【作者】 黎凯燕；

【导师】 李宜瑞；

【作者基本信息】 广州中医药大学 ， 中医儿科学， 2009， 博士

【摘要】 一、研究目的:探讨哮喘与过敏性鼻炎的关系;综合评价小儿喘咳液合苍耳子散的疗效及其优势,探讨并验证其抗哮喘气道重塑,防止不可逆性气道阻塞的作用及其对气道重塑主要相关生长因子、炎性介质及免疫调节作用的机制。二、研究方法(一)动物分组和造模:2-3个月的雄性Wistar大鼠135只,随机分为九组,每组各15只。哮喘空白组以生理盐水腹腔注射及雾化吸入。哮喘气道重塑模型分别于第1、8天腹腔注射新鲜配制的卵蛋白氢氧化铝溶液致敏。自第15天起对哮喘组动物进行超声雾化吸入卵蛋白溶液诱发哮喘,隔日1次,激发浓度分别是:第1、2周1%,第3、4周1.5%,第5、6周2%,第7、8周2.5%,每次激发时间为20分钟。(二)给药方法:从造模的第1天开始给予实验各组干预措施,一日1次,直至被处死,共给药70天。空白组、模型组给予生理盐水10ml/kg灌胃;西药组给予063mg/kg地塞米松灌胃给药;喘咳液小、中、大剂量组分别按10g/kg、5g/kg、2.5g/kg三个剂量给予小儿喘咳液(麻黄、桂枝、细辛、当归、五味子、蚤休等9味中药组成)灌胃给药;合方小、中、大剂量组分别按20g/kg、10g/kg、5g/kg三个剂量给予小儿喘咳合方灌胃给药(其中含小儿喘咳液及苍耳子散,苍耳子散药物由苍耳子、薄荷、白芷、辛夷组成)。三、结果1、一般情况:各组大鼠激发后,模型组、西药组、喘咳液组及合方组大鼠在吸入卵蛋白溶液后,约于1～2min开始出现较明显哮喘发作,空白对照组大鼠行动敏捷,未见异常。135只大鼠中有5只在引喘过程中死亡,其中咳喘液小剂量组及合方小剂量组在第二次致敏时各死亡1只,在引喘阶段,模型组死亡2只,西药组死亡1只,每组取10只,共90只大鼠进入结果分析。西药对照组大鼠从造模第12天开始出现多动,易激惹,咬人,21天时开始出现多毛及血便。至实验结束前,共有6只大鼠出现血便。而空白组、模型组、喘咳液各组及合方各组均无发现上述情况。2、过敏性鼻炎情况比较:在造模第21天,过敏性鼻炎叠加平均计分中模型组、西药组、喘咳液组和合方组较之空白组明显升高(P＜0.01);与模型组相比,喘咳液小剂量组计分有下降(P＜0.05),合方大剂量组的计分明显下降(P＜0.01)。在造模第70天,与空白组比较,模型组、喘咳液小剂量组计分大于五分的大鼠及比例增加(P＜0.05);与模型组比较,合方大剂量组计分大于五分的大鼠比例减少(P＜0.05)。平均计分中模型组、西药组、喘咳液各组和合方各组较之空白组明显升高(P＜0.01):西药组、喘咳液大剂量组与合方各剂量组的平均计分低于模型组(P＜0.05)。3、大鼠哮喘情况比较:在造模第15天,与模型组比较,合方大剂量组引喘时间明显延长,差异有非常显著性(P＜0.01);与西药组相比,合方大剂量组引喘时间延长,差异有显著性(P＜0.05);在实验第70天,与模型组比较,西药组、喘咳液大剂量组引喘时间增加,差异有显著性(P＜0.05);合方中、大剂量组引喘时间明显延长,差异有非常显著性(P＜0.01)。与自身前后对照,喘咳液中剂量组及合方中剂量组差异有显著性(P＜0.05),喘咳液大剂量组及合方大剂量组差异有非常显著性(P＜0.01)。合方大剂量组与喘咳液大剂量组比较,大鼠引喘时间延长,差异有显著性(P＜0.05)。在实验第70天,与模型组比较,合方小剂量组大鼠点头呼吸频度下降,差异有显著性(P＜0.05);西药组、喘咳液中、大剂量组及合方中、大剂量组大鼠点头呼吸频度明显下降,差异有非常显著性(P＜0.01)。4、血清细胞因子TGF-β1水平比较:与空白组相比,模型组大鼠血清TGF-β1水平明显上升,差异有非常显著性(P＜0.01)。与模型组相比,西药组、喘咳液各剂量组及合方各剂量组TGF-β1水平明显下降,差异有非常显著性(P＜0.01)。5、大鼠血清IFN-γ、IL-4水平及其比值比较:与空白组相比,模型组大鼠血清IFN-γ水平明显下降,差异有非常显著性(P＜0.01);与模型组相比,西药组、喘咳液及合方各剂量组IFN-γ水平明显上升,差异有非常显著性(P＜0.01);与西药组相比,喘咳液大剂量组及合方中、大剂量组IFN-γ水平明显上升,差异有非常显著性(P＜0.01)。与空白组相比,模型组及喘咳液小剂量组IL-4水平明显上升,差异有非常显著性(P＜0.01);西药组、喘咳液中、大剂量组及合方小、中剂量组IL-4水平上升,差异有显著性(P＜0.05)。与模型组相比,西药组、喘咳液各剂量组及合方小、中剂量组IL-4水平下降,差异有显著性(P＜0.05);合方大剂量组的IL-4水平明显下降,差异有非常显著性(P＜0.01);与西药组比较,合方大剂量组的IL-4水平下降,差异有显著性(P＜0.05);与喘咳液大剂量组相比,合方大剂量组IL-4水平下降,差异有显著性(P＜0.05)。与空白组相比,模型组大鼠血清IFN-γ/IL-4比值明显下降,差异有非常显著性(P＜0.01);喘咳液大剂量组及合方中、大剂量组大鼠血清IFN-γ/IL-4比值明显上升,差异有非常显著性(P＜0.01);与模型组相比,西药组、喘咳液及合方各剂量组IFN-γ/IL-4比值明显上升,差异有非常显著性(P＜0.01);与西药组相比,喘咳液大剂量组及合方中、大剂量组IFN-γ/IL-4比值明显上升,差异有非常显著性(P＜0.01)。6、大鼠血清ECP水平测定比较:与空白组相比,模型组大鼠血清ECP水平明显上升,差异有非常显著性(P＜0.01);与模型组相比,西药组、喘咳液大剂量组及合方大剂量组ECP水平下降,差异有显著性(P＜0.05)。7、大鼠体重增加情况比较:70天后与空白组、模型组、喘咳液及合方各剂量组比较,西药组体重增加明显,差异有显著性(P＜0.01)。8、支气管形态学观察:模型组气管上皮细胞水肿、脱落,气道壁不完整;腔内可见炎性渗出物和少量嗜酸性粒细胞、淋巴细胞、巨噬细胞;局部支气管黏膜下水肿,气道壁及气道平滑肌明显增厚,管腔狭窄;高倍镜下可见气道上皮下层纤维化改变,气道上皮下层可见大量梭形的成纤维细胞,平滑肌层可见肌成纤维细胞。杯状细胞增多,管腔内可见分泌物储留。西药组、喘咳液及合方各剂量组炎症减轻至消失,杯状细胞数量减少,气道平滑肌增厚及管腔狭窄减轻,高倍镜下可见纤维细胞减少。气道壁厚度比较,与空白组相比,模型组大鼠气道壁明显增厚,差异有非常显著性(P＜0.01);与模型组相比,西药组、喘咳液各剂量组及合方各剂量组气道壁厚度变薄,差异有非常显著性(P＜0.01)四、结论1、从临床症状、实验室指标以及支气管病理学检测三方面可以看出,采用卵蛋白氢氧化铝溶液致敏及卵蛋白溶液引喘建造的哮喘气道重塑模型取得成功。2、过敏性鼻炎是哮喘发生发展过程中重要的伴随因素,而且与气道重塑的形成密切相关。喘咳液组大鼠在哮喘缓解的同时,过敏性鼻炎的发生也较模型组有所下降,推测是由于过敏性鼻炎与哮喘存在共有的发病机制,在使用小儿喘咳液治疗哮喘的同时也截断了过敏性鼻炎的发生发展,从而使其发病率及症状计分随之下降。而运用小儿喘咳液合苍耳子散同时干预哮喘与过敏性鼻炎,较之单纯使用小儿喘咳液治疗哮喘,其在延长引喘时间、减少点头呼吸频度等方面有明显的优势,说明过敏性鼻炎的治疗,对于控制哮喘发生(大鼠引喘时间),减轻哮喘发作的程度(大鼠呼吸频度),有着重要的意义。3、大剂量的合方在预防和控制过敏性鼻炎症状、降低哮喘气道高反应性方面疗效较地塞米松对照组、喘咳液各剂量组及合方小、中剂量组更优,而改善呼吸困难方面则与西药组相当,优于喘咳液各剂量组及合方小、中剂量组,且其疗效随着治疗时间的延长而增加,但副作用更少,使用更加安全可靠。4、小儿喘咳液合苍耳子散能明显改善气道重塑大鼠支气管的病理改变,同时降低血清TGF-β1、IL-4、ECP水平,提高血清IFN-γ水平及失衡的IFN-γ/IL-4比值。我们推测小儿喘咳液合苍耳子散抗哮喘气道重塑的作用与其对细胞因子及炎症细胞的调节,调节免疫功能,减轻炎症反应有关。更多还原

