Experimental Study Of The Mechanism Of Effect Of Wuzisanhuang Formula On Steroid-Dependent Asthma Mouse Model

Objective:To observe the curative effects of Wuzisanhuang formula (WZSHF) on steroid-dependent asthma, study the pathogenes is of steroid-dependent asthma and the mechanism of effect of WZSHF on steroid-dependent asthma.Methods:According to random number table,120 mice weighed 18±5 g, half male and half female, were divided into 6 groups:blank group, model group, prednisone control group, low dose WZSHF group, middle dose WZSHF group, high dose WZSHF group. Asthma model mice in model group, prednisone control group, low dose WZSHF group, middle dose WZSHF group, and high dose WZSHF group were induced via intraperitoneal injection and ultrasonic atomizing inhalation ovalbumin (OVA) through sensitizing and excitation. Mice in blank group were given saline via intraperitoneal injection and ultrasonic atomizing inhalation. Mice in model group, prednisone control group, low dose WZSHF group, middle dose WZSHF group, and high dose WZSHF group were given steroid drug (prednisone) with dose of 3.9 mg/kg by gavages to duplicate steroid-dependent asthma mouse model before atomizing inhalation for 21 days, and decrease the dose by 0.325mg/kg/day to zero at the day 63. Asthma model mice in Low dose WZSHF group, middle dose WZSHF group, and high dose WZSHF group were given low, middle and high dose WZSHF granules at day 35 with dosage of 1ml/100g, while mice in blank group and model group were given saline by gavages until the day 63. Mice were decapitated and collected blood at day 64 to determine the concentration changes of corticotropin releasing hormone (CRH), adrenocorticotrophic hormone (ACTH) and Cortical (COR) in plasma by Radio immunoassay. Lung tissue of mice was taken to do HE staining and take photos. ICAM-1 expression in lung epithelial cells, eosinophil chemotactic activating factor and Eosinophil cationic protein in Lung homogenates were tested by Immunohistochemical method.Results:There were no significant differences of concentration of CRH, ACTH and COR between model group and normal group (P> 0.05). Concentration of CRH, ACTH and COR in prednisone control group decreased significantly by contrast with model group (P< 0.01).This implied that treatment of prednisone could decrease the concentration of CRH, ACTH and COR in plasma. CRH, ACTH and COR in plasma of three WZSHF group increased obviously (P< 0.01). Among these 3 groups, concentration of CRH, ACTH and COR in plasma of high dose WZSHF group was lower than the other two groups, while that of middle dose WZSHF group increased significantly.The result of HE staining showed that there is no change of form of lung tissues and no inflammatory cell infiltration in normal group. Bronchial epithelium and wall thickening, epithelial damage and shedding significantly, lumen stenosis, marked hyperplasia of goblet cells, bronchial lumen visible mucus plug blocking the cavity of a large number of inflammatory secretions, the large number of eosinophils and lymphocytes around large and medium-sized airways and blood vessels, mild bronchial and vascular smooth muscle hyperplasia, visible phagocytic cells increased obviously, expanding alveolar septa were seen in mice of model group. Bronchial epithelium and thickening of the wall, the destruction and shedding of epithelial, stenosis lumen and mildly moderate inflammatory infiltration were seen in the control group.Goblet cell hyperplasia alleviated compared with model group, infiltration of eosinophils and lymphocytes could be seen around the large and medium-sized airways and blood vessels, but inflammation reduced significantly compared with the model group, mild hyperplasia of bronchial and vascular smooth muscle, alveolar septa widened. Normal bronchial lumen, mild epithelial hyperplasia, a small amount of destruction with no obvious narrow lumen, a small amount of inflammation were seen in low dose WZSHF group, goblet cell hyperplasia seen to be lighter than the control group, a small amount of lymphocytes and eosinophils in the peribronchial with mild cell infiltrates, bronchial and vascular smooth muscle hyperplasia, mild widened alveolar septa were also seen in low dose WZSHF group. Normal bronchial lumen, mild epithelial hyperplasia, and no significant damage, no obvious narrow lumen, a small amount of inflammation were seen in middle dose WZSHF group. Goblet cell hyperplasia alleviated in middle dose WZSHF group compared with the control group. A small number of lymphocytes and eosinophils granulocyte infiltrates around bronchus, bronchial and vascular smooth muscle hyperplasia and alveolar septum expanding were not seen in middle dose WZSHF group. In high dose WZSHF group, normal bronchial lumen, the epithelium without apparent hyperplasia and significant disruption were seen. Obvious narrow lumen, inflammation, goblet cell decreased, peribronchial lymphocytes and eosinophils, bronchial and vascular smooth muscle hyperplasia, significantly widened alveolar septa were not found in high dose WZSHF group.Immunohistochemical detection results of expression of ICAM-lof lung epithelial cell: ICAM-1 expression of lung tissue epithelial cell was significantly enhanced in the asthma model group compared with normal group (P<0.01); Compared with the asthma model group, prednisone acetate enable slung epithelial cell ICAM-1 expression decreased significantly (P <0.01); compared with the asthma model group and the prednisone acetate group, low. medium and high dose WZSHF groups can significantly reduce ICAM-1 express ion of the lung epithelial cell (P<0.01). Moreover, high dose group of WZSHF reduced lung epithelial cell expression of ICAM-1 more significantly (P<0.01).Results of lung ho mo ge nates detection of eotaxin and Eosinophil Cationic Protein (ECP): Compared with normal group. eosinophil chemotactic activating factor (Eotaxin) and eosinophil cationic protein (ECP) in asthmatic model group increased significantly, the difference was significant (P<0.01); compared with the asthma model group, eosinophil cell chemotaxis activating factor (of Eotaxin) and eosinophil cell cationic protein (ECP) in prednisone acetate control group lowed significantly, with significant differences (P<0.01); compared with the asthma model group, eosinophil cell chemotaxis of activated factor (of Eotaxin) and eosinophil cell cationic protein (ECP) of low, medium and high dose WZSHF groups significant reduced significantly, and that of medium and high dose WZSHF groups reduce more obvious, differences were very significant (P<0.01).Conclusion:1. Bronchial asthma mouse model was established successfully by use of ovalbumin (OVA) sensitized and challenged.2. Dexamethasone treatment of asthma made the hypothalamus-pituitary-adrenal (HPA) axis function severely suppressed.3. Bronchial asthma mouse mode established by ovalbumin (OVA) was represented with varying degrees of inflammation and airway remodeling.4. WZSHF could improve the function of hypothalamic-pituitary-adrenal (HPA) axis impaired by hormone prednisone acetate intervention in asthmatic mice led significantly.5. The result of pathology implied that prednisone acetate can significantly reduce the infiltration of asthmatic airway inflammatory cells in mice, but does not improve the airway remodeling.6. WZSHF not only reduced infiltration of airway inflammatory cell of hormone-dependent asthma mouse significantly, but also can significantly improve airway remodeling of asthmatic mouse.7. The results of concentration of measuring eotaxin and ECP by lung tissue homogenates indicated that prednisone acetate could reduce the amount of ECP and eotaxin in lung of asthmatic model mice.8. The result that prednisone reduced ICAM-1 expression in lung tissue epithelial cell significantly, suggesting that prednisone acetate had the effects of relieving inflammation of airway.9. Plus WZSHF treatment in the process of withdrawal Prednisone intervention in the treatment of asthma mouse model can significantly reduce levels of ECP and eotaxin in the asthmatic mice lung, and decrease the expression of ICAM-1 of epithelial cell significantly, present better efficacy than pure acetic prednisone intervention (P<0.01).