吸入布地奈德对哮喘大鼠气道炎症及重塑影响的研究
摘要
目的:支气管哮喘(bronchial astham)是世界医学界公认的四大顽症之一,被列为十大死亡原因之最。目前,随着人们生活环境的改变,发病率逐年上升。其临床表现为突然而反复发作的喘息,呼吸困难,胸闷和咳嗽。气道炎症与气道重塑是支气管哮喘发病的病理基础。本实验通过建立哮喘模型,观察哮喘大鼠吸入布地奈德后血清中LI-25与IL-12比值的变化,探讨哮喘时免疫失衡与NF-κB表达的动态关系。观察哮喘大鼠吸入布地奈德后气道NF-κB,MMP-9表达,探讨哮喘时气道重塑与NF-κB的关系。
方法:
1动物与药品
Wistar雄性大鼠30只,由河北省实验动物中心提供(实验动物质量合格证编号:903010);布地奈德由阿斯利康制药公司生产;免疫组化试剂盒,一抗cox2兔抗IgG多克隆抗体、购于boshide公司,SP9001试剂盒及DAB显色剂购于北京中衫生物技术有限公司。酶联免疫试剂盒购于美国RB公司;其他实验药品由河北医科大学免疫教研室提供。
2模型制备及实验分组
用随机数字法将30只雄Wistar大鼠随机分为3组:正常对照组、哮喘模型组、布地奈德治疗组,每组10只。哮喘大鼠模型的制备分致敏和激发两步。除正常对照组外,其余2组于第1天、第8天、第15天分3次腹腔注射抗原液1ml(含卵蛋白100mg、氢氧化铝干粉100mg)致敏,正常对照组用等量的生理盐水进行致敏;第21天开始将大鼠放入自制的不完全封闭的有机玻璃箱内,哮喘模型组和布地奈德治疗组予1%卵蛋白(OVA)溶液用空气压缩泵喷雾激发,每天1次,每次20分钟,连续激发7天,布地奈德治疗组于激发后,均采用布地奈德雾化吸入治疗,方法为0.5mg/ml布地奈德加入1ml生理盐水中,放入压缩雾化器中雾化吸入,bid。支气管哮喘组激发后以生理盐水雾化吸入,对照组雾化生理盐水作为对照。上述处理共持续7天。各组于末次诱发哮喘后24小时内麻醉,分离右侧股动脉,留取血标本,取血清-70℃保存,备做酶联免疫。右肺组织灌入4%多聚甲醛至右肺膨胀,然后结扎右支气管,固定,取右肺中叶组织,备做免疫组化。
4指标的检测
4.1 IL-12与IL-25的检测
应用ELISA方法检测大鼠血清IL-12及IL-25的含量,其操作步骤以试剂说明书进行。
4.2免疫组化法检测哮喘肺组织中NF-κB与MMP-9的表达
采用SP9001通用二步法试剂盒检测肺组织中NF-κB与MMP-9的表达,光镜观察,阳性部位呈棕黄色。设定阳性比率,并行统计学处理。
结果:
1哮喘大鼠模型鉴定
模型组大鼠在吸入OVA后不久即出现躁动不安,搔抓颜面皮毛等过敏症状,继而大鼠俯伏不动,出现呼吸急促,呼吸困难(明显的腹式呼吸),部分大鼠可闻及响亮的呼气相喘鸣音,口鼻流出粘液,严重者可出现呼吸不规则,反复激发后大鼠毛色失去光泽,精神不振和反应迟钝。
2酶联免疫检测结果
2.1血清IL-25水平的变化
哮喘组血清IL-25含量是(40.71±3.87)pg/ml,激素治疗组血清IL-25含量是(30.43±1.48)pg/ml,对照组血清IL-25的含量是(25.96±1.74)pg/ml,三者比较有统计学意义(P<0.05)。并且哮喘组血清IL-25含量(40.71±3.87)pg/ml高于激素治疗组(30.43±1.48)pg/ml,两者比较有统计学差异,明显高于对照组(25.96±1.74)pg/ml,两者比较有统计学差异(P<0.05),激素治疗组与对照组之间没有显著性差异(P>0.05)。
2.2血清IL-12水平的变化
哮喘组血清IL-12含量是(25.91±3.20)pg/ml,激素治疗组血清IL-12含量是(29.42.±2.43)pg/ml,对照组血清IL-12的含量是(33.71±2.69)pg/ml,三者比较有统计学意义(P<0.05)。并且哮喘组血清IL-12含量(25.91±3.20)pg/ml较激素治疗组降低,两者比较有统计学差异(P<0.05),较对照组明显降低,有统计学差异(P<0.05),激素治疗组与对照组之间有统计学意义(P<0.05)。
3各组大鼠组织标本的制备
2.3血清IL-25/ IL-12比值变化
模型组血清中IL-25/ IL-12比值与正常组相比明显升高,差异有显著性(P<0.01);经激素治疗后,血清中IL-25/ IL-12比值与模型组相比降低,与正常组相比升高,差异亦均有显著性(P<0.01)。
3切片结果的鉴定
3.1光镜结果哮喘大鼠细支气管痉挛、收缩呈菊花状,管腔内有分泌物,细支气管、小血管周围见明显炎性细胞浸润,以嗜酸细胞、淋巴细胞为主,亦见中性粒细胞;肺泡间隔增宽,有纤维化,大量淋巴细胞、巨噬细胞浸润。治疗组大鼠细支气管无明显痉挛、收缩,管腔内无分泌物,管壁周围炎性细胞浸润明显减少,肺泡壁间隔无增宽。
3.2免疫组化检测结果
NF-κB和MMP-9在模型组、激素治疗组和正常对照组大鼠肺组织中均有阳性表达,并主要在细胞核表达,少量在细胞浆表达。
3.2.1肺组织内NF-κB表达的变化
模型组肺组织内NF-κB的表达与正常组相比明显升高,差异有显著性(P<0.01);经激素治疗后,大鼠肺组织内NF-κB的表达与模型组相比降低,与正常组相比升高,差异均有显著性(P<0.01)。
3.2.2肺组织内MMP-9表达的变化
模型组肺组织内MMP-9的表达与正常组相比明显升高,差异有显著性(P<0.01);经激素治疗后,大鼠肺组织内MMP-9的表达与模型组相比降低,与正常组相比升高,差异均有显著性(P<0.01)。
4各指标之间的相关性
应用Spearman等级相关法分析发现:IL-25与IL-12之间存在负相关(r=-0.754, P<0.01),IL-25与NF-κB之间存在正相关(r=0.812, P<0.01),IL-12与NF-κB之间存在负相关(r=-0.747, P<0.01),MMp-9与与NF-κB之间存在正相关(r=0.797, P<0.01)。
结论:
1哮喘时,Th1/Th2免疫平衡破坏,T细胞向Th2方向偏移,Th2优势反应在哮喘发病机制中有重要意义。
2哮喘组大鼠NF-κB支气管肺组织中表达明显高于对照组,说明哮喘气道炎症及重塑过程中NF-κB具有重要作用。
3哮喘组大鼠NF-κB活性增强,IL-25水平上升,IL-12水平下降,IL-25/IL-12升高,相关分析示:NF-κB与Th2类细胞因子IL-25成正相关,与Th1类细胞因子IL-12成负相关。
4哮喘组大鼠NF-κB活化与MMP-9支气管肺组织中表达明显高于对照组,支气管黏膜增生肥厚明显,表明两者参与气道重塑的过程,相关分析示两者存在正相关。
5布地奈德作为治疗哮喘的有效药物,可以抑制NF-κB活性,使IL-25水平降低,IL-12水平升高,改善减轻气道炎症。使MMP-9水平降低,改善气道重塑。可作为治疗哮喘的首选。为临床治疗哮喘提供理论依据。
Objective: Bronchial asthma is recognized as one of the world's four major chronic medical profession. The leading cause of death was listed as the most. Now, with people's lives change, the incidence rate increase every year. The clinical manifestations is sudden onset recurrent wheezing, difficulty breathing, chest tightness and coughing. The pathological basis of bronchial asthma is airway inflammation and airway remodeling. In this study, through the establishment of asthma model, we observe inhaled budesonide in asthmatic rat the LI-25/ IL-12 ratio change and explore the relationship between the immune imbalance and the expression of NF-κB dynamic in asthma. Observation of inhaled budesonide airway NF-κB, MMP-9 expression in asthmatic rat, we explore relationship between airway remodeling and NF-κB.