【Abstract】 Objective:To evaluate the relationships between Asthma and Allergic Rhinitis; Integrative evaluation of the therapeutic effects and predominance of Xiao Er Chuan Ke Ye combined with Cang Er Zi San;Explore and validate its functions of anti-asthma airway remodeling and prevention in irreversible airway obstruction,as well as its regulating mechanism to the main airway remodeling-related growth factor,inflammation medium and immunity.Research methods:1.Group division and Modeling:One hundred and thirty five male Wistar rats (aged 2-3 months) were randomly divided into nine groups with 15 rats each group:Blank group was intraperitoneal injected and with inhalation of saline. Asthma with airway remodeling model group was sensitized by intraperitoneal injection of fresh albumin and aluminum hydroxide solution on the first,eighth day respectively,from the fifteenth day on,introduced asthma attack by means of ultrasonic aerosol inhalation of albumin solution,once every two days, 20 minutes each time,and the inspired concentration are:the first,second week 1%;the third,fourth week 1.5%;the fifth,sixth week 2%;2.5%in the seventh,eighth week.2.Medication method:From the first day on give each group experimental interventions,once per day,until they are executed,a total of 70 days of interventions.Blank group,model group were given oral saline 10ml/kg; Western medicine group were given oral administration of dexamethasone 0.63mg/kg;Xiao Er Chuan Ke Ye low,medium and high-dose group was respectively given Xiao Er Chuan Ke Ye(Composed of Mahuang,Guizhi,Xixin,Danggui,Wuweizi, Zaoxiu etc.nine Chinese Herbs) oral administration 10g / kg,5g/kg, 2.5g/kg;Combination group low,medium and high-dose group was given respectively Xiao Er Chuan Ke Combination(Composed of Xiao Er Chuan Ke Ye and Cang Er Zi San,half each,Cang Er Zi San was composed of Cangerzi, Bohe,Baizi,Xinyi) 20g/kg,10g/kg,5g/kg.Result:1.General situations:1～2 minutes after inhaled albumin solution,Model group, Western Medicine group,Chuan Ke Ye group and Combination group rats began to appear symptoms of asthma attack,while blank group’ s activities were quick and prompt,nothing unusual was detected.There were a total of five rats died of asthma attack:one in each of Chuan Ke Ye low-dose group and Combination low-dose group in the second sensitization stage;during asthma attack two died in Model group and one in Western group.Choose 10 each group,a total of 90 rats entered the final analysis.From the twelfth day on the rats in Western medicine group began to appear hyperactive,aggressive,bite others; from the twenty-first day on began to appear