补肺益肾方对慢性阻塞性肺疾病模型大鼠肺泡表面活性蛋白A、D及肺泡结构的影响

Effects of Bufei Yishen Formula on Surfactant-associated Proteins A,D and Alveolar Structure of COPD Rats

目的:观察补肺益肾方对慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)模型大鼠肺泡表面活性蛋白(Surfactant-associated Proteins,SP)-A、SP-D及肺泡结构的影响。方法:将36只COPD大鼠随机分为空白组,模型组,补肺益肾高剂量组、中剂量组、低剂量组,氨茶碱组,采用香烟暴露联合反复细菌感染制备COPD稳定期大鼠模型,空白组、模型组给予生理盐水灌胃,补肺益肾高剂量组、中剂量组、低剂量组分别给予补肺益肾方7.4 g·(kg·d)^(-1)、3.7g·(kg·d)^(-1)、1.85 g·(kg·d)^(-1)灌胃,氨茶碱组给予氨茶碱54 mg·(kg·d)^(-1)灌胃。治疗12周后取材,检测肺组织病理、肺泡Ⅱ型上皮细胞超微结构,荧光定量PCR检测肺组织SP-A、SP-D基因表达,激光共聚焦检测肺组织SP-A、SP-D阳性表达。结果:肺组织病理示,空白组大鼠肺组织结构基本正常;模型组大鼠肺泡壁部分断裂融合,细支气管壁增厚,管壁周围结缔组织增生、炎症细胞浸润,气道内黏液分泌;补肺益肾高剂量组、中剂量组、低剂量组和氨茶碱组均有不同程度减轻,补肺益肾中剂量组减轻尤为明显。肺组织超微结构示,空白组大鼠肺组织肺泡Ⅱ型细胞中可见线粒体丰富,线粒体嵴排列整齐,板层小体完整;模型组Ⅱ型细胞线粒体较小,线粒体嵴较为清晰,但板层小体脱颗粒、空泡化;补肺益肾高剂量组Ⅱ型细胞中线粒体丰富,线粒体嵴清晰,板层小体较少但结构完整;补肺益肾中剂量组Ⅱ型细胞中线粒体丰富且排列整齐,线粒体嵴较为清晰,板层小体丰富且结构完整,细胞边缘绒毛完整;补肺益肾低剂量组和氨茶碱组Ⅱ型细胞中线粒体较少,结构不清晰,板层小体少且部分空泡化。荧光定量PCR示,与空白组比较,模型组肺组织SP-A、SP-D mRNA显著降低(P≤0.01);与模型组比较,补肺益肾高剂量组、中剂量组,氨茶碱组SP-A、SP-D mRNA均显著升高(P≤0.01),补肺益肾低剂量组SP-A mRNA升高(P≤0.01);与补肺益肾高剂量组、中剂量组比较,低剂量组和氨茶碱组SPA、SP-D mRNA均显著降低(P≤0.01)。激光共聚焦显示,与空白组比较,模型组肺组织SP-A、SP-D阳性表达细胞数明显降低(P≤0.01);与模型组比较,补肺益肾高剂量组、中剂量组和氨茶碱组SP-A、SP-D均升高(P≤0.01);与补肺益肾高剂量组比较,中剂量组SP-A、SP-D均升高(P≤0.05或P≤0.01);与补肺益肾中剂量组比较,补肺益肾低剂量组和氨茶碱组SP-A、SP-D均降低(P≤0.01)。结论:补肺益肾方能够改善COPD模型大鼠肺泡Ⅱ型上皮细胞中板层小体的结构,调节肺泡表面活性蛋白的合成和分泌,从而维持肺泡结构稳定,以补肺益肾中剂量尤为显著。

Objective： To explore the effect of Bufei Yishen Formula on Surfactant-associated Proteins A,D and Alveolar Structure of rats with chronic obstructive pulmonary disease（ COPD）. Methods： 36 rats were randomized into control group,model group,Bufei Yishen high-dose group,Bufei Yishen middle-dose group,Bufei Yishen low-dose group and aminophyline group. COPD rats were induced by cigarette-smoke and bacterial exposures,and were administrated with normal saline,Bufei Yishen Formula,with 7. 4 g,3. 7 g and 1. 85 g·（kg·d）^-1 in each dose,and the aminophyline group was given aminophylline,with 54 mg·（kg·d）^-1 in each dose. The treatment lasted for 12 weeks and the rats were sacrificed. Lung pathology,ultrastructure of alveolar type Ⅱ epithelial cell,SP-A,D mRNA and protein expression in lung tissue were measured. Results： Lung pathology： There is no marked pulmonary impairment was observed in the control rats,while rats in model group showed severe pathological such as alveolar destruction,alveolar cavity expansion,bronchiole stenosis,inflammatory cells infiltration and mucosal hyperplasia. The pathological changes were alleviated in the treatment groups at different degrees,especially in Bufei Yishen middle-dose groups. The ultrastructure of alveolar type Ⅱ epithelial cell： In Control group,abundant mitochondria and lamellar bodies arranged in concentric circles or in parallel were present in the cytoplasm of type Ⅱ alveoli epithelial cells. In Model group,the limited and small mitochondria,degranulated and cavitated lamellar bodies were observed in type Ⅱ alveoli epithelial cells. In Bufei Yishen high-dose group,the rich mitochondria and clear mitochondrial cristae,as well as few lamellar bodies with complete structure were observed in type Ⅱ alveoli epithelial cells. In Bufei Yishen middle-dose group,the rich mitochondria with clear mitochondrial cristae,abundant,complete lamellar bodies,and complete marginal villi were observed in type Ⅱ alveoli epithelial cells. In Bufei Yishen low-dose and aminophyline groups,the limited mitochondria and lamellar bodies were observed. QPCR： Compared with that of the control group,SPA,SP-D mRNA were decreased significantly in model group（ P≤0. 01）. Compared with that of the model group,SP-A,SP-D mRNA in Bufei Yishen high-dose group,middle-dose group,and aminophyline group increased significantly（ P≤0. 01）. Compared with that of the Bufei Yishen high-dose group and middle-dose group,SP-A and SP-D mRNA in Bufei Yishen low-dose and aminophyline group decreased significantly（ P≤0. 01）. Laser scanning confocal microscope： Compared with that of the control group,the SP-A,SP-D positive cells were decreased significantly in model group（ P≤0. 01）; Compared with that of the model group,SPA,SP-D positive cells in Bufei Yishen high-dose,middle-dose,and aminophyline groups increased significantly（ P≤0. 01）. Compared with that of the Bufei Yishen high-dose group,SP-A,SP-D positive cells in Bufei Yishen middle-dose increased（ P≤0. 05 or P≤0. 01）. Compared with that of the Bufei Yishen middle-dose groups,SP-A,SP-D positive cells in Bufei Yishen low-dose group and aminophyline group decreased significantly（ P≤0. 01）. Conclusion： Bufei Yishen Formular can improve the lamellar body of alveolar type Ⅱ epithelial cell,regulate the synthesis and secretion of surfactant-associated proteins and maintain alveolar structure stability,in which the effect of Bufei Yishen Formula middle-dose is the most significant.