Methods:
1 Animals and drugs
We selected 30 wistar mouse, provided by the Experimental Animal Center of Hebei Province (experimental animal quality certification number: 90301 );Budesonid produced by AstraZeneca Pharmaceuticals; Immunohistochemistry kit, one anti-cox2 rabbit anti-IgG polyclonal antibody, purchased in boshide company, SP9001 kit and DAB reagent purchased in Bio-Technology ,Beijing T-shirt. Enzyme immunoassay kit purchased in the United States RB; other experimental drugs provide by immunity teaching of the Hebei Medical University.
2 Model preparation and experimental groups
the law use random numbers and 30 male Wistar rats were randomly divided into three groups:normal control group,asthma model group, budesonide-treated group,10 in each group.Preparation of asthma model group, two-step:sub-sensitized and challenged. In addition to the normal control group, the other two groups were injectioned antigen solution 1ml (including egg protein, 100mg, aluminum hydroxide powder 100mg) by intraperitoneal sensitized on 1 day, 8 day, 15 day three times. and normal control group with equal of saline to sensitize; the 21 days, the rats were placed not completely closed self-made plexiglass box, asthma model group and the budesonide treatment group were Sucked in 1% ovalbumin (OVA) solution by the air compression pump spray Fog excitation,once a day,each20 minutes,continuously 7 days.The budesonide treatment group after excitation are used to budesonide inhalation treatment ,method 0.5mg/ml adding to 1ml saline. Budesonide compressed into the inhalation nebulizer, bid. Bronchial asthma group inhalation the saline after excitation. normal control group were stimulated with saline for the treatment. This last 7 days.
3 The preparation of tissue samples
In each groups, in the Last 24 hours after induced asthma, intraperitoneal injection of anesthesia, the right femoral artery get blood samples. the serum -70℃preservation, prepared to do enzyme-linked immunosorbent . ligation of the left lung, the right lung was slowly poured into 4% paraformaldehyde for the right lung inflated. then ligating of the right bronchus and placing the fixedan.taking right middle lobe organization prepared to do immunohistochemistry.
4 Target detection
4.1 IL-12 and IL-25 detection
Application of ELISA to detect serum IL-12 and IL-25 levels, their manual steps carry out the reagen.
4.2 TO detect NF-kB and MMP-9 expression in the lung tissue with immunohistochemistry
SP9001 Universal two-step method using to detect NF-kBand MMP-9 expression in the lung tissue, light microscopic observation, the positive part of brownish yellow. Set-positive rate, parallel statistically.
Results:
1 Identification of rat model of asthma
The model group rats after inhalation of OVA appear restless, scratch facial fur and other allergic symptoms, then bowed down and not move , shortness of breath, difficulty breathing ( the notable abdominal breathing), and some rats were audible and louddly expiratory wheezing, nose and mouth out of mucus,severe cases,irregular breathing could occur.Repeatedly stimulated, fur dull, lassitude and unresponsiveness.
2 Enzyme-link ed immunosorbent assay results
2.1 The serum IL-25 levels
The serum IL-25 concentration (40.71±3.87) pg/ml in asthma group, The serum IL-25 concentration (30.43±1.48) pg/ml in hormone therapy group, The serum IL-25 concentration (25.96±1.74) pg/ml in control group ,three groups have more statistically significant (P <0.05). And asthma group serum IL-25 levels(40.71±3.87) higher than the hormone therapy group (30.43±1.48) pg/ml, their have more significant differences. Asthma group serum IL-25 levels (40.71±3.87) significantly higher (25.96±1.74) pg /ml than the control group, their have more significant difference (P <0.05), The hormone therapy group and the control group was no significant difference. (P> 0.05).
2.2 The serum IL-12 levels
The serum IL-12 concentration (25.91±3.20) pg/ml in asthma group, The serum IL-12 concentration (29.42.±2.43) pg/ml in hormone therapy. serum IL-12 concentration (33.71±2.69) pg/ml in control group, three groups have more statistically significant (P <0.05). And asthma group serum IL-12 levels (25.91±3.20) pg/ml lowwer than the hormone therapy group,Their have a statistically significant difference (P <0.05), asthma group serum IL-12 levels (25.91±3.20) pg / ml significantly lower than the control group, their have significant difference (P <0.05),the hormone therapy and the control group were statistically significant (P <0.05).
2.3 Serum IL-25 / IL-12 ratio of change
Compared with the normal group, asthma group serum IL-25 / IL-12 ratio was significantly higher, the difference was significant (P <0.01); After hormone treatment, serum IL-25 / IL-12 ratio, compared with asthma group, ratio decreased; compared with the control group increased, the difference also was significant (P <0.01).
3 Identification of biopsy results
3.1 Light microscopy
Asthmatic rat bronchioles had spasm,contraction, lumen containing secretions,bronchioles, the small blood vessels around the apparent infiltration of inflammatory cells,including eosinophils, lymphocyte predominance and neutrophils;alveolar interval widened, fibrosis, a large number of lymphocytes, macrophages infiltration. Treated rats had no significant bronchiole spasm, contraction, non-intraluminal secretions, wall significantly reduced infiltration of inflammatory cells ,without interval widened.
3.2 Immunohistochemistry results
NF-κB and MMP-9 in the asthmiatic group, hormone therapy group and normal control rats were positive expression in lung tissue and were mainly expressed in the nucleus, a small amount of expression in the cytoplasm.
3.2.1 Lung tissue NF-κB expression
Asthmatic group, the lung tissue expression of NF-κB was significantly higher compared with the normal group, the difference was significant (P <0.01); After hormone treatment, the rat lung tissue expression of NF-κB compared with Asthmatic group lower , when compared with the normal group increased, the differences were significant (P <0.01)
3.2.2 Lung tissue MMP-9 expression
Asthmatic group,The lung tissue expression of MMP-9 was significantly higher compared with the normal group,the difference was significant (P <0.01); After hormone treatment, the rat lung tissue expression of MMP-9 compared with the Asthmatic group reduced,compared with the control group increased, the differences were significant (P <0.01).
4 The correlation between the various indicators
Application of Spearman Rank Correlation analysis showed that:IL-25 and IL-12 were negative correlation (r =- 0.754, P <0.01), IL-25 and NF-κB were positive correlation (r = 0.812, P < 0.01), IL-12 and NF-κB were negative correlation (r =- 0.747, P <0.01), MMP-9 and NF-κB were positive correlation (r = 0.797, P <0.01).
Conclusion:
1 The immune balance of Th1/Th2 was broken,and it migrant to Th2 direction.Th2 dominant reaction play an important role in the pathogenesis of asthma.
