盐酸氨溴索对慢性哮喘大鼠气道炎症及杯状细胞增生的影响探析

1、盐酸氨溴索对慢性哮喘大鼠气道炎症及杯状细胞增生的影响探析    【摘要】目的:观察药物盐酸氨溴索对慢性哮喘大鼠气道炎症及杯状细胞增生(GCH)的影响,探讨在哮喘防治中的作用.方法:用卵白蛋白致敏、激发建立大鼠哮喘模型,54只Wistar大鼠随机分为3组:正常对照组、哮喘组、盐酸氨溴索治疗组(简称治疗组),每组18只.激发哮喘后测定气道内压的变化;支气管肺泡灌洗液(BALF)内细胞计数、分类计数,HE染色及AB-PAS染色观察肺组织病理改变及杯状细胞增生状况.结果:治疗组气道内压增加的百分数明显低于哮喘组(P<0.01);治疗组BALF细胞总数及炎症

2、细胞数明显低于哮喘组(P道,未见粘液潴留.结论:盐酸氨溴索可抑制哮喘大鼠气道GCH,改善哮喘气道炎症,对哮喘的防治有一定作用.【关键词】哮喘 氨溴索 杯状细胞Effect of ambroxol on airway inflam-mation and goblet cell hyperplasia ofchronic asthmatic rat 【Abstract】AIM:To evaluate the protective effect of ambroxolin asthma, we observed the effect of ambroxol on airway in-fla

3、mmation and airway goblet cell hyperplasia (GCH) of chronicasthmatic rat.METHODS:Rat asthma models were estab-lished with the ovalbumin sensitization and provocation method.Fifty-four Wistar rats were divided into three groups randomly:Normal control group (n=18);blank asthma group (n=18);ambroxol g

4、roup (n=18). Sensitized rats were treatedwith ambroxol daily (10mg·kg-1·d-1) during the period of al-lergen and provocation. The change in intratra-cheal pressure of sensitized rats was recorded. The total anddifferential white blood cell counting of bronchial alveolarlavage fluid (BALF) w

5、as calculated. Lung tissue sections werestained with hematoxylin and eosin for general morphology andAlician Blue-Periodic Acid Schiff (AB-PAS)for identification ofgoblet cells. The pathologic changes were observed under anoptical microscope.RESULTS:Percentage of increased intra-tracheal pressure in

6、 ambroxol group was significantly lower thanthat in the blank asthma group (P<0.01). The bronchoconstriction and inflammatory cells infiltrationsurrounding bronchi in the asthma group were observed. Amarked and extensive airway GCH and mucus retention in air-way lumen in the blank asthma group we

7、re also observed. Theairway inflammation and GCH were significantly alleviated andno mucus retention in the airway lumen was observed in Am-broxol group. Mildly GCH was restricted in the larger airways.CONCLUSION:Ambroxol can alleviate the airway inflammationand suppress GCH in chronic asthmatic rat

8、s. Ambroxol is valu-able in the prophylaxis and treatment of asthma.【Keywords】asthma; ambroxol; goblet cells 0引言 气道上皮杯状细胞增生(goblet cell hyperplasia,GCH)是哮喘的病理特征之一,是哮喘时气道上皮最明确的改变. GCH可引起气道上皮增厚,糖蛋白分泌增加,导致气道管腔狭窄,气道阻力增加.盐酸氨溴索(Ambroxol,商品名mucosulvan),是一种常用的祛痰剂,近年来其对呼吸系统的保护作用倍受关注,它可以抑制粘液腺和杯状细胞中酸

9、性糖蛋白合成.我们应用盐酸氨溴索对慢性哮喘大鼠进行干预,观察其长期作用对哮喘大鼠GCH及气道炎症的影响.1材料和        方法 1.1材料雄性Wistar大鼠54只,体质量(250±50) g,由第二军医大学实验动物中心提供.卵白蛋白为美国Sigma公司产品.盐酸氨溴索片由德国勃林格英格翰公司提供.阿尔辛蓝(Alcian blue 8GX)为美国Amersco公司产品. EJD-IV型生物信号处理系统系第二军医大学生理教研室研制.定容型动物呼吸机DH-1408型为浙江大学医学院医学仪器试验

10、厂生产.1.2方法1.2.1动物分组及模型制备用卵白蛋白致敏、激发建立大鼠哮喘模型.参照文献1稍加改变复制哮喘大鼠模型:哮喘组,予免疫原液1 mL(含卵蛋白100 mg、氢氧化铝100 mg)胸前皮下注射致敏,同时ip气管炎疫苗(含5×1012个灭活菌),第15日起予1g·L-1卵蛋白超声雾化吸入,每日20 min,连续7 d.盐酸氨溴索治疗组致敏前1 wk即开始ig给予盐酸氨溴索片10 mg·kg-1·d-1,2次·d-1ig喂食,与哮喘组同时致敏、激发,处理亦相同,但实验中一直服用盐酸氨溴索,共连续服用4 wk.正常对照组予生理盐水胸前sc

