两种肾阳虚证支气管哮喘病证结合动物模型的建立与评价

Establishment and Evaluation of Two Animal Models of Bronchial Asthma with Disease-Syndrome Combination of Kidney-Yang Deficiency Syndromes

目的评价两种肾阳虚证支气管哮喘病证结合动物模型造模方法。方法将54只Wistar大鼠分为空白组、哮喘组、激素组、激素+哮喘组、手术组、手术+哮喘组,每组9只。激素组、激素+哮喘组大鼠采用氢化可的松注射液15 mg/(kg·d)皮下注射,手术组、手术+哮喘组采用切除双侧全部肾上腺复制为肾阳虚证候模型。哮喘组、激素+哮喘组、手术+哮喘组采用卵清白蛋白(OVA)致敏诱发哮喘。观察各组大鼠一般状态、行为学、肺功能及肺泡灌洗液中白细胞分类,同时检测血清下丘脑-垂体-肾上腺(HPA)轴功能指标[包括促肾上腺皮质激素释放激素(CRH)、肾上腺皮质激素释放激素(ACTH)、皮质醇(CORT)、干扰素-γ(INF-γ)、免疫球蛋白E(Ig E)]及肺组织各项炎症因子水平;观察肺组织病理改变。结果激素+哮喘组、手术+哮喘组肺泡灌洗液中白细胞、嗜酸性细胞总数升高,CRH、ACTH、CORT分泌减少;血清总Ig E、OVA特异性Ig E含量升高;肺组织内INF-γ表达分泌减少,白细胞介素4(IL-4)表达分泌增多(P<0.05或P<0.01);肺组织病理显示支气管出现大量炎症浸润、气道黏膜水肿。激素+哮喘组与手术+哮喘组比较,在肺泡灌洗液炎症细胞检测上无明显差异,后者HPA轴功能指标下降更加明显,前者大鼠肺组织的病理改变更加明显。结论两种肾阳虚证支气管哮喘动物模型均能够反映肾阳虚证支气管哮喘的发病特点,但采用切除双侧全部肾上腺方法的动物模型对HPA轴功能影响更明显。

Objective To evaluate two modeling methods of animal model of bronchial asthma with disease-syndrome combination of kidney-yang deficiency syndrome. Methods Fifty-four Wistar rats were divided into a blank group,an asthma group,a hormone group,a hormone ＋ asthma group,an operation group,and an operation ＋ asthma group,with 9 rats in each. Rats in the hormone groupand the hormone ＋ asthma group were given 15 mg /（ kg·d）of hydrocortisone by subcutaneous injection,while the operation group and the operation ＋ asthma group received bilateral total adrenalectomy to establish the kidney-yang deficiency syndrome model. The asthma group,the hormone ＋asthma group,and the operation ＋ asthma group were given ovalbumin（ OVA） sensitization to induce asthma. General state,behavior,lung function and leukocyte classification in bronchoalveolar lavage fluid in each group were observed. At the same time,indexes of the function of hypothalamic-pituitary-adrenal axis in serum [including corticotropin-releasing hormone（ CRH）,adrenocorticotropic hormone（ ACTH）,cortisol（ CORT）,interferon-γ（ INF-γ）,immune globulin E（ Ig E） ],and levels of each inflammatory factor in lung tissue were examined,and pathologic changes in the lung tissue were observed. Results In the hormone ＋ asthma group and the operation ＋ asthma group,total number of leukocyte and eosinophils in bronchoalveolar lavage fluid increased,while the excretion of CRH,ACTH and CORT decreased; levels of total serum Ig E and OVA-specific Ig E increased; expression and excretion of INF-γ in lung tissues decreased while the expression and excretion of interleukin-4（ IL-4） increased（ P 〈0. 05 or P 〈 0. 01）; There were large amounts of inflammation infiltrations and mucosa edema of air passage in bronchus. There was no significant difference in inflammatory cells in bronchoalveolar lavage fluid when comparing the hormone ＋ asthma group and the operation ＋ asthma group. Indexes of HPA axis function in the operation ＋ asthma group decreased more remarkably while pathologic changes in lung tissue in the hormone ＋ asthma group were more remarkable. Conclusion Both two animal models of bronchial asthma with kidney-yang deficiency syndrome could reflect clinical characters of bronchial asthma of kidney-yang deficiency syndrome. But animal model by bilateral total adrenalectomy seems to have more remarkable influence on HPA axis function.