hairy and bloody stool.Till the end of the experiment,a total of six rats appeared bloody stool,while the Blank group,Model group,Chuan Ke Ye groups and Combination groups had no signs of above.2.Comparison of Allergic Rhinitis situation:On the twenty-first day,the allergic rhinitis average superimposed scoring in model group,western medicine group,Chuan Ke Ye group and combination group was significantly higher than blank group(P＜0.01);compare to model group,Chuan Ke Ye low-dose group was lower(P＜0.05),combination high-dose group was significantly lower (P＜0.01).On the seventieth day,compared to the blank group,the percentage of rats scoring above five in model group,Chuan Ke Ye low-dose group increased (P＜0.05);compared to the model group,the percentage of rats scoring above five in combination high-dose group decreased(P＜0.05).The average score in model group,western medicine group,Chuan Ke Ye groups and combination groups were significantly higher than blank group(P＜0.01);the average scoring in western medicine group,Chuan Ke Ye high-dose group and combination groups were lower than model group(P＜0.05).3.Comparison of asthma attack:On the fifteenth day,compared with the model group,the introducing time of asthma in combination high-dose group was significantly prolonged,the difference was significant(P＜0.01);compared with the western medicine group,the introducing time of asthma in combination high-dose group was prolonged,the difference was significant(P＜0.05);on the seventieth day of the experiment,compare with the model group,the introducing time of asthma in Western medicine group and Chuan Ke Ye high-dose group prolonged,the difference was significant(P＜0.05);combination medium, high-dose group significantly prolonged,the difference was significant(P＜0.01).Before and after self-comparison,Chuan Ke Ye medium dose group and combination medium group had significant difference(P＜0.05),Chuan Ke Ye high dose and combination high-dosegroup had significant difference(P＜0.01). Compare with combination high-dose group and Chuan Ke Ye high-dose group,the introducing time of asthma has been prolonged,the difference was significant (P＜0.05).On the seventieth day of the experiment,compared with the model group,the nodding breath frequency of combination low-dose group decreased, the difference was significant(P＜0.05);Western medicine group,Chuan Ke Ye medium,high-dose group and combination medium,high-dose decreased,the difference was significant(P＜0.01).4.Comparison of serum TGF-β1 levels:compare with the blank group,the serum TGF-β1 levels in model group significantly increased,the difference was significant(P＜0.01).Compared with model group,the level of TGF-β1 in western medicine group,all Chuan Ke Ye groups and combination groups had significantly decreased,the difference was significant(P＜0.01).5.Comparison of serum IFN-γ,IL-4 level and their ratio:compare with the blank group,the serum IFN-γlevel in model group significantly decreased, the difference was