2 The percentage of NF-κB in asthma mice was notable higher than that of the control group.It hint that plays an important role in the process of the airway inflammation and remodeling of asthma.
3 In asthma group.the positive percentage of NF-κB and the level of IL-25 increase signicantly,but the level of IL-12 decreased, the ratio of IL-25/IL-12 increase, correlation analysis showed: NF-κB and IL-25 were positively correlated:NF-κB and IL-12 were negative correlation.
4 Asthma, NF-κB activation and MMP-9 expression in bronchopulmonary tissues were significantly higher than control group.Bronchial mucosal hyperplasia hypertrophy significantly, indicating that both participate in the process of airway remodeling,correlation analysis showed a positive correlation between the two.
5 Budesonide as the effective drug treatment of asthma can inhibit NF-κB activity,so that it is IL-25 levels decrease, IL-12 levels increased, and reduce airway inflammation;Budesonide also can reduce the level of MMP-9 and improve airway remodeling.Can be used as first choice for treatment of asthma and provide a theoretical basis for the treatment of asthma.
方法:
1动物与药品
Wistar雄性大鼠30只,由河北省实验动物中心提供(实验动物质量合格证编号:903010);布地奈德由阿斯利康制药公司生产;免疫组化试剂盒,一抗cox2兔抗IgG多克隆抗体、购于boshide公司,SP9001试剂盒及DAB显色剂购于北京中衫生物技术有限公司。酶联免疫试剂盒购于美国RB公司;其他实验药品由河北医科大学免疫教研室提供。
2模型制备及实验分组
用随机数字法将30只雄Wistar大鼠随机分为3组:正常对照组、哮喘模型组、布地奈德治疗组,每组10只。哮喘大鼠模型的制备分致敏和激发两步。除正常对照组外,其余2组于第1天、第8天、第15天分3次腹腔注射抗原液1ml(含卵蛋白100mg、氢氧化铝干粉100mg)致敏,正常对照组用等量的生理盐水进行致敏;第21天开始将大鼠放入自制的不完全封闭的有机玻璃箱内,哮喘模型组和布地奈德治疗组予1%卵蛋白(OVA)溶液用空气压缩泵喷雾激发,每天1次,每次20分钟,连续激发7天,布地奈德治疗组于激发后,均采用布地奈德雾化吸入治疗,方法为0.5mg/ml布地奈德加入1ml生理盐水中,放入压缩雾化器中雾化吸入,bid。支气管哮喘组激发后以生理盐水雾化吸入,对照组雾化生理盐水作为对照。上述处理共持续7天。各组于末次诱发哮喘后24小时内麻醉,分离右侧股动脉,留取血标本,取血清-70℃保存,备做酶联免疫。右肺组织灌入4%多聚甲醛至右肺膨胀,然后结扎右支气管,固定,取右肺中叶组织,备做免疫组化。
4指标的检测
4.1 IL-12与IL-25的检测
应用ELISA方法检测大鼠血清IL-12及IL-25的含量,其操作步骤以试剂说明书进行。
4.2免疫组化法检测哮喘肺组织中NF-κB与MMP-9的表达
采用SP9001通用二步法试剂盒检测肺组织中NF-κB与MMP-9的表达,光镜观察,阳性部位呈棕黄色。设定阳性比率,并行统计学处理。
结果:
1哮喘大鼠模型鉴定
模型组大鼠在吸入OVA后不久即出现躁动不安,搔抓颜面皮毛等过敏症状,继而大鼠俯伏不动,出现呼吸急促,呼吸困难(明显的腹式呼吸),部分大鼠可闻及响亮的呼气相喘鸣音,口鼻流出粘液,严重者可出现呼吸不规则,反复激发后大鼠毛色失去光泽,精神不振和反应迟钝。
2酶联免疫检测结果
2.1血清IL-25水平的变化
哮喘组血清IL-25含量是(40.71±3.87)pg/ml,激素治疗组血清IL-25含量是(30.43±1.48)pg/ml,对照组血清IL-25的含量是(25.96±1.74)pg/ml,三者比较有统计学意义(P<0.05)。并且哮喘组血清IL-25含量(40.71±3.87)pg/ml高于激素治疗组(30.43±1.48)pg/ml,两者比较有统计学差异,明显高于对照组(25.96±1.74)pg/ml,两者比较有统计学差异(P<0.05),激素治疗组与对照组之间没有显著性差异(P>0.05)。
2.2血清IL-12水平的变化
哮喘组血清IL-12含量是(25.91±3.20)pg/ml,激素治疗组血清IL-12含量是(29.42.±2.43)pg/ml,对照组血清IL-12的含量是(33.71±2.69)pg/ml,三者比较有统计学意义(P<0.05)。并且哮喘组血清IL-12含量(25.91±3.20)pg/ml较激素治疗组降低,两者比较有统计学差异(P<0.05),较对照组明显降低,有统计学差异(P<0.05),激素治疗组与对照组之间有统计学意义(P<0.05)。
3各组大鼠组织标本的制备
2.3血清IL-25/ IL-12比值变化
模型组血清中IL-25/ IL-12比值与正常组相比明显升高,差异有显著性(P<0.01);经激素治疗后,血清中IL-25/ IL-12比值与模型组相比降低,与正常组相比升高,差异亦均有显著性(P<0.01)。
3切片结果的鉴定
3.1光镜结果哮喘大鼠细支气管痉挛、收缩呈菊花状,管腔内有分泌物,细支气管、小血管周围见明显炎性细胞浸润,以嗜酸细胞、淋巴细胞为主,亦见中性粒细胞;肺泡间隔增宽,有纤维化,大量淋巴细胞、巨噬细胞浸润。治疗组大鼠细支气管无明显痉挛、收缩,管腔内无分泌物,管壁周围炎性细胞浸润明显减少,肺泡壁间隔无增宽。
3.2免疫组化检测结果
NF-κB和MMP-9在模型组、激素治疗组和正常对照组大鼠肺组织中均有阳性表达,并主要在细胞核表达,少量在细胞浆表达。
3.2.1肺组织内NF-κB表达的变化
模型组肺组织内NF-κB的表达与正常组相比明显升高,差异有显著性(P<0.01);经激素治疗后,大鼠肺组织内NF-κB的表达与模型组相比降低,与正常组相比升高,差异均有显著性(P<0.01)。
3.2.2肺组织内MMP-9表达的变化
模型组肺组织内MMP-9的表达与正常组相比明显升高,差异有显著性(P<0.01);经激素治疗后,大鼠肺组织内MMP-9的表达与模型组相比降低,与正常组相比升高,差异均有显著性(P<0.01)。
4各指标之间的相关性
应用Spearman等级相关法分析发现:IL-25与IL-12之间存在负相关(r=-0.754, P<0.01),IL-25与NF-κB之间存在正相关(r=0.812, P<0.01),IL-12与NF-κB之间存在负相关(r=-0.747, P<0.01),MMp-9与与NF-κB之间存在正相关(r=0.797, P<0.01)。
结论:
1哮喘时,Th1/Th2免疫平衡破坏,T细胞向Th2方向偏移,Th2优势反应在哮喘发病机制中有重要意义。
2哮喘组大鼠NF-κB支气管肺组织中表达明显高于对照组,说明哮喘气道炎症及重塑过程中NF-κB具有重要作用。
3哮喘组大鼠NF-κB活性增强,IL-25水平上升,IL-12水平下降,IL-25/IL-12升高,相关分析示:NF-κB与Th2类细胞因子IL-25成正相关,与Th1类细胞因子IL-12成负相关。
4哮喘组大鼠NF-κB活化与MMP-9支气管肺组织中表达明显高于对照组,支气管黏膜增生肥厚明显,表明两者参与气道重塑的过程,相关分析示两者存在正相关。
5布地奈德作为治疗哮喘的有效药物,可以抑制NF-κB活性,使IL-25水平降低,IL-12水平升高,改善减轻气道炎症。使MMP-9水平降低,改善气道重塑。可作为治疗哮喘的首选。为临床治疗哮喘提供理论依据。
Objective: Bronchial asthma is recognized as one of the world's four major chronic medical profession. The leading cause of death was listed as the most. Now, with people's lives change, the incidence rate increase every year. The clinical manifestations is sudden onset recurrent wheezing, difficulty breathing, chest tightness and coughing. The pathological basis of bronchial asthma is airway inflammation and airway remodeling. In this study, through the establishment of asthma model, we observe inhaled budesonide in asthmatic rat the LI-25/ IL-12 ratio change and explore the relationship between the immune imbalance and the expression of NF-κB dynamic in asthma. Observation of inhaled budesonide airway NF-κB, MMP-9 expression in asthmatic rat, we explore relationship between airway remodeling and NF-κB.
