鹽酸氨溴索對(duì)慢性哮喘大鼠氣道炎癥及杯狀細(xì)胞增生的影響探析

1、鹽酸氨溴索對(duì)慢性哮喘大鼠氣道炎癥及杯狀細(xì)胞增生的影響探析    【摘要】目的:觀察藥物鹽酸氨溴索對(duì)慢性哮喘大鼠氣道炎癥及杯狀細(xì)胞增生(GCH)的影響,探討在哮喘防治中的作用.方法:用卵白蛋白致敏、激發(fā)建立大鼠哮喘模型,54只Wistar大鼠隨機(jī)分為3組:正常對(duì)照組、哮喘組、鹽酸氨溴索治療組(簡(jiǎn)稱治療組),每組18只.激發(fā)哮喘后測(cè)定氣道內(nèi)壓的變化;支氣管肺泡灌洗液(BALF)內(nèi)細(xì)胞計(jì)數(shù)、分類計(jì)數(shù),HE染色及AB-PAS染色觀察肺組織病理改變及杯狀細(xì)胞增生狀況.結(jié)果:治療組氣道內(nèi)壓增加的百分?jǐn)?shù)明顯低于哮喘組(P<0.01);治療組BALF細(xì)胞總數(shù)及炎癥

2、細(xì)胞數(shù)明顯低于哮喘組(P道,未見粘液潴留.結(jié)論:鹽酸氨溴索可抑制哮喘大鼠氣道GCH,改善哮喘氣道炎癥,對(duì)哮喘的防治有一定作用.【關(guān)鍵詞】哮喘 氨溴索 杯狀細(xì)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 氣道上皮杯狀細(xì)胞增生(goblet cell hyperplasia,GCH)是哮喘的病理特征之一,是哮喘時(shí)氣道上皮最明確的改變. GCH可引起氣道上皮增厚,糖蛋白分泌增加,導(dǎo)致氣道管腔狹窄,氣道阻力增加.鹽酸氨溴索(Ambroxol,商品名mucosulvan),是一種常用的祛痰劑,近年來其對(duì)呼吸系統(tǒng)的保護(hù)作用倍受關(guān)注,它可以抑制粘液腺和杯狀細(xì)胞中酸

9、性糖蛋白合成.我們應(yīng)用鹽酸氨溴索對(duì)慢性哮喘大鼠進(jìn)行干預(yù),觀察其長(zhǎng)期作用對(duì)哮喘大鼠GCH及氣道炎癥的影響.1材料和        方法 1.1材料雄性Wistar大鼠54只,體質(zhì)量(250±50) g,由第二軍醫(yī)大學(xué)實(shí)驗(yàn)動(dòng)物中心提供.卵白蛋白為美國(guó)Sigma公司產(chǎn)品.鹽酸氨溴索片由德國(guó)勃林格英格翰公司提供.阿爾辛藍(lán)(Alcian blue 8GX)為美國(guó)Amersco公司產(chǎn)品. EJD-IV型生物信號(hào)處理系統(tǒng)系第二軍醫(yī)大學(xué)生理教研室研制.定容型動(dòng)物呼吸機(jī)DH-1408型為浙江大學(xué)醫(yī)學(xué)院醫(yī)學(xué)儀器試驗(yàn)

10、廠生產(chǎn).1.2方法1.2.1動(dòng)物分組及模型制備用卵白蛋白致敏、激發(fā)建立大鼠哮喘模型.參照文獻(xiàn)1稍加改變復(fù)制哮喘大鼠模型:哮喘組,予免疫原液1 mL(含卵蛋白100 mg、氫氧化鋁100 mg)胸前皮下注射致敏,同時(shí)ip氣管炎疫苗(含5×1012個(gè)滅活菌),第15日起予1g·L-1卵蛋白超聲霧化吸入,每日20 min,連續(xù)7 d.鹽酸氨溴索治療組致敏前1 wk即開始ig給予鹽酸氨溴索片10 mg·kg-1·d-1,2次·d-1ig喂食,與哮喘組同時(shí)致敏、激發(fā),處理亦相同,但實(shí)驗(yàn)中一直服用鹽酸氨溴索,共連續(xù)服用4 wk.正常對(duì)照組予生理鹽水胸前sc

11、及ip,第15日起予生理鹽水超聲霧化吸入,每日20 min,連續(xù)7 d.1.2.2氣道內(nèi)壓測(cè)定末次激發(fā)前各組大鼠隨機(jī)取5只檢測(cè)激發(fā)前后氣道內(nèi)壓的變化,戊巴比妥鈉ip麻醉,氣管插管,Y形管分別連接定容呼吸機(jī)、壓力傳感器及生物信號(hào)處理系統(tǒng),先記錄未激發(fā)時(shí)氣道內(nèi)壓,待壓力平穩(wěn)后各組分別予10 g·L-1生理鹽水及卵蛋白氧氣驅(qū)動(dòng)霧化激發(fā)20 min,激發(fā)后持續(xù)記錄氣道內(nèi)壓60 min,觀察氣道內(nèi)壓變化,計(jì)算記錄過程中氣道內(nèi)壓的最大增幅作為氣道內(nèi)壓變化的指標(biāo).計(jì)算公式:氣道內(nèi)壓增加的百分?jǐn)?shù)=(激發(fā)后氣道內(nèi)壓-激發(fā)前氣道內(nèi)壓)/激發(fā)前氣道內(nèi)壓×100%.1.2.3病理學(xué)檢查每組隨機(jī)取5

