COPD肺气虚证大鼠肺组织结构变化及爱罗咳喘宁方干预研究
发布时间：2019-06-28 16:42
【摘要】：目的研究爱罗咳喘宁方对慢性阻塞性肺疾病(Chronic Obstructive Pulmonary Disease,COPD)肺气虚证模型大鼠的肺功能、炎症、肺组织结构重塑的影响。明确该方药对COPD的治疗作用及其机制,为此方药的临床应用及新药开发提供实验依据。方法将50只SD大鼠随机分5组、每组10只动物,包括正常组、模型组、爱罗咳喘宁低剂量(7.76g·kg-1·d-1)组、中剂量(15.52g·kg-1·d-1)组和高剂量(31.04g·kg-1·d-1)组。以烟熏加气管滴注脂多糖(lipopolysaccharide,LPS)的方法制备COPD肺气虚证大鼠模型,以肺功能和病理改变作为评价COPD肺气虚证大鼠模型制备是否成功的依据。造模成功后灌胃给药；实验中观察并记录各组大鼠一般情况、检测肺功能[用力肺活量(forced vital capacity, FVC)、一秒用力呼气容积(forced expiratory volume in one second, FEV1)和二者的百分比(FEV1/FVC)]。酶联免疫吸附测定法(Enzyme-Linked ImmunoSorbent Assay ELISA)检测大鼠肺组织匀浆液和支气管肺泡灌洗液(bronchoalveolar lavage fluid, BALF)中的炎症介质白细胞介素-6(interleukin 6, IL-6)和可溶性细胞间黏附分子-1(soluble intercellular cell adhesion molecule-1 sICAM-1)的含量,光镜下观察大鼠肺组织结构。免疫组织化学(Immunohistochemistry,IHC)检测大鼠肺组织中与细胞外基质(extracellular matrix,ECM)沉积有关的转化生长因子-β l(transforming growth factor-β 1,TGF-βI)及其受体(TGF-β RI), Smad3, Smad4, Smad6和Smad7蛋白,基质金属蛋白酶-9(matrix metalloproteinases,MMP-9),金属蛋白酶组织抑制剂-1(tissueinhibitor of metalloproteinase,TIMP-1),以及Ⅰ型胶原和Ⅲ型胶原蛋白表达。实时荧光定量PCR (Quantitative Real-time PCR,RT-PCR)法检测肺组织中Smad3, Smad4, Smad6和Smad7 mRNA表达。结果与正常组相比,模型组大鼠FVC, FEV1和FEV1/FVC均显著降低(P0.01),BALF及肺组织匀浆液中IL6, sICAM-1含量均显著升高(均P0.01)；模型组肺组织Smad3 mRNA, Smad4 mRNA的表达量显著升高(P0.05), Smad6 mRNA, Smad7 mRNA表达均显著降低(P0.05),Smad3,Smad4蛋白的表达显著增强(P0.01), Smad6, Smad7蛋白的表达显著减弱(P0.01)；模型组肺组织中TGF-β1及TGF-βRI的表达显著增强(P0.01), MMP-9, TIMP-1的表达显著增强(P0.01),Ⅰ型、Ⅲ型胶原蛋白的表达显著增强(P0.01)。与模型组相比,爱罗咳喘宁低、中、高剂量组FVC, FEV1和FEV1/FVC均有不同程度升高(P0.0 1),肺组织匀浆液及BALF中IL6, sICAM-1含量均降低(P0.01),其中爱罗咳喘宁中剂量组降低明显(P0.01)；爱罗咳喘宁高、中、低剂量组Smad3 mRNA, Smad4 mRNA的表达量均下降(P0.05), Smad6mRNA, Smad7 mRNA的表达量显著升高(P0.05) Smad3, Smad4蛋白的表达有明显减弱(P0.01)Smad6, Smad7蛋白的表达有明显增强(P0.01)；爱罗咳喘宁低、中、高剂量组TGF-β1及TGF-β RI的蛋白表达有明显减弱(P0.01),MMP-9, TIMP-1的表达有明显减弱(P0.01),Ⅰ型、Ⅲ型胶原蛋白的表达明显减弱(P0.01)镜下观,模型组细支气管腔内有痰栓,黏膜上皮变性、坏死,部分脱落。管壁周围及肺间质增厚、大量炎细胞浸润；细支气管周围肺泡间质明显增厚,肺泡内见渗出物。与模型组相比较,爱罗咳喘宁中剂组细支气管黏膜有不同程度的修复,黏膜上皮排列较为规整,炎细胞基本消失；细支气管周围肺泡间质变薄,局部有淋巴细胞浸润；爱罗咳喘宁高剂量组和低剂量组恢复均差于中剂量组。结论1爱罗咳喘宁方能明显改善COPD肺气虚证大鼠肺功能。2爱罗咳喘宁方可有效治疗COPD肺气虚证大鼠炎症反应、保护肺组织,缓解肺气虚咳、痰等症状。其机制可能与降低细胞因子IL-6, sICAM-I的分泌,抑制炎症反应,阻止病变进程有关。3爱罗咳喘宁方能有效抑制细支气管结构重塑,改善肺通气,具有止咳平喘作用。其机制可能是通过降低Smad3的基因表达,提高Smad6, Smad7的基因表达,协调Smad4的基因表达,抑制TGF-β1及其受体的表达,进而抑制MMP-9,并协调性抑制TIMP-1的蛋白表达,缓解细支气管及肺组织结构重塑。
[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，
本文编号：2507454
[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，
本文编号：2507454
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