11、及ip,第15日起予生理盐水超声雾化吸入,每日20 min,连续7 d.1.2.2气道内压测定末次激发前各组大鼠随机取5只检测激发前后气道内压的变化,戊巴比妥钠ip麻醉,气管插管,Y形管分别连接定容呼吸机、压力传感器及生物信号处理系统,先记录未激发时气道内压,待压力平稳后各组分别予10 g·L-1生理盐水及卵蛋白氧气驱动雾化激发20 min,激发后持续记录气道内压60 min,观察气道内压变化,计算记录过程中气道内压的最大增幅作为气道内压变化的指标.计算公式:气道内压增加的百分数=(激发后气道内压-激发前气道内压)/激发前气道内压×100%.1.2.3病理学检查每组随机取5

12、只大鼠于末次激发后行病理检查,每叶肺取样进行脱水,石蜡包埋连续切片,片厚5m,行HE染色及阿尔辛蓝-糖原染色(AB-PAS染色). AB-PAS染色:切片入10 g·L-1阿尔辛蓝(pH 2.5)染色30 min, 10 mL·L-1过碘酸氧化10 min,再入Schiffs液暗处加盖30 min左右,作苏木素核衬染,中性粘液呈红色,酸性粘液物质呈蓝色,混合性粘液呈紫红色,气道上皮细胞胞质被染成蓝色或紫红色的为杯状细胞.光镜下观察,按Ceng2的方法每只大鼠观察5张不同部位的切片,每张切片观察12个高倍视野的杯状细胞总数,其中叶支气管、小支气管、细支气管或终末细支气管各4个

13、视野,计算其平均值.1.2.4支气管肺泡灌洗液计数及分类其余大鼠末次激发后行支气管肺泡灌洗,收集支气管肺泡灌洗液(BALF)并记录回收量.用血球计数板行BALF细胞计数并计算细胞总数,将BALF 4离心(2500 r·min-1, 8 min),取沉渣适量涂片,采用瑞氏染色法进行染色,镜检300个细胞按照形态学标准进行细胞分类计数.统计学处理:应用统计软件SPSS进行数据分析,数据用x±s表示,采用完全随机设计资料的方差分析和LSD-t检验检测样本均数差异的显著性.2结果 2.1气道内压的变化各组大鼠激发前气道内压无显著差异(P>0.05).正常对照组超声雾

14、化生理盐水后气道内压平均最大增幅为(18.4±4.7)%, 10min左右恢复至正常;哮喘组大鼠卵蛋白激发后气道内压迅速增加,在5 min内达到高峰,平均最大增幅为(119.2±30.1)%, 30 min左右逐渐恢复至正常.治疗组气道内压平均最大增幅为(58.8±19.0)%,明显低于哮喘组.气道内压最大增幅在各组之间均有显著差异(P<0.01).2.2肺组织HE染色所见对照组支气管、肺组织结构正常,无支气管收缩征象,亦未见炎症细胞浸润.哮喘组支气管粘膜上皮增厚,管腔明显狭窄,可见明显支气管痉挛征象,管壁及其周围大量炎细胞浸润,除中性粒细胞及淋巴细胞外,可

15、见大量嗜酸性粒细胞.治疗组支气管粘膜上皮无明显增厚,管腔狭窄的程度及发生频率均较哮喘组明显减轻,管壁及其周围少量炎细胞浸润,支气管腔内无粘液栓形成.2.3AB-PAS染色所见哮喘组杯状细胞增生明显,从支气管到终末细支气管,每一级气道均可见杯状细胞,支气管、叶支气管及小支气管杯状细胞大量增生.随机观察50个终末细支气管,38%也可见杯状细胞,细支气管腔内可见粘液栓形成(Fig 1A, B,C).而对照组杯状细胞主要分布于支气管、叶支气管及小支气管,细支气管杯状细胞很少,终末细支气管未见杯状细胞,支气管腔内未见粘液栓形成(Fig1D).治疗组GCH较哮喘组减轻,杯状细胞主要分布于支气管、叶支气管及

16、小支气管,少数细支气管及终末细支气管也可见杯状细胞,6%的终末细支气管也可见杯状细胞,但杯状细胞数目很少,细支气管腔内未见粘液栓形成(Fig 1E, F).各组大鼠气道杯状细胞增生情况(各视野杯状细胞平均数),哮喘组(105±21)和治疗组(43±6),均显著高于对照组(22±6),(P<0.01);哮喘组和治疗组间差异也有显著性(P<0.01).A: Asthmatic rats, extensive GCH develop in the epithelium of bronchia×200; B: Asthmatic rats, mucous plug in small airway×400; C:Asthmatic rats: Moderate GCH develop in the epithelium of bronchiole×400; D: Normal rats, normal epithelium bronchia with little gobletcell×400; E: Treated rats, little GCH develop in the epithelium ofbronchia×400; F: Treated