宣肺健脾中藥對(duì)哮喘大鼠支氣管-肺組織病理學(xué)和轉(zhuǎn)化生長(zhǎng)因子-β

1、宣肺健脾中藥對(duì)哮喘大鼠支氣管-肺組織病理學(xué)和轉(zhuǎn)化生長(zhǎng)因子-1的影響         11-04-29 15:27:00     編輯：studa20              作者：馬佐英,何山,袁衛(wèi)玲,郭茂娟,王學(xué)嶺【摘要】  目的 觀察宣肺健脾中藥哮逐平對(duì)支氣管哮喘大鼠的治療作用,探討其可能的作用機(jī)制。方法 SPF級(jí)雄性Wistar大

2、鼠32只,隨機(jī)分為正常對(duì)照組(A組)、哮喘模型組(B組)、地塞米松組(C組)、哮逐平組(D組),每組8只。除A組外,其余組動(dòng)物建立哮喘模型。HE染色,觀察支氣管-肺組織病理學(xué)改變,以醫(yī)學(xué)圖像分析軟件測(cè)量支氣管基底膜周徑(Pbm)、支氣管壁厚度(Wat)和平滑肌厚度(Wam)。免疫組化PV二步法染色,應(yīng)用上述圖像分析軟件檢測(cè)支氣管-肺組織轉(zhuǎn)化生長(zhǎng)因子-1(TGF-1)的積分光密度(IOD)。結(jié)果 與A組相比,B組Wat、Wam和支氣管-肺組織TGF-1含量明顯增加,差異有統(tǒng)計(jì)學(xué)意義(P0.01)。經(jīng)藥物干預(yù)后,C、D組Wat、Wam減少及支氣管-肺組織TGF-1含量顯著降低(P0.05)。D組W

3、at、Wam和TGF-1減少尤為明顯,與B組相比,差異有統(tǒng)計(jì)學(xué)意義(P0.05),其中Wat、Wam與A組相比,差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。與C組相比,D組TGF-1含量明顯下降,差異有統(tǒng)計(jì)學(xué)意義(P0.01)。結(jié)論 宣肺健脾中藥可能通過(guò)抑制哮喘大鼠支氣管-肺組織TGF-1的過(guò)度表達(dá)而影響平滑肌增殖、減輕氣道壁增厚,干預(yù)氣道重塑。 【關(guān)鍵詞】  宣肺健脾中藥；哮逐平；哮喘；氣道重塑；轉(zhuǎn)化生長(zhǎng)因子-1；大鼠Abstract：Objective To observe the effect of bronchial asthma in rats treated by Chuanzhup

4、ing, a prescription which has the function of dispersing lung Qi and invigorating spleen Qi, and explore its possible mechanism. Method Thirty-two male Wistar rats of SPF level were divided into four groups randomly：control group (A), asthma group (B), Dexamethasone group (C) and Xiaozhuping group (

5、D). Except group A, the others were established animal model of asthma. After drawing materials, hematoxylin and eosin (HE) staining were performed. The pathological changes in bronchopulmonary tissue of rats were detected. The basementmembrane perimeter (Pbm), the airwaywall area (Wat) and the smoo

6、th musclewall area (Wam) were measured and calculated by using image analysis system. The concentration of transforming growth factor-1 (TGF-1) in bronchopulmonary tissue was observed by immunohistochemical method and the integral optical density (IOD values) of it was measured. Results In bronchopu

7、lmonary tissue of group B, Wat, Wam and the concentration of TGF-1 increased, and were significantly different from that in group A (P0.01). In group C and D, Wat, Wam and the concentration of TGF-1 decreased, and the concentration of TGF-1 was significantly different from that in group B (P0.05). I

8、n group D, Wat and Wam were significantly different from that in group B (P0.05), and were not significantly different from that in group A (P0.05). In group D, the concentration of TGF-1 decreased and was significantly different from that in group C (P0.01). Conclusion In treatment of asthma, Xiaoz

9、huping with the function of dispersing lung Qi and invigorating spleen Qi may affect airway remodeling by inhibiting the expression of TGF-1, suppressing smooth muscle proliferation and decreasing the thickness of bronchial wall.Key words：herbs of dispersing lung Qi and invigorating spleen Qi；Xiaozh