Methods:
1 Animals and drugs
We selected 30 wistar mouse, provided by the Experimental Animal Center of Hebei Province (experimental animal quality certification number: 90301 );Budesonid produced by AstraZeneca Pharmaceuticals; Immunohistochemistry kit, one anti-cox2 rabbit anti-IgG polyclonal antibody, purchased in boshide company, SP9001 kit and DAB reagent purchased in Bio-Technology ,Beijing T-shirt. Enzyme immunoassay kit purchased in the United States RB; other experimental drugs provide by immunity teaching of the Hebei Medical University.
2 Model preparation and experimental groups
the law use random numbers and 30 male Wistar rats were randomly divided into three groups:normal control group,asthma model group, budesonide-treated group,10 in each group.Preparation of asthma model group, two-step:sub-sensitized and challenged. In addition to the normal control group, the other two groups were injectioned antigen solution 1ml (including egg protein, 100mg, aluminum hydroxide powder 100mg) by intraperitoneal sensitized on 1 day, 8 day, 15 day three times. and normal control group with equal of saline to sensitize; the 21 days, the rats were placed not completely closed self-made plexiglass box, asthma model group and the budesonide treatment group were Sucked in 1% ovalbumin (OVA) solution by the air compression pump spray Fog excitation,once a day,each20 minutes,continuously 7 days.The budesonide treatment group after excitation are used to budesonide inhalation treatment ,method 0.5mg/ml adding to 1ml saline. Budesonide compressed into the inhalation nebulizer, bid. Bronchial asthma group inhalation the saline after excitation. normal control group were stimulated with saline for the treatment. This last 7 days.
3 The preparation of tissue samples
In each groups, in the Last 24 hours after induced asthma, intraperitoneal injection of anesthesia, the right femoral artery get blood samples. the serum -70℃preservation, prepared to do enzyme-linked immunosorbent . ligation of the left lung, the right lung was slowly poured into 4% paraformaldehyde for the right lung inflated. then ligating of the right bronchus and placing the fixedan.taking right middle lobe organization prepared to do immunohistochemistry.
4 Target detection
4.1 IL-12 and IL-25 detection
Application of ELISA to detect serum IL-12 and IL-25 levels, their manual steps carry out the reagen.
4.2 TO detect NF-kB and MMP-9 expression in the lung tissue with immunohistochemistry
SP9001 Universal two-step method using to detect NF-kBand MMP-9 expression in the lung tissue, light microscopic observation, the positive part of brownish yellow. Set-positive rate, parallel statistically.
Results:
1 Identification of rat model of asthma
The model group rats after inhalation of OVA appear restless, scratch facial fur and other allergic symptoms, then bowed down and not move , shortness of breath, difficulty breathing ( the notable abdominal breathing), and some rats were audible and louddly expiratory wheezing, nose and mouth out of mucus,severe cases,irregular breathing could occur.Repeatedly stimulated, fur dull, lassitude and unresponsiveness.
2 Enzyme-link ed immunosorbent assay results
2.1 The serum IL-25 levels
The serum IL-25 concentration (40.71±3.87) pg/ml in asthma group, The serum IL-25 concentration (30.43±1.48) pg/ml in hormone therapy group, The serum IL-25 concentration (25.96±1.74) pg/ml in control group ,three groups have more statistically significant (P <0.05). And asthma group serum IL-25 levels(40.71±3.87) higher than the hormone therapy group (30.43±1.48) pg/ml, their have more significant differences. Asthma group serum IL-25 levels (40.71±3.87) significantly higher (25.96±1.74) pg /ml than the control group, their have more significant difference (P <0.05), The hormone therapy group and the control group was no significant difference. (P> 0.05).
2.2 The serum IL-12 levels
The serum IL-12 concentration (25.91±3.20) pg/ml in asthma group, The serum IL-12 concentration (29.42.±2.43) pg/ml in hormone therapy. serum IL-12 concentration (33.71±2.69) pg/ml in control group, three groups have more statistically significant (P <0.05). And asthma group serum IL-12 levels (25.91±3.20) pg/ml lowwer than the hormone therapy group,Their have a statistically significant difference (P <0.05), asthma group serum IL-12 levels (25.91±3.20) pg / ml significantly lower than the control group, their have significant difference (P <0.05),the hormone therapy and the control group were statistically significant (P <0.05).
2.3 Serum IL-25 / IL-12 ratio of change
Compared with the normal group, asthma group serum IL-25 / IL-12 ratio was significantly higher, the difference was significant (P <0.01); After hormone treatment, serum IL-25 / IL-12 ratio, compared with asthma group, ratio decreased; compared with the control group increased, the difference also was significant (P <0.01).
3 Identification of biopsy results
3.1 Light microscopy
Asthmatic rat bronchioles had spasm,contraction, lumen containing secretions,bronchioles, the small blood vessels around the apparent infiltration of inflammatory cells,including eosinophils, lymphocyte predominance and neutrophils;alveolar interval widened, fibrosis, a large number of lymphocytes, macrophages infiltration. Treated rats had no significant bronchiole spasm, contraction, non-intraluminal secretions, wall significantly reduced infiltration of inflammatory cells ,without interval widened.
3.2 Immunohistochemistry results
NF-κB and MMP-9 in the asthmiatic group, hormone therapy group and normal control rats were positive expression in lung tissue and were mainly expressed in the nucleus, a small amount of expression in the cytoplasm.
3.2.1 Lung tissue NF-κB expression
Asthmatic group, the lung tissue expression of NF-κB was significantly higher compared with the normal group, the difference was significant (P <0.01); After hormone treatment, the rat lung tissue expression of NF-κB compared with Asthmatic group lower , when compared with the normal group increased, the differences were significant (P <0.01)
3.2.2 Lung tissue MMP-9 expression
Asthmatic group,The lung tissue expression of MMP-9 was significantly higher compared with the normal group,the difference was significant (P <0.01); After hormone treatment, the rat lung tissue expression of MMP-9 compared with the Asthmatic group reduced,compared with the control group increased, the differences were significant (P <0.01).
