宣肺健脾中藥對哮喘大鼠支氣管-肺組織病理學和轉化生長因子-β

1、宣肺健脾中藥對哮喘大鼠支氣管-肺組織病理學和轉化生長因子-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,王學嶺【摘要】  目的 觀察宣肺健脾中藥哮逐平對支氣管哮喘大鼠的治療作用,探討其可能的作用機制。方法 SPF級雄性Wistar大

2、鼠32只,隨機分為正常對照組(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,其余組動物建立哮喘模型。HE染色,觀察支氣管-肺組織病理學改變,以醫(yī)學圖像分析軟件測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,應用上述圖像分析軟件檢測支氣管-肺組織轉化生長因子-1(TGF-1)的積分光密度(IOD)。結果 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)計學意義(P0.01)。經藥物干預后,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)計學意義(P0.05),其中Wat、Wam與A組相比,差異無統(tǒng)計學意義(P0.05)。與C組相比,D組TGF-1含量明顯下降,差異有統(tǒng)計學意義(P0.01)。結論 宣肺健脾中藥可能通過抑制哮喘大鼠支氣管-肺組織TGF-1的過度表達而影響平滑肌增殖、減輕氣道壁增厚,干預氣道重塑。 【關鍵詞】  宣肺健脾中藥；哮逐平；哮喘；氣道重塑；轉化生長因子-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構改變,除炎性細胞浸潤、痙攣、導致氣道狹窄外,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,此種改變稱為氣道重塑1。已有較多的資料顯示,轉化生長因子-1(TGF-1)是哮喘氣道重構的主要調控因子,氣道管壁增厚、平滑肌增生以及膠原沉積等氣道重構的特征與TGF-1的表達呈正相關2-3。本實驗以卵蛋白(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。通過觀測支氣管

11、-肺組織病理學的改變和TGF-1的表達,探討宣肺健脾中藥對哮喘氣道重塑的影響及其可能的作用機制。1  材料與方法1.1  動物SPF級雄性Wistar大鼠32只,體重150180 g,天津市山川紅實驗動物科技有限公司提供,許可證號：SCXK(津)2009- 001。1.2  藥物、主要試劑及儀器哮逐平由天津中醫(yī)藥大學保康醫(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,過濾,濃縮至每毫升含原藥材2.25 g,密封于無菌量瓶中,4 儲存?zhèn)溆?。醋酸地塞米松片由天津力生制藥股份有限公司生產(批號0708015),研

12、細末,用蒸餾水配制成濃度為0.187 5 mg/mL混懸液。OVA為美國Sigma公司產品(批號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(批號200901)；氫氧化鋁由天津市化學試劑三廠生產(批號070621)。兔抗鼠TGF-1多克隆抗體(產品編號：sc-146)購自美國Santa Cruz Biotechnology公司。二步法免疫組化檢測試劑和濃縮型DAB試劑盒均購自北京中杉金橋生物技術有限公司。亞都超聲波醫(yī)用霧化器(型號：YC-Y800B)為北京亞都科技股份有限公司產品。Lecia DM3000自動化正置顯微鏡圖像采集系統(tǒng)由德國Leica Microsystem

13、s公司提供。1.3  動物分組及造模正常飼養(yǎng)1周后,將大鼠隨機分為正常對照組(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,其余組哮喘動物模型復制參照文獻4-6方法進行。每只大鼠于實驗第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,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、反應遲鈍等表現(xiàn)判定造模成功。A組以等量生理鹽水進行致敏和激發(fā),方法同上。造模成功當日開始給藥,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14 d。其間每日給藥后30 min各誘發(fā)哮喘1次。1.4  標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  指標檢測1.5.1  支氣管-肺組織病理學觀察

15、  切片行常規(guī)蘇木精-伊紅(HE)染色,觀察病理學改變。支氣管壁厚度(Wat)和平滑肌厚度(Wam)的檢測7-8：200倍光學顯微鏡下挑選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,再以專業(yè)圖像分析軟件(Image-Pro Plus 6.0)測量基底膜周徑(Pbm)、支氣管總面積(Wat1)、管腔面積(Wat2)、平滑肌外緣內氣管面積(Wam1)、平滑肌內緣內氣管面積(Wam2),并用Pbm標準化,即分別以(Wat1Wat2)/Pbm和(Wam1Wam2)/Pbm來表示Wat(m2/m)和Wam(m2/m)。1.5.2  支氣管-肺組織轉化生長因子-1含量檢測&

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以滅活內源性過氧化物酶,磷酸鹽緩沖液(PBS)沖洗3次各2 min,滴加一抗,4 過夜,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,蘇木精復染5 min,蒸餾水沖洗,1%鹽酸酒精分色,自來水沖洗,載玻片蒸干,二甲苯透明,中性樹膠封片。顯微鏡下觀察,組織切片中染成棕黃色者為陽性。每張切片于200倍光鏡下采集5個具有代表性的互不重疊視野,應用上述圖像分析軟件測量每個視野的積分光密度(IOD),以I

17、OD數(shù)值大小反映TGF-1表達的多少。1.6  統(tǒng)計學方法所有數(shù)據(jù)以x±s表示,采用SPSS 11.5統(tǒng)計軟件進行處理,組間比較用方差分析,以0.05為檢驗水準,P0.05為有統(tǒng)計學意義。2  結果2.1  各組大鼠支氣管-肺組織病理學改變情況A組支氣管、肺組織無炎性細胞浸潤,支氣管壁完整,平滑肌厚度正常,細胞排列規(guī)整,管腔內無脫落上皮細胞。B組支氣管壁及其周圍肺組織中有大量以嗜酸性粒細胞、淋巴細胞為主的炎性細胞浸潤,黏液腺增生,黏膜皺襞增多,支氣管腔內可見黏液栓和炎性細胞,周圍組織水腫。支氣管平滑肌層明顯增厚,排列紊亂,管腔狹窄。C組支氣管周圍嗜酸性粒細胞及其他炎性細胞浸潤減少,氣管壁和平滑肌增厚明顯減輕,黏膜結構較