水平有关。
According to the survey of epidemiology the incidence rate and the death rate of asthma is increasing since 1960s. Together with the long course and the difficulty of cure, asthma brings heavy economic burden to society. So it attracts much attentions of countries around the world.
Asthma is a kind disease of chronic airway inflammation, and airway hyperreactivity (AHR) and reversible airstream restriction are related to airway inflammation closely, and glucocorticoid is the principle choice for asthma treatment. However, many recent researches suggest that there is abnormal GCR (glucocorticoid receptor) level including quantity reduction and functional defect in asthma patients with and without glucocorticoid treated, and reason of abnormal GCR standard is still confirmed. It is truism that glucocorticoid will lead to further descent of GCR standard. So, it is urgent to seek theraputic drugs for asthma in essence not simply improving asthmatic symptom.
In the opinions of my advisor professor Wu Weiping, the position of asthma is the lung, it is related to the liver closely. "Wind", "phlegm", "Qi", "stasis", "asthenia" are the main pathogenesis of asthma, and disharmony of the liver and lung, disturbance of Qi and blood is the key point of pathogenesis. So regulate the function the liver and the lung, adjust Qi and blood is in common use when treat asthma, and it's effective in clinic. The complex prescription of traditional Chinese madicine梄iao Chuan King granule created on the basis of the theory and has been used in clinic for many years shows definite therapeutic effects. This prescription gained good effects not only in mild and moderate asthma but also in severe glucocorticoid dependend asthma by reducing the oral dosage of glucocorticoid.
The purpose of this study is finding out the pharmacological mechanism of Xiao Chuan King granule treating ashtma. During the experiment, we repeat animal models of allergic asthmatic rats by ovalbumin mixture intraperitoneal injection, ovalbumin acting as allergen, aluminium hydroxide and bordetella pertussis acting as immune adjuvent. Using Bu Shen Fang Chuan pill as the control medicament, search possible pharmacological effect of Xiao Chuan King granule on GCR.
1 observing asthmatic rats' common state, inspecting pulmonary function of asthmatic rats, observing morphology of pulmonary tissues under microscope suggest, that the asthmatic rats appeared typical symptoms of asthma including fast respiratory frequency > lower pulmonary ventilation function and typical pathologic changes of lung tissues. Xiao Chuan King granule can (1) effectively improve rats' asthmatic symptoms. (2) lower asthmatic rats' respiratory frequency. (3) improve pulmonary ventilation function by reducing resistance of expiration and resistance of inspiration, rising pulmonary compliance, rising maximum ventilatory volume, appreciably rising 0. 3FEV% , appreciably rising peak expiratory flow and appreciably rising forced expiratory flow 25% -75%. (4) improve asthmatic rats morphological abnormal by decreasing lymphocytic cells and eosinophilic granulocytes soakage.
2 Examining asthmatic rats' IL-4 level in serum and BALF suggest, that asthmatic rats' IL-4 level is increasing in serum, especially in BALF, and Xiao Chuan Ning granule can reduce the IL-4 level in serum and in BALF.
3 Examining GCR maximum specific binding in asthmatic rats' lung tissues, GCR mRNA expression and cortisol concentration in serum suggest, that GCR maximum specific binding reduce obviously as well as GCRmRNA expression, while cortisol concentration in serum reduce obviously. Xiao Chuan Ning granule can increase GCR maximum specific binding reduce obviously, GCRmRNA expression, and cortisol concentration in serum.
4 By intervented with dexamethasone, asthmatic rats represent typically asthmatic appearance later but badly little by little, respiratory frequency becoming fast obviously, pulmonary ventilation function reducing and lung tissues appearing typical pathomorphologic changes. Xiao Chuan
引文
1．吕国平，崔德健，郭英江，等．介绍一种建立大鼠哮喘模型的实验方法．中华结核和呼吸杂志，1995，18(6)：377
2. Maryo. Amdur, Jere Mead. Mechanisms of respiration in anaesthetized guinea pigs. Am J Physiol, 1968,192:364～368
3. Karol MH. Animal models of occupational asthma. Eur Respir J, 1994, (7): 555
4．潘慧云，朱元珏，王曾礼．大鼠支气管哮喘模型的建立及其肺功能的改变．汕头大学医学院学报，1998，11(增)：10～12
5．苗会，薛全福，庄逢源，等．哮喘大鼠动物模型的制备．基础医学与临床，1998，18(1)：72～78
6. Elwood W, Lotvali JO, Barnes PJ, et al. Charaterization of allergeninduced bronchial hyperresponsiveness and airway inflammation in actively sensitized brown-norway rats. J Allergy Clin Immunol, 1991, 88:951～960
2. Maryo. Amdur, Jere Mead. Mechanisms of respiration in anaesthetized guinea pigs. Am J Physiol, 1968,192:364～368
3. Karol MH. Animal models of occupational asthma. Eur Respir J, 1994, (7): 555
4．潘慧云，朱元珏，王曾礼．大鼠支气管哮喘模型的建立及其肺功能的改变．汕头大学医学院学报，1998，11(增)：10～12
5．苗会，薛全福，庄逢源，等．哮喘大鼠动物模型的制备．基础医学与临床，1998，18(1)：72～78
6. Elwood W, Lotvali JO, Barnes PJ, et al. Charaterization of allergeninduced bronchial hyperresponsiveness and airway inflammation in actively sensitized brown-norway rats. J Allergy Clin Immunol, 1991, 88:951～960