4 The correlation between the various indicators
Application of Spearman Rank Correlation analysis showed that:IL-25 and IL-12 were negative correlation (r =- 0.754, P <0.01), IL-25 and NF-κB were positive correlation (r = 0.812, P < 0.01), IL-12 and NF-κB were negative correlation (r =- 0.747, P <0.01), MMP-9 and NF-κB were positive correlation (r = 0.797, P <0.01).
Conclusion:
1 The immune balance of Th1/Th2 was broken,and it migrant to Th2 direction.Th2 dominant reaction play an important role in the pathogenesis of asthma.
2 The percentage of NF-κB in asthma mice was notable higher than that of the control group.It hint that plays an important role in the process of the airway inflammation and remodeling of asthma.
3 In asthma group.the positive percentage of NF-κB and the level of IL-25 increase signicantly,but the level of IL-12 decreased, the ratio of IL-25/IL-12 increase, correlation analysis showed: NF-κB and IL-25 were positively correlated:NF-κB and IL-12 were negative correlation.
4 Asthma, NF-κB activation and MMP-9 expression in bronchopulmonary tissues were significantly higher than control group.Bronchial mucosal hyperplasia hypertrophy significantly, indicating that both participate in the process of airway remodeling,correlation analysis showed a positive correlation between the two.
5 Budesonide as the effective drug treatment of asthma can inhibit NF-κB activity,so that it is IL-25 levels decrease, IL-12 levels increased, and reduce airway inflammation;Budesonide also can reduce the level of MMP-9 and improve airway remodeling.Can be used as first choice for treatment of asthma and provide a theoretical basis for the treatment of asthma.
引文
1 Nightingale JA, Rogers DF, Hart LA, et al. Effect of inhaled endotoxin on induce sputum in normal, atopic and atopic asthmatic subject .Am J Respir Cirt care Med,1998,158 (5 Pt 1)::1585-1592
2王玲,张霞,薛玉文,等.哮喘患者白细胞介素IL-12和IL-13的变化及糖皮质激素对其影响,中华内科杂志,2003,01:24-36
3 Chen XQ ,Yang J,Hu sp,et al.Increased expression of CD86 and reduced production of IL-12 and IL-10 by monocyte derived dendritic cells from allergic and their effect on TH1 and Th2-type cytokine balance.Respiration,2006,73(1):34-40
4 Tomitab K. Lim S, Hanazawa T,et al. Attenuated production of intracellular IL-12 and IL-10 in monoctyes from patients with severe asthma.clin Immunol 2002,102(3):258-266
5 Ye Yl,huang Wc,Lee YL,et al.Interleukir 12 inhibits eotaxin secertion of cultured primary lung cells and alleviates airway inflammation in vivo.Cytokine,2002,19(2):76-84
6 Kim TS,Dekruyff Rh,Rupper R,et al.An ovalbumir IL-12 fusion protein is more effective than ovallbumin plus free recombinant IL-12 in inducing a Thelper cell type 1dominated immune response and inhibitting angigen-specific LgE production.J Immunol,1997,158(9):4137-4144
7 Sharkhuu T, Matthaei KI, Forbes E,et al. Mechanism of interleukin-25 (IL-17E)-induced pulmonary inflammation and airways hyper-reactivity. Clinical and Experimental Allergy, 2006,36(12): 1575~1583
8 Hurst SD, Muchamuel T, Gorman DM, et al. New IL-17 family members promote Th1 or Th2 responses in the lung: in vivo function of the novel cytokine IL-25. J Immunol, 2002,169(1):443~453
9 Kim MR, Manoukian R, Yeh R, et al. Transgenic overexpression of human IL-17E results in eosinophilia, B-lymphocyte hyperplasia, and altered antibody production. Blood, 2002,100(7):2330~2340
10 Pan G, French D, Mao W, et al. Forced expression of murine IL-17Einduces growth retardation, jaundice, a Th2-biased response, and multiorgan inflammation in mice. J Immunol,2001,167(11):6559-6567
11 Tamachi T, Maezawa Y, Ikeda K, et al. IL-25 enhances allergic airway inflammation by amplifying a Th2 celldependent pathway in mice. J Allergy Clin Immunol ,2006,118(1):606-614
12 Tamachi T , Maezawa Y, Ikeda K,et al. Interleukin 25 in allergic airway inflammation. Int Arch Allergy Immunol ,2006,140(suppl 1):59~62.
13 Wong CK, Li PW, Lam CWK, et al. Intracellular JNK,p38 MAPK and NF-κB regulate IL-25 induced release of cytokines and chemokines from costimulated T helper lymphocytes. Immunology Letters,2007,112(2):82-91
14 Wang YH, Angkasekwinai P, Lu N, et al. IL-25 augments type 2 immune responses by enhancing the expansion and functions of TSLP-DC–activated Th2 memory cells.JEM , 2007,204(8): 1837~1847
15 Barnes PJ,Karin M..Nuclear factor-kB:a pivotal transcription factor inchoric inflammation disease. New England Journal of Medicine,1997,336(10):1066-1071
16 Hart LA, Krishnan VL,Adock LM,et al.Activation and location of transcription factor,nuclear factor-kB in asthma.Am J Respir Cirt Care Med,1998,158 (5 Ptl):1585-1592
17 Rin F, Bbu S. Bioiogy of human Th1 cells. Gchin Immunol,1999,15(3):121-129
18 Chen ZJ,Parent L,Maniatis T.Site-specific phosphorylation of I-κB bya novel ubiquitnination dependent protein kinase activity.cell,1996,84(6):853-862
19 Woolley Kl,Gibon PG,Carty K,et al. Eosinophil apoptosis and the resolution of airway inflammation in asthma. Am J Respir crit care Med,1996,154(1):237-243
20 Ruile A,Cai ZZ,pretolani M,et al. Fas mediated apoptosisin cultured human eosinophils.Blood,1999,87(4):2282-2290
21 Yamasaki K,A sai T,Shimizu M,et al.Inhibition of NF-kappaB activationusing cis-elementdecoy of Nf-KappaB binding site reduces neointimal from ation in porcine balbon-injured coronary artery model.Model Gene Ther,2003,10(4):356-364
22 YC Lee,HB Lee,Yk Rhee,et al.The involvement of matrix metalloproteinase-9 in airway inflammation of patients with acute asthma. Clin Exp Allergy,2001,31(10):1623-1630
23 Belleguic C,Corbel M,Germain N,et al.Increased release of matrix metalloproteinase-9 in the plasma of acute severe asthmatic patients.Clin Exp Allergy,2003,32(2):217-223
24 Wu YG, Wang LX.Function of MMP/TIMP on airway remodeling of bronchial asthma and treating effects of zhichuan capsule.zhong xi yi jie xue Bao,2004,2(6):435-439
25 Alkinson JJ,Senior RM,Matrix metalloproteinase-9 in lung remodeling.Am J Respir Cell Mol Biol,2003,28(1)12-24
26 xu shuyun,xu yongjian,zhang zhengxiang et al.Effect of nuclear factor-κB on airway remoding in asthmatic rats.J Huazhong Univ Sci Technolog Med Sci,2004,24(1):13-18
27马秀琴,黄茂,德伟等.哮喘大鼠肺组织中NF-κB与MMP-9表达的关系.江苏医药,2006,07:684-685
28 Ebert H , Schulze M, Engels C, et al. Glucocorticod-mediated suppression of cytokine-induced matrixm etalloproteinase-9 expression in rat mesangal cell: involvement of nuclear factor-kappaB and E transcription factors.Mol Endocrinol,2002, 16(8):752-766