12、只大鼠于末次激發(fā)后行病理檢查,每葉肺取樣進(jìn)行脫水,石蠟包埋連續(xù)切片,片厚5m,行HE染色及阿爾辛藍(lán)-糖原染色(AB-PAS染色). AB-PAS染色:切片入10 g·L-1阿爾辛藍(lán)(pH 2.5)染色30 min, 10 mL·L-1過碘酸氧化10 min,再入Schiffs液暗處加蓋30 min左右,作蘇木素核襯染,中性粘液呈紅色,酸性粘液物質(zhì)呈藍(lán)色,混合性粘液呈紫紅色,氣道上皮細(xì)胞胞質(zhì)被染成藍(lán)色或紫紅色的為杯狀細(xì)胞.光鏡下觀察,按Ceng2的方法每只大鼠觀察5張不同部位的切片,每張切片觀察12個(gè)高倍視野的杯狀細(xì)胞總數(shù),其中葉支氣管、小支氣管、細(xì)支氣管或終末細(xì)支氣管各4個(gè)

13、視野,計(jì)算其平均值.1.2.4支氣管肺泡灌洗液計(jì)數(shù)及分類其余大鼠末次激發(fā)后行支氣管肺泡灌洗,收集支氣管肺泡灌洗液(BALF)并記錄回收量.用血球計(jì)數(shù)板行BALF細(xì)胞計(jì)數(shù)并計(jì)算細(xì)胞總數(shù),將BALF 4離心(2500 r·min-1, 8 min),取沉渣適量涂片,采用瑞氏染色法進(jìn)行染色,鏡檢300個(gè)細(xì)胞按照形態(tài)學(xué)標(biāo)準(zhǔn)進(jìn)行細(xì)胞分類計(jì)數(shù).統(tǒng)計(jì)學(xué)處理:應(yīng)用統(tǒng)計(jì)軟件SPSS進(jìn)行數(shù)據(jù)分析,數(shù)據(jù)用x±s表示,采用完全隨機(jī)設(shè)計(jì)資料的方差分析和LSD-t檢驗(yàn)檢測(cè)樣本均數(shù)差異的顯著性.2結(jié)果 2.1氣道內(nèi)壓的變化各組大鼠激發(fā)前氣道內(nèi)壓無顯著差異(P>0.05).正常對(duì)照組超聲霧

14、化生理鹽水后氣道內(nèi)壓平均最大增幅為(18.4±4.7)%, 10min左右恢復(fù)至正常;哮喘組大鼠卵蛋白激發(fā)后氣道內(nèi)壓迅速增加,在5 min內(nèi)達(dá)到高峰,平均最大增幅為(119.2±30.1)%, 30 min左右逐漸恢復(fù)至正常.治療組氣道內(nèi)壓平均最大增幅為(58.8±19.0)%,明顯低于哮喘組.氣道內(nèi)壓最大增幅在各組之間均有顯著差異(P<0.01).2.2肺組織HE染色所見對(duì)照組支氣管、肺組織結(jié)構(gòu)正常,無支氣管收縮征象,亦未見炎癥細(xì)胞浸潤(rùn).哮喘組支氣管粘膜上皮增厚,管腔明顯狹窄,可見明顯支氣管痙攣征象,管壁及其周圍大量炎細(xì)胞浸潤(rùn),除中性粒細(xì)胞及淋巴細(xì)胞外,可

15、見大量嗜酸性粒細(xì)胞.治療組支氣管粘膜上皮無明顯增厚,管腔狹窄的程度及發(fā)生頻率均較哮喘組明顯減輕,管壁及其周圍少量炎細(xì)胞浸潤(rùn),支氣管腔內(nèi)無粘液栓形成.2.3AB-PAS染色所見哮喘組杯狀細(xì)胞增生明顯,從支氣管到終末細(xì)支氣管,每一級(jí)氣道均可見杯狀細(xì)胞,支氣管、葉支氣管及小支氣管杯狀細(xì)胞大量增生.隨機(jī)觀察50個(gè)終末細(xì)支氣管,38%也可見杯狀細(xì)胞,細(xì)支氣管腔內(nèi)可見粘液栓形成(Fig 1A, B,C).而對(duì)照組杯狀細(xì)胞主要分布于支氣管、葉支氣管及小支氣管,細(xì)支氣管杯狀細(xì)胞很少,終末細(xì)支氣管未見杯狀細(xì)胞,支氣管腔內(nèi)未見粘液栓形成(Fig1D).治療組GCH較哮喘組減輕,杯狀細(xì)胞主要分布于支氣管、葉支氣管及

16、小支氣管,少數(shù)細(xì)支氣管及終末細(xì)支氣管也可見杯狀細(xì)胞,6%的終末細(xì)支氣管也可見杯狀細(xì)胞,但杯狀細(xì)胞數(shù)目很少,細(xì)支氣管腔內(nèi)未見粘液栓形成(Fig 1E, F).各組大鼠氣道杯狀細(xì)胞增生情況(各視野杯狀細(xì)胞平均數(shù)),哮喘組(105±21)和治療組(43±6),均顯著高于對(duì)照組(22±6),(P<0.01);哮喘組和治療組間差異也有顯著性(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