【摘要】：目的研究愛羅咳喘寧方對慢性阻塞性肺疾病(Chronic Obstructive Pulmonary Disease,COPD)肺氣虛證模型大鼠的肺功能、炎癥、肺組織結(jié)構重塑的影響。明確該方藥對COPD的治療作用及其機制,為此方藥的臨床應用及新藥開發(fā)提供實驗依據(jù)。方法將50只SD大鼠隨機分5組、每組10只動物,包括正常組、模型組、愛羅咳喘寧低劑量(7.76g·kg-1·d-1)組、中劑量(15.52g·kg-1·d-1)組和高劑量(31.04g·kg-1·d-1)組。以煙熏加氣管滴注脂多糖(lipopolysaccharide,LPS)的方法制備COPD肺氣虛證大鼠模型,以肺功能和病理改變作為評價COPD肺氣虛證大鼠模型制備是否成功的依據(jù)。造模成功后灌胃給藥；實驗中觀察并記錄各組大鼠一般情況、檢測肺功能[用力肺活量(forced vital capacity, FVC)、一秒用力呼氣容積(forced expiratory volume in one second, FEV1)和二者的百分比(FEV1/FVC)]。酶聯(lián)免疫吸附測定法(Enzyme-Linked ImmunoSorbent Assay ELISA)檢測大鼠肺組織勻漿液和支氣管肺泡灌洗液(bronchoalveolar lavage fluid, BALF)中的炎癥介質(zhì)白細胞介素-6(interleukin 6, IL-6)和可溶性細胞間黏附分子-1(soluble intercellular cell adhesion molecule-1 sICAM-1)的含量,光鏡下觀察大鼠肺組織結(jié)構。免疫組織化學(Immunohistochemistry,IHC)檢測大鼠肺組織中與細胞外基質(zhì)(extracellular matrix,ECM)沉積有關的轉(zhuǎn)化生長因子-β l(transforming growth factor-β 1,TGF-βI)及其受體(TGF-β RI), Smad3, Smad4, Smad6和Smad7蛋白,基質(zhì)金屬蛋白酶-9(matrix metalloproteinases,MMP-9),金屬蛋白酶組織抑制劑-1(tissueinhibitor of metalloproteinase,TIMP-1),以及Ⅰ型膠原和Ⅲ型膠原蛋白表達。實時熒光定量PCR (Quantitative Real-time PCR,RT-PCR)法檢測肺組織中Smad3, Smad4, Smad6和Smad7 mRNA表達。結(jié)果與正常組相比,模型組大鼠FVC, FEV1和FEV1/FVC均顯著降低(P0.01),BALF及肺組織勻漿液中IL6, sICAM-1含量均顯著升高(均P0.01)；模型組肺組織Smad3 mRNA, Smad4 mRNA的表達量顯著升高(P0.05), Smad6 mRNA, Smad7 mRNA表達均顯著降低(P0.05),Smad3,Smad4蛋白的表達顯著增強(P0.01), Smad6, Smad7蛋白的表達顯著減弱(P0.01)；模型組肺組織中TGF-β1及TGF-βRI的表達顯著增強(P0.01), MMP-9, TIMP-1的表達顯著增強(P0.01),Ⅰ型、Ⅲ型膠原蛋白的表達顯著增強(P0.01)。與模型組相比,愛羅咳喘寧低、中、高劑量組FVC, FEV1和FEV1/FVC均有不同程度升高(P0.0 1),肺組織勻漿液及BALF中IL6, sICAM-1含量均降低(P0.01),其中愛羅咳喘寧中劑量組降低明顯(P0.01)；愛羅咳喘寧高、中、低劑量組Smad3 mRNA, Smad4 mRNA的表達量均下降(P0.05), Smad6mRNA, Smad7 mRNA的表達量顯著升高(P0.05) Smad3, Smad4蛋白的表達有明顯減弱(P0.01)Smad6, Smad7蛋白的表達有明顯增強(P0.01)；愛羅咳喘寧低、中、高劑量組TGF-β1及TGF-β RI的蛋白表達有明顯減弱(P0.01),MMP-9, TIMP-1的表達有明顯減弱(P0.01),Ⅰ型、Ⅲ型膠原蛋白的表達明顯減弱(P0.01)鏡下觀,模型組細支氣管腔內(nèi)有痰栓,黏膜上皮變性、壞死,部分脫落。管壁周圍及肺間質(zhì)增厚、大量炎細胞浸潤；細支氣管周圍肺泡間質(zhì)明顯增厚,肺泡內(nèi)見滲出物。與模型組相比較,愛羅咳喘寧中劑組細支氣管黏膜有不同程度的修復,黏膜上皮排列較為規(guī)整,炎細胞基本消失；細支氣管周圍肺泡間質(zhì)變薄,局部有淋巴細胞浸潤；愛羅咳喘寧高劑量組和低劑量組恢復均差于中劑量組。結(jié)論1愛羅咳喘寧方能明顯改善COPD肺氣虛證大鼠肺功能。2愛羅咳喘寧方可有效治療COPD肺氣虛證大鼠炎癥反應、保護肺組織,緩解肺氣虛咳、痰等癥狀。其機制可能與降低細胞因子IL-6, sICAM-I的分泌,抑制炎癥反應,阻止病變進程有關。3愛羅咳喘寧方能有效抑制細支氣管結(jié)構重塑,改善肺通氣,具有止咳平喘作用。其機制可能是通過降低Smad3的基因表達,提高Smad6, Smad7的基因表達,協(xié)調(diào)Smad4的基因表達,抑制TGF-β1及其受體的表達,進而抑制MMP-9,并協(xié)調(diào)性抑制TIMP-1的蛋白表達,緩解細支氣管及肺組織結(jié)構重塑。
[Abstract]:Objective To study the effects of the lung function, inflammation and the remodeling of lung tissue in the model of chronic obstructive pulmonary disease (COPD). The treatment effect and mechanism of the formula on COPD are clear, and the experimental basis for the clinical application of the prescription and the development of new drugs is provided. Methods Fifty SD rats were randomly divided into 5 groups, including normal group, model group, low dose (7.76 g 路 kg-1 路 d-1) group, middle dose (15.52 g 路 kg-1 路 d-1) group and high dose (31.04 g 路 kg-1 路 d-1) group. The model of the lung-qi deficiency of COPD was prepared by using the method of lipopolysaccharides (LPS) and the function of the lung and the pathological changes as the basis for evaluating the success of the model of the model of the lung-qi deficiency of COPD. The rats were given intragastric administration after the establishment of the model; the general conditions of each group were observed and recorded in the experiment, and the lung function[forced vital capacity, FVC), one second forced expiratory volume in one second, and the percentage of both (FEV1/ FVC)] were detected. Enzyme-linked immunosorbent assay (ELISA) was used to detect the content of interleukin-6 (IL-6) and soluble intercellular adhesion molecule-1 (ICAM-1) in the rat lung tissue homogenate and broncho-alveolar lavage fluid (BALF). The structure of lung tissue was observed under light microscope. Immunohistochemistry (IHC) was used to detect the transformation growth factor-1, TGF-(I) and its receptor (TGF-TRI), Smad3, Smad4, Smad6 and Smad7 protein, matrix metalloproteinase-9 (matrix metalloproteinases-9) related to the deposition of extracellular matrix (ECM) in rat lung tissue. MMP-9, the inhibitor of metalloproteinase-1 (TIMP-1), and the expression of type I collagen and type 鈪，

本文編號：2507454