significant(P＜0.01);compare with the model group,the IFN-γlevels in Western medicine group,Chuan Ke Ye and combination groups significantly increased,the difference was significant(P＜0.01);compare with the western medicine group,the IFN-γlevels in Chuan Ke Ye high-dose group and combination medium,high-dose group significantly increased,the difference was significant(P＜0.01).Compare with the blank group,IL-4 levels in model group and Chuan Ke Ye low-dose group significantly increased,the difference was significant(P＜0.01);IL-4 levels of Western medicine group, Chuan Ke Ye medium,high dose group and combination low,medium dose increased, the difference was significant(P＜0.05).Compared with model group,IL-4 level of western medicine group,all Chuan Ke Ye groups and combination low, medium dose group decreased,the difference was significant(P＜0.05); combination high-dose group IL-4 levels significantly decreased,the difference was significant(P＜0.01);compare with the western medicine group, level of IL-4 in combination high-dose group decreased,the difference was significant(P＜0.05).Compare with Chuan Ke Ye high dose group,IL-4 levels of combination high dose group decreased,the difference was significant(P＜0.05).Compare with the blank group,serum IFN -γ/IL-4 ratio of model group significantly decreased,the difference was significant(P＜0.01);serum IFN -γ/IL-4 ratio of Chuan Ke Ye high-dose group and combination medium,high dose group increase significantly,the difference was significant(P＜0.01); Compared with model group,IFN-γ/IL-4 radio of western medicine group,all Chuan Ke Ye and combination groups significantly increased,the difference was significant(P＜0.01);compare with the western medicine group, IFN-γ/IL-4 radio of Chuan KeYe high-dose group and combination medium, high-dose group significantly increased,the difference was significant(P＜0.01).6.Comparison of serum ECP level:Compare with blank group,serum ECP levels of model group significantly increased,the difference was significant(P＜0.01);Compared with model group,ECP level of western medicine group,Chuan Ke Ye high-dose group and combination high-dose group decreased,the difference was significant(P＜0.05).7.Comparison of weight increase:After 70 days compare the blank group, model group,all Chuan Ke Ye and combination groups,Western Medicine group significantly gained weight,the difference was significant(P＜0.01).8.Bronchial morphological observation:The tracheal epithelia of model group rats edema,fall off and the airway wall is incomplete;a small amount of inflammatory exudates,eosinophils,lymphocytes,maerophages can be seen in the cavity;local bronchial submucosal edema,airway wall and airway smooth muscle obviously thicken,luminal stenosis;under high-power microscope fibrosis changes can be observed in the airway sub-epithelial cells,under the airway epithelium a large number of spindle-shaped fibroblasts can be found, myofibroblast can be observed in the smooth muscle layer.Number of goblet cells,secretion can be seen