29 Vignola AM. And R.L. Effects of low doses of inhaled fluticasone propionate on inflammation and remodelling in persistent-mild asthma[J]Allergy,2005,60(12):1511-1517
30陈强,刘建梅,李丽,等.吸入糖皮质激素对支气管哮喘患儿气道重塑的影响,中华哮喘杂志,2009,3(2):14-15
31 Gibson PG,Sahos N,Fakc K.Acte Anti-inflammatory effects of inhaled budesonide in asthma: a randomized controlled trial. Am Respir Crit are Med,2001,163(1):32-36
32尹文,袁静,黄杨,等.急性肺损伤核因子—κB的活性变化及糖皮质激素的干预研究.中国急救医学,2002,22(9):497-498
33 Hart L,Sam Lim, PETER J, et al. Effects of Inhaled Corticosteroid Therapy on Expression and DNA-Binding Activity of Nuclear Factor B in Asthma Am J Respir Crit care Med,2000,161(1):224-231
34雷向阳,梁仁.糖皮质激素抵抗性哮喘的新进展.广东药学院学报,2005,23(3):21-24
1 Global strategy for asthma management andprenention. 2008(update)[EB/OL],hppt://www.ginasthma.org
2中华医学会儿科学分会呼吸学组.儿童支气管哮喘与防治指南.中华儿科杂志,2008,46(10):745-753
3 Rytil? , A. S. Pelkonen, T. Metso, et al. Induced sputum in childrenwith newly diagnosed mild asthma:the effect of 6 monthsof treatment with budesonide or disodium cromoglycate. Allergy,2004,59(8):839-844
4 Stelmach I, P.Majak, W.Stelmach, et al. The effect of montelukast and different dose of budesonide on lgE serum levels and clinical parameters in children with newly diagnosed asthma .Pulm-Pharmacol Ther,2005,18(5):374-380
5 Olof Selroos,Staffan Edsb cker ,Christer Hultquist, et al. Once-daily inhaled budesonide for the treatment of asthma: Clinical evidence and pharmacokinetic explanation. Asthma, 2004,41(8): 771-790
6 Nick P Adams, Janine C Bestall, Toby J Lasserson, et al. Fluticasone versus placebo for chronic asthna in adults and children.Cochraane-Database-syst-Rev,2005,4(4):31-35
7 Masoli M, Weatherall M , Holt S, et al. Systematic review of the dose-response relation of inhaled fluticasonepropionate.Aych-Dis-child,2004,89(10):902-907
8 Mahachoklert wattana, Direkwattanachai C, Choubtum L, et al.
9 Decreased cortisol response to insulin induced hypoglycaemia in asthmatics treated with inhaled fluticasone propionate.Arch-Dis-child, 2004,89(11):1055-1058
10 O'Connor B,Bonnaud G,Haahtela T,et al.Dose-ranging study of Mometasone furoate dry powder inhaler in the treatment of moderate pers-istent asthma using fluticasone propionate as an active comparator. Ann All ergy Asthma Immunol,2001,86(4):397-404
11 Bousquet J,D'IJrzoA,Hebert J ,et al. Comparison of the efficacy and safety of mometasone furoate dry powder inhaler to budesonide Turbuhaler. Eur RespirJ,2000,16(5):808-816
12 Ayne DN,Rogers AV,Adelroth,et al.Early thicking of the reticular basement membrane in children with difficult asthma.Am J Respir crit care Med,2003,167(1):78-82
13 Jekins HA,Cool C,Szefler SJ,et al.histopathology of severe childhoodasthma:a case series.chest,2003,124(1):32-41
14 Hashimoto M,Tanaka H,Abe S.Quantitative analysis of bronchial wall vasularity in the medium and small airway of patients with asthma and COPD.Chest ,2005,127(3):965-972
15 Hoshio M,Takahashi M,Takai Y,et al. Inhaled corticosteoids decrease vascularity of the bronchial mucosa in patients with asthma.Ciln ExpAllergy,2001,31(5):722-730
16 Chetta A, Zanini A, Foresi A,et al. Vascular component of airway remodling in asthma is reduced by high dose of fluticasone.Am J Respir crit care Med,2003,167(5):751-757
17 Xie S,Sukkar MB,Issa R,et al.Mechanisms of induction of airway smooth muscle hyperplasia by transforming growth factor-beta.Am J physiolLungCellMolPhysiol,2007,293(1):245-253
18 Vignola AM, Chanea P,Chiappara G,et al.Transforming growth factor-beta expression in mucosal biopsies in asthma and chronic bronchitis.Am J Respir Crit Care Med,1997,156(2 Pt1):591-599
19陈强,刘建梅,李丽,等.吸入糖皮质激素对支气管哮喘患儿气道重塑的影响.中华哮喘杂志,2009,3(2):14-15
20 Bousquet J, Jeffery PK,Busse WW,et al.Asthma.From bronchoconstriction to airways inflammation and remodeling.Am Respir crit care Med,2000,161(5):1720-1745
21 Gibson PG, Sahos N, Fakcs K.Acute anti-inflammatory effects of inhaled budesonide in asthma; a randomized controlled trial. Am J Respir Crit care Med,2001,163(1):32-36
22 Sagara H,Okada T,Okumura K,et al.Activation of TGF-beta/smad2 signaling is associated with airway remodeling in asthma. J Allergy Clin Immunl,2002,110(no.2 Pt 1):249-254
23 Li G,Wang S,Gelehrter TD.Identification of glucocorticoid receptor domains involed in transrep ression of transforming growth factor-beta action.J Biol Chem,2003,278(43):41779-41788
24 Pelaia G,Cuda G,Vatrella A,et al.Effects of transforming growth factor-beta and budesonide on mitogen-activated protein kinase activation and apoptosis in airway epithelial cells. Am J Respir Cell Mol Biol,2003,29(1):12-18
25张春兰.雾化吸入治疗在呼吸系统疾病中的应用.安徽卫生职业技术学院学报,2008,7(1):3-25
26朱锦萍,赵荣升.吸入给药及其临床应用评价.临床药物治疗杂志, 2008,6(1):49-52
27 Molimard M, GuenoléE, Duvauchelle T, et al. A randomized,double-bline, double-dummy,safety crossover tial comparing cumulative dosesup to 96 microg of formnoterol delivered via an HFA-134a-propelled pMDI vs same cumulative doses of formoterol DPI and placebo in asthmatic patients. Respiration,2005,72(suppl 1):28-34
28于永丰,潘玉娟,曲政海,等.长期吸入糖皮质激素对支气管哮喘儿童血清皮质醇,生长激素,胰岛素抑生长因子系统的影响.实用儿科临床杂志, 2007,22(4):275-276
29梁慧,赵德育,秦铭,等.毛细支气管炎患儿吸入激素治疗后血清皮质醇的变化.陕西医学杂志,2008,37(9):1144-1146
30 Reis TF,Altman LC,Chervisky P, et al.A once-daily leukotrienee rceptor antagonist in the teratment of chronic asthma.Intnet Med,1998,158(11):1213-1220
31 Leef JA,Busse W,PealmanD,et a1. A leukotriene receptor antagonist for the Teratment of mild asthma and excercist induced bornchoconstrictions. New Eng J Med,1998,339(3):147
32 Beeh KM,BeierJ,Konrmann O,et al.Efficacy and safety of salmeteorl( 50 microgram)and fluticasone(250 microgram)in a single inhaler device (diskus)in patients with mild to moderate asthma.Pneumologie,2002,56(2):91-97
33 BusseW ,New man SP.Evolution of dry powdet inhaler design formulation and performance.Respir Med,2002,96(5):293-304
34 Brown PH. Greening AP,Crompton GK. Large volume spacer devicesand the influence of high dose beclomethasone dipropionate on hypothalamo-pituitary -adrenal axis function .Thorax,1993,48(3):233-238
35 Jackson LD,Polygenis D,Mclvor RA,et a1.Comparative efficacy and safety of inhaled corticosteroids inasthma.Clin Pharmacol,1999,6(1):26-37
36 Greening AP,Ind Pw,Northfield M,et al.Added salmeterol versus hjgher—dose corticosteroid in asthma patients with symptoms on existinginhaled corticosteroid . Lancet,1994,344(2):219-224
37 Baraniuk J N,Ali M,Brody D,et al.Glucocorticoids induce beta2一adrenergic receptor function in human nasal mucosa.Am Respircri care med,1997,155(5):704-7l0
