| Chapter 1.Comparison of different challenge methods for making rat asthma modelsObjective:To explore the challenge methods for establishing rat asthma model.Methods:Thirty male Sprague-Dawley(SD)rats of 4 weeks old were divided into two groups randomly,10 as control group and 20 as treated group.The rats of control group were given intraperitoneal injection with 1 ml 0.9%sodium chloride instead of 10% ovalbumin(OVA)in the sensitized stage,and 0.9%sodium chloride instead of OVA was inhaled through ultrasonic method in the challenge stage.The rats in treated group were given intraperitoneal injection with 1 ml 10%OVA mixed with 10%aluminum hydroxide gel and the inactivated Bacillus pertussis on day 1,then divided into two groups randomly two weeks later,2%OVA was inhaled to sensitize the rats with pump and ultrasonic methods respectively.The rat asthma model was established successfully,and the two challenge methods for making asthma rat models were compared.Results:1.Evaluation of asthma model:compared with the control group,the rats inhaled 2%OVA with pump and ultrasonic methods appeared visible respiratory distress signs (dysphoria,polypnea,contraction of accessory respiratory muscles and cyanosis).The lung histopathology showed typical airway inflammation, and the differential cell counts in BALF(Bronchial alveolar lavage fluid) indicated that the rat asthma model was established successfully.2.The time that the rats appeared asthma syndrome after challenge with the pump method were earlier than that with ultrasonic method,and the number of rats with asthma syndrome in pump group were more than that in ultrasonic group at the same challenge duration(P＜0.01).But they had no significant difference in lung histopathology and differential cell counts in BALF.Conclusion:1.OVA mixed with aluminum hydroxide gel and the inactivation Bacillus pertussis sensitized rats successfully, and inhaling 2%OVA with pump and ultrasonic methods to challenge established the rat asthma model successfully.2.The pump method is superior to the traditional ultrasonic method to challenge the rat asthma model.Chapter 2.The protective effect of budesonide to the airway inflammation in rat asthma modelObjective:To explore the protective effect of budesonide(BUD)to the airway inflammation in rat asthma model.Method:Forty male SD rats of 4 weeks old were divided into four groups randomly,including control group,model group,BUD group and Dexamethasone(DXM) group.The established method for the model group(pump group) described as before(chapter one).The control group were challenged with pump inhaled method,the followed steps were described as chapter one also.The BUD and DXM groups were treated with BUD and DXM respectively before they were challenged,and the rats in these two groups were sacrificed 24h later after the last challenge.Then the total cell number and percentage of differential cells including eosinophils (EOS)in the bronchial alveolar lavage fluid(BALF)were detected,and the lung tissue histopathology was analyzed.Results:1.The rats in both BUD group and DXM group appeared typical asthma syndrome after challenged,but the symptoms in these rats of both groups were alleviated than those in model group.The inflammatory cells and the percentage of EOS in these groups were increased than those in control group significantly(P＜0.01),and were decreased than those in model group significantly(P＜0.01),but there were no significant difference between the BUD group and DXM group(P＞0.05).2.The total cells number,percentage of EOS and neutrophil in BALF of model group, BUD group and DXM group rats were increased than those in control group significantly(P＜0.01),but those in the BUD group and the DXM group were decreased than those in the model group significantly(P＜0.01),but there were no significant difference in total cell numbers and percentages between the rats of BUD group and the DXM group(P＞0.05).Conclusion:The BUD inhaled can alleviate the airway inflammation of asthma model rats,which has the similar effect as DXM administrated systemically. Chapter 3 The effect of budesonide on the regulation of heme oxygenase-1 on asthma model ratsObjective:To investigate the expression characteristics of heme oxygenase-1(HO-1)gene and protein and the effect of BUD to the HO-1 expression on lung tissue of asthma model rats,and to provide some laboratorial data for treating asthma with BUD.Methods:Fifty male SD rats of 4 weeks old were divided into 5 groups randomly,including control group,model group,hemin group,BUD group and DXM group. The methods for establishing rats model were described as that in the chapter two.The rats of Hemin,BUD and DXM groups were treated with Heroin,BUD and DXM respectively before they were challenged, and these rats were sacrificed 24h later after the last challenging.Then the arterial blood,alveolar lavage fluids and lung tissue of each group rats were taken,and the amount of COHb,total cells number of alveolar lavage fluids and the percentage of differential cells were detected.The expression of HO-1 protein on lung tissue was detected with immunohistochemistry,the expression of HO-1 mRNA was detected with RT-PCR,the airway inflammation situation was analyzed with histopathology.Results:1.The challenged rats treated with BUD,DXM and hemin appeared typical asthma syndrome,but which were alleviated than that in model group.The inflammatory cells and the percentage of EOS were increased significantly than that in control group(P＜0.01), and those were decreased significantly in Heimin group(The inflammatory cells P＜0.05,EOS P＜0.01),BUD and DXM group(P＜0.01)than those in model group,but there were no significant differences among the rats of BUD,DXM and hemin groups(all P＞0.05).2.The total cells number,percentage of EOS and neutrophil in BALF in the rats of model,BUD,DXM,Hemin groups were increased significantly than that in control group(all P＜0.01),and those in BUD group,DXM group and Heimin group decreased significantly than those in model group(all P＜0.01),but there were no significant differences detected among the rats of BUD,DXM and Heimin groups (all P＞0.05).but there were no significant differences among these three groups(all P＞0.05).3.Immunehistochemistry study showed:the expression of HO-1 was up-regulated in model group rats than in that in control group rats(P＜0.01),but that expressed only a little in control group rats.As compared with model group,the HO-1 expression increased significantly after treated with BUD,DXM and Hemin respectively(all P＜0.01).The positive expression of HO-1 protein can be seen in macrophage,airway epithelia cells and smooth muscle cells. 4.The RT-PCR showed:The mRNA expression of HO-1 was increased significantly in Hemin group rats(P＜0.01),BUD group rats and DXM group rats(all P＜0.05)than in that in model group rats and in control group(all P＜0.05).5.As compared with control group rats,the expression of COHb were increased significantly in model group rats, BUD group rats,DXM group rats and Hemin group rats(all P＜0.01);as compared with model group rats,no significant difference between model group and BUD group(P＞0.05),but the expressions of COHb were increased significantly in DXM and Hemin group(P＜0.05),there were no significant difference between DXM group rats and Hemin group rats(P＞0.05).Conclusion:1.The expression of HO-1 protein and mRNA were up-regulated in lung tissue of asthma model rats,the activity of HO-1 in arterial blood were enhanced,the expressions also increased significantly after adding the HO-1 challenger hemin,and the typical asthma syndrome and airway inflammation were alleviated, which showed the protective effect of HO-1 on asthmatic rats.2.BUD and DXM can up-regulate the expressions of HO-1 protein and mRNA, and enhance its activity.3.The protective effect of BUD and DXM on the airway inflammation of the asthmatic rats may be related to the up-regulation of HO-1.
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