in the lumen.Airway inflammation in Western medicine group,all Chuan Ke Ye and combination groups had been reduced to disappear.Number of goblet cells decreased,both airway smooth muscle thickening and luminal stenosis have been reduced,under high-power microscope can observe the fiber cells decrease.Compared with the blank group,the airway wall thickness of model group obviously more thickening,the difference was significant(P＜0.01);Compared with model group,the airway wall thickness of western medicine group,all Chuan Ke Ye and combination groups are less thickening,the difference was significant(P＜0.01).Conclusions:1.According to the clinical symptoms,laboratory examinations and bronchial pathology observations,the models of Asthma with airway remodeling had been successfully made by means of ovalbumin and aluminum hydroxide solution sensitization and ovalbumin solution introducing asthma.2.Allergic rhinitis is not only the important accompanied factor during the occurrence and development of asthma,but also has close relationship with the development of airway remodeling.During the remission of asthma,the occurrence of allergic rhinitis in Chuan Ke Ye Group decreased,we presume allergic rhinitis and asthma have the same pathogenesis,when treating asthma with Xiao Er Chuan Ke Ye the occurrence and development of allergic rhinitis have been cut off at the same time,thus the radio of allergic rhinitis and symptoms scoring decreased too.The simultaneity use of Xiao Er Chuan Ke Ye and Cang Er Zi San intervening asthma and allergic rhinitis,comparing simply use Xiao Er Chuan Ke Ye,has the predominance in prolonging time of introducing asthma and reducing the frequency of nodding breathe,which indicated that the treatment of allergic rhinitis has great importance in asthma control(time of introducing rat asthma) and lessen the degree of asthma(frequency of rat nodding breath).3,The combination high-dose group has better therapeutic effect than dexamethasone group(western medicine group),all Chuan Ke Ye groups and combination low,medium group in the prevention and control of allergic rhinitis symptoms,reduced airway hyper responsiveness,while in improving breath difficulties combination high-dose group has similar effect with western medicine group,and better effect than all Chuan Ke Ye groups and combination low,medium group.Its efficacy increased with the prolonging of treatment time,but fewer side effects,which makes the application safer and more reliable.4,Xiao Er Chuan Ke Ye combining with Cang Er Zi San can obviously improve the pathological changes in rats with bronchial airway remodeling,reducing the serum TGF-β1,IL-4,ECP levels,increasing the level of serum IFN-γand the imbalance IFN-γ/ IL-4 ratio.We can presume the anti-airway remodeling effects of Xiao Er Chuan Ke Ye combining with Cang Er Zi San have close relationships with its regulation role to the cytokines and inflammatory cells, improving immunity function and thus reducing the inflammatory response.更多还原