2王玲,张霞,薛玉文,等.哮喘患者白细胞介素IL-12和IL-13的变化及糖皮质激素对其影响,中华内科杂志,2003,01:24-36
3 Chen XQ ,Yang J,Hu sp,et al.Increased expression of CD86 and reduced production of IL-12 and IL-10 by monocyte derived dendritic cells from allergic and their effect on TH1 and Th2-type cytokine balance.Respiration,2006,73(1):34-40
4 Tomitab K. Lim S, Hanazawa T,et al. Attenuated production of intracellular IL-12 and IL-10 in monoctyes from patients with severe asthma.clin Immunol 2002,102(3):258-266
5 Ye Yl,huang Wc,Lee YL,et al.Interleukir 12 inhibits eotaxin secertion of cultured primary lung cells and alleviates airway inflammation in vivo.Cytokine,2002,19(2):76-84
6 Kim TS,Dekruyff Rh,Rupper R,et al.An ovalbumir IL-12 fusion protein is more effective than ovallbumin plus free recombinant IL-12 in inducing a Thelper cell type 1dominated immune response and inhibitting angigen-specific LgE production.J Immunol,1997,158(9):4137-4144
7 Sharkhuu T, Matthaei KI, Forbes E,et al. Mechanism of interleukin-25 (IL-17E)-induced pulmonary inflammation and airways hyper-reactivity. Clinical and Experimental Allergy, 2006,36(12): 1575~1583
8 Hurst SD, Muchamuel T, Gorman DM, et al. New IL-17 family members promote Th1 or Th2 responses in the lung: in vivo function of the novel cytokine IL-25. J Immunol, 2002,169(1):443~453
9 Kim MR, Manoukian R, Yeh R, et al. Transgenic overexpression of human IL-17E results in eosinophilia, B-lymphocyte hyperplasia, and altered antibody production. Blood, 2002,100(7):2330~2340
10 Pan G, French D, Mao W, et al. Forced expression of murine IL-17Einduces growth retardation, jaundice, a Th2-biased response, and multiorgan inflammation in mice. J Immunol,2001,167(11):6559-6567
11 Tamachi T, Maezawa Y, Ikeda K, et al. IL-25 enhances allergic airway inflammation by amplifying a Th2 celldependent pathway in mice. J Allergy Clin Immunol ,2006,118(1):606-614
12 Tamachi T , Maezawa Y, Ikeda K,et al. Interleukin 25 in allergic airway inflammation. Int Arch Allergy Immunol ,2006,140(suppl 1):59~62.
13 Wong CK, Li PW, Lam CWK, et al. Intracellular JNK,p38 MAPK and NF-κB regulate IL-25 induced release of cytokines and chemokines from costimulated T helper lymphocytes. Immunology Letters,2007,112(2):82-91
14 Wang YH, Angkasekwinai P, Lu N, et al. IL-25 augments type 2 immune responses by enhancing the expansion and functions of TSLP-DC–activated Th2 memory cells.JEM , 2007,204(8): 1837~1847
15 Barnes PJ,Karin M..Nuclear factor-kB:a pivotal transcription factor inchoric inflammation disease. New England Journal of Medicine,1997,336(10):1066-1071
16 Hart LA, Krishnan VL,Adock LM,et al.Activation and location of transcription factor,nuclear factor-kB in asthma.Am J Respir Cirt Care Med,1998,158 (5 Ptl):1585-1592
17 Rin F, Bbu S. Bioiogy of human Th1 cells. Gchin Immunol,1999,15(3):121-129
18 Chen ZJ,Parent L,Maniatis T.Site-specific phosphorylation of I-κB bya novel ubiquitnination dependent protein kinase activity.cell,1996,84(6):853-862
19 Woolley Kl,Gibon PG,Carty K,et al. Eosinophil apoptosis and the resolution of airway inflammation in asthma. Am J Respir crit care Med,1996,154(1):237-243
20 Ruile A,Cai ZZ,pretolani M,et al. Fas mediated apoptosisin cultured human eosinophils.Blood,1999,87(4):2282-2290
21 Yamasaki K,A sai T,Shimizu M,et al.Inhibition of NF-kappaB activationusing cis-elementdecoy of Nf-KappaB binding site reduces neointimal from ation in porcine balbon-injured coronary artery model.Model Gene Ther,2003,10(4):356-364
22 YC Lee,HB Lee,Yk Rhee,et al.The involvement of matrix metalloproteinase-9 in airway inflammation of patients with acute asthma. Clin Exp Allergy,2001,31(10):1623-1630
23 Belleguic C,Corbel M,Germain N,et al.Increased release of matrix metalloproteinase-9 in the plasma of acute severe asthmatic patients.Clin Exp Allergy,2003,32(2):217-223
24 Wu YG, Wang LX.Function of MMP/TIMP on airway remodeling of bronchial asthma and treating effects of zhichuan capsule.zhong xi yi jie xue Bao,2004,2(6):435-439
25 Alkinson JJ,Senior RM,Matrix metalloproteinase-9 in lung remodeling.Am J Respir Cell Mol Biol,2003,28(1)12-24
26 xu shuyun,xu yongjian,zhang zhengxiang et al.Effect of nuclear factor-κB on airway remoding in asthmatic rats.J Huazhong Univ Sci Technolog Med Sci,2004,24(1):13-18
27马秀琴,黄茂,德伟等.哮喘大鼠肺组织中NF-κB与MMP-9表达的关系.江苏医药,2006,07:684-685
28 Ebert H , Schulze M, Engels C, et al. Glucocorticod-mediated suppression of cytokine-induced matrixm etalloproteinase-9 expression in rat mesangal cell: involvement of nuclear factor-kappaB and E transcription factors.Mol Endocrinol,2002, 16(8):752-766
29 Vignola AM. And R.L. Effects of low doses of inhaled fluticasone propionate on inflammation and remodelling in persistent-mild asthma[J]Allergy,2005,60(12):1511-1517
30陈强,刘建梅,李丽,等.吸入糖皮质激素对支气管哮喘患儿气道重塑的影响,中华哮喘杂志,2009,3(2):14-15
31 Gibson PG,Sahos N,Fakc K.Acte Anti-inflammatory effects of inhaled budesonide in asthma: a randomized controlled trial. Am Respir Crit are Med,2001,163(1):32-36
32尹文,袁静,黄杨,等.急性肺损伤核因子—κB的活性变化及糖皮质激素的干预研究.中国急救医学,2002,22(9):497-498
33 Hart L,Sam Lim, PETER J, et al. Effects of Inhaled Corticosteroid Therapy on Expression and DNA-Binding Activity of Nuclear Factor B in Asthma Am J Respir Crit care Med,2000,161(1):224-231
34雷向阳,梁仁.糖皮质激素抵抗性哮喘的新进展.广东药学院学报,2005,23(3):21-24
1 Global strategy for asthma management andprenention. 2008(update)[EB/OL],hppt://www.ginasthma.org
2中华医学会儿科学分会呼吸学组.儿童支气管哮喘与防治指南.中华儿科杂志,2008,46(10):745-753
3 Rytil? , A. S. Pelkonen, T. Metso, et al. Induced sputum in childrenwith newly diagnosed mild asthma:the effect of 6 monthsof treatment with budesonide or disodium cromoglycate. Allergy,2004,59(8):839-844
4 Stelmach I, P.Majak, W.Stelmach, et al. The effect of montelukast and different dose of budesonide on lgE serum levels and clinical parameters in children with newly diagnosed asthma .Pulm-Pharmacol Ther,2005,18(5):374-380
5 Olof Selroos,Staffan Edsb cker ,Christer Hultquist, et al. Once-daily inhaled budesonide for the treatment of asthma: Clinical evidence and pharmacokinetic explanation. Asthma, 2004,41(8): 771-790
6 Nick P Adams, Janine C Bestall, Toby J Lasserson, et al. Fluticasone versus placebo for chronic asthna in adults and children.Cochraane-Database-syst-Rev,2005,4(4):31-35
7 Masoli M, Weatherall M , Holt S, et al. Systematic review of the dose-response relation of inhaled fluticasonepropionate.Aych-Dis-child,2004,89(10):902-907
