一种慢性阻塞性肺疾病合并气管支气管软化症模型制备方法及调补肺肾方干预效果评价

目的建立一种慢性阻塞性肺疾病(COPD)合并气管支气管软化症模型并评价调补肺肾方保护气管软骨细胞退变的机制。方法实验分为空白组、模型组、模型+调补肺肾方组。采用流式细胞分析方法确定中药调补肺肾方和香烟烟雾提取物(CSE)的最适浓度,采用最适浓度CSE处理气管软骨细胞制作COPD细胞模型,然后加入白细胞介素-1β(IL-1β)建立软骨细胞退变模型。造模成功后,模型+调补肺肾方组中加入调补肺肾方干预48 h,空白组、模型组不予特殊处理。甲苯胺蓝染色和免疫组化染色观察各组软骨细胞退变情况,Western blot法检测各组软骨细胞中p-p38、caveolin-1表达情况,RT-PCR法检测各组软骨细胞中基质金属蛋白酶3(MMP-3)、caveolin-1 mRNA表达情况。结果调补肺肾方最适干预浓度为1%,CSE最适建模浓度为2.5%。模型组甲苯胺蓝染色显示软骨细胞染色明显变浅,细胞狭长,多为单层生长,胞内可见较多空泡形成,核仁变形,细胞形态不规则,细胞明显退变;免疫组化法显示Ⅱ型胶原表达明显减弱,基本为阴性。模型+调补肺肾方组甲苯胺蓝染色显示软骨细胞呈浅蓝色,核仁较清晰,胞内空泡较少,细胞形态相对规则,退变明显减轻;免疫组化法显示Ⅱ型胶原表达减弱,在较多软骨细胞质及胞外可见。模型组软骨细胞中p-p38、caveolin-1表达水平和MMP-3、caveolin-1 mRNA表达水平均明显高于空白组(P均<0.05),模型+调补肺肾方组均明显低于模型组(P均<0.05)。结论采用CSE联合IL-1β处理气管软骨细胞能成功制备COPD合并气管支气管软化症模型,调补肺肾方能降低软骨细胞中p-p38、caveolin-1、MMP-3的表达,从而抑制气管软骨细胞退变。

A method for preparing a model of chronic obstructive pulmonary disease combined with tracheobronchomalacia and evaluation on the intervention effect of Tiaobufeishen Decoction

Objective It is to establish a model of chronic obstructive pulmonary disease(COPD)combined with tracheobronchomalacia and to evaluate the mechanism of Tiaobufeishen Decoction in protecting tracheal chondrocytes from degeneration.Methods The experiment was divided into blank group,model group,model+Tiaobufeishen Decoction group.Flow cytometry was used to determine the optimal concentration of Tiaobufeishen Decoction and cigarette smoke extract(CSE).The optimal concentration of CSE was used to treat tracheal chondrocytes to make COPD cell models,and then interleukin-1β(IL-1β)was added to establish a model of chondrocyte degeneration.After successful model building,the model+Tiaobufeishen Decoction group was treated with Tiaobufeishen Decoction for 48 h,and the blank group and model group were not given special treatment.Toluidine blue staining and immunohistochemical staining were used to observe the degeneration of chondrocytes in each group.Western blot was used to detect the expression of p-p38 and caveolin-1 in the chondrocytes of each group,and the expression of matrix metalloproteinases-3(MMP-3),caveolin-1 mRNA in the chondrocytes of each group were detected by RT-PCR.Results The optimal intervention concentration of Tiaobufeishen Decoction was 1%,and the optimal modeling concentration of CSE was 2.5%.Toluidine blue staining in the model group showed that chondrocyte staining was obviously lighter,the cells were narrow and long,and most of them grew in a single layer,there were more vacuoles in the cells,deformed nucleoli,irregular cell morphology,and obvious cell degeneration;immunohistochemical method showed that the expression of type Ⅱ collagen was significantly weakened,which was basically negative.Toluidine blue staining in the model+Tiaobufeishen Decoction group showed that chondrocytes were light blue,nucleoli were clearer,intracellular vacuoles were less,cell morphology was relatively regular,and degeneration was significantly reduced;immunohistochemistry showed that the expression of type Ⅱ collagen was weakened and could be found in more chondrocyte cytoplasm and extracellular.The expression levels of p-p38 and caveolin-1 and the mRNA expression levels of MMP-3 and caveolin-1 in chondrocytes in the model group were significantly higher than those in the blank group(all P<0.05),the model+Tiaobufeishen Decoction group was significantly lower than the model group(all P<0.05).Conclusion Treating tracheal chondrocytes with CSE combined with IL-1β can successfully prepare COPD combined with tracheobronchomalacia model.Tiaobufeishen Decoction can reduce the expression of p-p38,caveolin-1 and MMP-3 in chondrocytes,thereby inhibiting the trachea degeneration of chondrocytes.