10、uping；asthma；airway remodeling；TGF-1；rat近年研究發(fā)現(xiàn),幾乎所有的支氣管哮喘患者均有不同程度的氣道壁結(jié)構(gòu)改變,除炎性細(xì)胞浸潤(rùn)、痙攣、導(dǎo)致氣道狹窄外,還表現(xiàn)為氣道壁增厚,氣道平滑肌增生、肥大,基底膜增厚、玻璃樣變等,此種改變稱(chēng)為氣道重塑1。已有較多的資料顯示,轉(zhuǎn)化生長(zhǎng)因子-1(TGF-1)是哮喘氣道重構(gòu)的主要調(diào)控因子,氣道管壁增厚、平滑肌增生以及膠原沉積等氣道重構(gòu)的特征與TGF-1的表達(dá)呈正相關(guān)2-3。本實(shí)驗(yàn)以卵蛋白(OVA)、氫氧化鋁干粉和滅活百日咳桿菌疫苗致敏大鼠,造成哮喘大鼠模型,將具有宣肺健脾、利氣祛痰之功效的哮逐平用于哮喘大鼠的治療。通過(guò)觀測(cè)支氣管

11、-肺組織病理學(xué)的改變和TGF-1的表達(dá),探討宣肺健脾中藥對(duì)哮喘氣道重塑的影響及其可能的作用機(jī)制。1  材料與方法1.1  動(dòng)物SPF級(jí)雄性Wistar大鼠32只,體重150180 g,天津市山川紅實(shí)驗(yàn)動(dòng)物科技有限公司提供,許可證號(hào)：SCXK(津)2009- 001。1.2  藥物、主要試劑及儀器哮逐平由天津中醫(yī)藥大學(xué)?？滇t(yī)院藥房提供,藥物基本組成：麻黃、杏仁、桔梗、川貝母、枇杷葉、橘紅、茯苓、半夏、厚樸、枳殼、神曲等,水煎,過(guò)濾,濃縮至每毫升含原藥材2.25 g,密封于無(wú)菌量瓶中,4 儲(chǔ)存?zhèn)溆?。醋酸地塞米松片由天津力生制藥股份有限公司生產(chǎn)(批號(hào)0708015),研

12、細(xì)末,用蒸餾水配制成濃度為0.187 5 mg/mL混懸液。OVA為美國(guó)Sigma公司產(chǎn)品(批號(hào)A5253)；滅活百日咳桿菌疫苗由北京天壇生物制品股份有限公司生產(chǎn)(批號(hào)200901)；氫氧化鋁由天津市化學(xué)試劑三廠生產(chǎn)(批號(hào)070621)。兔抗鼠TGF-1多克隆抗體(產(chǎn)品編號(hào)：sc-146)購(gòu)自美國(guó)Santa Cruz Biotechnology公司。二步法免疫組化檢測(cè)試劑和濃縮型DAB試劑盒均購(gòu)自北京中杉金橋生物技術(shù)有限公司。亞都超聲波醫(yī)用霧化器(型號(hào)：YC-Y800B)為北京亞都科技股份有限公司產(chǎn)品。Lecia DM3000自動(dòng)化正置顯微鏡圖像采集系統(tǒng)由德國(guó)Leica Microsystem

13、s公司提供。1.3  動(dòng)物分組及造模正常飼養(yǎng)1周后,將大鼠隨機(jī)分為正常對(duì)照組(A組)、哮喘模型組(B組)、地塞米松組(C組)、哮逐平組(D組),每組8只。除A組外,其余組哮喘動(dòng)物模型復(fù)制參照文獻(xiàn)4-6方法進(jìn)行。每只大鼠于實(shí)驗(yàn)第1日和第8日分別腹腔注射抗原液1 mL (含OVA 100 mg、滅活百日咳桿菌疫苗5×109個(gè)和氫氧化鋁干粉100 mg),第15日將致敏大鼠置于50 cm×40 cm×30 cm密閉玻璃容器內(nèi),給予1% OVA生理鹽水懸液超聲霧化吸入加以激發(fā),中等霧量,每日1次,每次約20 min,以大鼠出現(xiàn)煩躁不安、呼吸急促、噴嚏或嗆咳、輕度