8 Mahachoklert wattana, Direkwattanachai C, Choubtum L, et al.
9 Decreased cortisol response to insulin induced hypoglycaemia in asthmatics treated with inhaled fluticasone propionate.Arch-Dis-child, 2004,89(11):1055-1058
10 O'Connor B,Bonnaud G,Haahtela T,et al.Dose-ranging study of Mometasone furoate dry powder inhaler in the treatment of moderate pers-istent asthma using fluticasone propionate as an active comparator. Ann All ergy Asthma Immunol,2001,86(4):397-404
11 Bousquet J,D'IJrzoA,Hebert J ,et al. Comparison of the efficacy and safety of mometasone furoate dry powder inhaler to budesonide Turbuhaler. Eur RespirJ,2000,16(5):808-816
12 Ayne DN,Rogers AV,Adelroth,et al.Early thicking of the reticular basement membrane in children with difficult asthma.Am J Respir crit care Med,2003,167(1):78-82
13 Jekins HA,Cool C,Szefler SJ,et al.histopathology of severe childhoodasthma:a case series.chest,2003,124(1):32-41
14 Hashimoto M,Tanaka H,Abe S.Quantitative analysis of bronchial wall vasularity in the medium and small airway of patients with asthma and COPD.Chest ,2005,127(3):965-972
15 Hoshio M,Takahashi M,Takai Y,et al. Inhaled corticosteoids decrease vascularity of the bronchial mucosa in patients with asthma.Ciln ExpAllergy,2001,31(5):722-730
16 Chetta A, Zanini A, Foresi A,et al. Vascular component of airway remodling in asthma is reduced by high dose of fluticasone.Am J Respir crit care Med,2003,167(5):751-757
17 Xie S,Sukkar MB,Issa R,et al.Mechanisms of induction of airway smooth muscle hyperplasia by transforming growth factor-beta.Am J physiolLungCellMolPhysiol,2007,293(1):245-253
18 Vignola AM, Chanea P,Chiappara G,et al.Transforming growth factor-beta expression in mucosal biopsies in asthma and chronic bronchitis.Am J Respir Crit Care Med,1997,156(2 Pt1):591-599
19陈强,刘建梅,李丽,等.吸入糖皮质激素对支气管哮喘患儿气道重塑的影响.中华哮喘杂志,2009,3(2):14-15
20 Bousquet J, Jeffery PK,Busse WW,et al.Asthma.From bronchoconstriction to airways inflammation and remodeling.Am Respir crit care Med,2000,161(5):1720-1745
21 Gibson PG, Sahos N, Fakcs K.Acute anti-inflammatory effects of inhaled budesonide in asthma; a randomized controlled trial. Am J Respir Crit care Med,2001,163(1):32-36
22 Sagara H,Okada T,Okumura K,et al.Activation of TGF-beta/smad2 signaling is associated with airway remodeling in asthma. J Allergy Clin Immunl,2002,110(no.2 Pt 1):249-254
23 Li G,Wang S,Gelehrter TD.Identification of glucocorticoid receptor domains involed in transrep ression of transforming growth factor-beta action.J Biol Chem,2003,278(43):41779-41788
24 Pelaia G,Cuda G,Vatrella A,et al.Effects of transforming growth factor-beta and budesonide on mitogen-activated protein kinase activation and apoptosis in airway epithelial cells. Am J Respir Cell Mol Biol,2003,29(1):12-18
25张春兰.雾化吸入治疗在呼吸系统疾病中的应用.安徽卫生职业技术学院学报,2008,7(1):3-25
26朱锦萍,赵荣升.吸入给药及其临床应用评价.临床药物治疗杂志, 2008,6(1):49-52
27 Molimard M, GuenoléE, Duvauchelle T, et al. A randomized,double-bline, double-dummy,safety crossover tial comparing cumulative dosesup to 96 microg of formnoterol delivered via an HFA-134a-propelled pMDI vs same cumulative doses of formoterol DPI and placebo in asthmatic patients. Respiration,2005,72(suppl 1):28-34
28于永丰,潘玉娟,曲政海,等.长期吸入糖皮质激素对支气管哮喘儿童血清皮质醇,生长激素,胰岛素抑生长因子系统的影响.实用儿科临床杂志, 2007,22(4):275-276
29梁慧,赵德育,秦铭,等.毛细支气管炎患儿吸入激素治疗后血清皮质醇的变化.陕西医学杂志,2008,37(9):1144-1146
30 Reis TF,Altman LC,Chervisky P, et al.A once-daily leukotrienee rceptor antagonist in the teratment of chronic asthma.Intnet Med,1998,158(11):1213-1220
31 Leef JA,Busse W,PealmanD,et a1. A leukotriene receptor antagonist for the Teratment of mild asthma and excercist induced bornchoconstrictions. New Eng J Med,1998,339(3):147
32 Beeh KM,BeierJ,Konrmann O,et al.Efficacy and safety of salmeteorl( 50 microgram)and fluticasone(250 microgram)in a single inhaler device (diskus)in patients with mild to moderate asthma.Pneumologie,2002,56(2):91-97
33 BusseW ,New man SP.Evolution of dry powdet inhaler design formulation and performance.Respir Med,2002,96(5):293-304
34 Brown PH. Greening AP,Crompton GK. Large volume spacer devicesand the influence of high dose beclomethasone dipropionate on hypothalamo-pituitary -adrenal axis function .Thorax,1993,48(3):233-238
35 Jackson LD,Polygenis D,Mclvor RA,et a1.Comparative efficacy and safety of inhaled corticosteroids inasthma.Clin Pharmacol,1999,6(1):26-37
36 Greening AP,Ind Pw,Northfield M,et al.Added salmeterol versus hjgher—dose corticosteroid in asthma patients with symptoms on existinginhaled corticosteroid . Lancet,1994,344(2):219-224
37 Baraniuk J N,Ali M,Brody D,et al.Glucocorticoids induce beta2一adrenergic receptor function in human nasal mucosa.Am Respircri care med,1997,155(5):704-7l0