14、紫紺、腹肌抽搐、反應(yīng)遲鈍等表現(xiàn)判定造模成功。A組以等量生理鹽水進(jìn)行致敏和激發(fā),方法同上。造模成功當(dāng)日開(kāi)始給藥,C、D組分別給予地塞米松0.75 mg/(kg·d)和哮逐平22.5 g/(kg·d)灌胃,給藥劑量均為10 mL/kg,A、B組給予等量生理鹽水灌胃,均每日1次,共計(jì)14 d。其間每日給藥后30 min各誘發(fā)哮喘1次。1.4  標(biāo)本采集末次霧化激發(fā)24 h后,用3%戊巴比妥鈉30 mg/kg腹腔注射麻醉大鼠。開(kāi)胸,取右肺中葉組織,4%多聚甲醛固定,常規(guī)石蠟包埋制片,切片厚5 m。1.5  指標(biāo)檢測(cè)1.5.1  支氣管-肺組織病理學(xué)觀察

15、  切片行常規(guī)蘇木精-伊紅(HE)染色,觀察病理學(xué)改變。支氣管壁厚度(Wat)和平滑肌厚度(Wam)的檢測(cè)7-8：200倍光學(xué)顯微鏡下挑選3支有完整橫斷面的中小支氣管,采用圖像采集系統(tǒng)獲取圖像,再以專(zhuān)業(yè)圖像分析軟件(Image-Pro Plus 6.0)測(cè)量基底膜周徑(Pbm)、支氣管總面積(Wat1)、管腔面積(Wat2)、平滑肌外緣內(nèi)氣管面積(Wam1)、平滑肌內(nèi)緣內(nèi)氣管面積(Wam2),并用Pbm標(biāo)準(zhǔn)化,即分別以(Wat1Wat2)/Pbm和(Wam1Wam2)/Pbm來(lái)表示W(wǎng)at(m2/m)和Wam(m2/m)。1.5.2  支氣管-肺組織轉(zhuǎn)化生長(zhǎng)因子-1含量檢測(cè)&

16、#160; 采用免疫組化PV二步法染色：石蠟切片常規(guī)脫蠟、水化,3%H2O2孵育10 min以滅活內(nèi)源性過(guò)氧化物酶,磷酸鹽緩沖液(PBS)沖洗3次各2 min,滴加一抗,4 過(guò)夜,PBS沖洗3次各2 min,滴加二抗,37 孵育30 min,PBS沖洗3次各2 min,二氨基聯(lián)苯胺(DAB)顯色,室溫下顯色520 min,蒸餾水沖洗,蘇木精復(fù)染5 min,蒸餾水沖洗,1%鹽酸酒精分色,自來(lái)水沖洗,載玻片蒸干,二甲苯透明,中性樹(shù)膠封片。顯微鏡下觀察,組織切片中染成棕黃色者為陽(yáng)性。每張切片于200倍光鏡下采集5個(gè)具有代表性的互不重疊視野,應(yīng)用上述圖像分析軟件測(cè)量每個(gè)視野的積分光密度(IOD),以I

17、OD數(shù)值大小反映TGF-1表達(dá)的多少。1.6  統(tǒng)計(jì)學(xué)方法所有數(shù)據(jù)以x±s表示,采用SPSS 11.5統(tǒng)計(jì)軟件進(jìn)行處理,組間比較用方差分析,以0.05為檢驗(yàn)水準(zhǔn),P0.05為有統(tǒng)計(jì)學(xué)意義。2  結(jié)果2.1  各組大鼠支氣管-肺組織病理學(xué)改變情況A組支氣管、肺組織無(wú)炎性細(xì)胞浸潤(rùn),支氣管壁完整,平滑肌厚度正常,細(xì)胞排列規(guī)整,管腔內(nèi)無(wú)脫落上皮細(xì)胞。B組支氣管壁及其周?chē)谓M織中有大量以嗜酸性粒細(xì)胞、淋巴細(xì)胞為主的炎性細(xì)胞浸潤(rùn),黏液腺增生,黏膜皺襞增多,支氣管腔內(nèi)可見(jiàn)黏液栓和炎性細(xì)胞,周?chē)M織水腫。支氣管平滑肌層明顯增厚,排列紊亂,管腔狹窄。C組支氣管周?chē)人嵝粤＜?xì)胞及其他炎性細(xì)胞浸潤(rùn)減少,氣管壁和平滑肌增厚明顯減輕,黏膜結(jié)構(gòu)較