调补肺肾三法通过抑制ERK1/2信号通路改善COPD大鼠气道黏液高分泌  

Beneficial effects of Tiao-Bu Fei-Shen therapies on airway mucus hypersecretion in chronic obstructive pulmonary disease rats via inhibition of ERK1/2 signaling pathway

作　　者：李高峰 刘淑娟 李亚[2,3] 李素云[3,4] 范正媛 沈婷婷 LI Gaofeng;LIU Shujuan;LI Ya;LI Suyun;FAN Zhengyuan;SHEN Tingting(the First Clinic School,Henan University of Chinese Medicine,Zhengzhou 450046,China;Chinese Medicine Pharmacology(Respiratory)Laboratory,the First Affiliated Hospital of Henan University of Chinese Medicine,Henan Key Laboratory of Traditional Chinese Medicine for the Prevention and Treatment of Respiratory Diseases,Zhengzhou 450000,China;Respiratory Department of the First Affiliated Hospital of Henan University of Chinese Medicine,Zhengzhou 450000,China;Co-Construction Collaborative Innovation Center for Chinese Medicine and Respiratory Diseases by Henan&Education Ministry of China,Henan University of Chinese Medicine,Zhengzhou 450046,China)

机构地区：[1]河南中医药大学第一临床医学院,郑州450046 [2]河南中医药大学第一附属医院中药药理(呼吸)实验室,河南省呼吸病防治中医药重点实验室,郑州450000 [3]河南中医药大学第一附属医院呼吸科,郑州450000 [4]河南中医药大学呼吸疾病中医药防治省部共建协同创新中心,郑州450046

出　　处：《中国实验动物学报》2024年第4期411-422,共12页Acta Laboratorium Animalis Scientia Sinica

基　　金：国家自然科学基金(82074413,82374416);国家重点研发计划项目(2018YFC1704801);河南省级科技研发计划联合基金项目(222301420081)。

摘　　要：目的 探究调补肺肾三法对慢性阻塞性肺疾病(chronic obstructive pulmonary disease, COPD)稳定期大鼠气道黏液高分泌的影响及作用机制。方法 90只大鼠随机分为对照(Control)组、模型(COPD)组、补肺健脾(Bu-Fei Jian-Pi Formula, BJF)组、补肺益肾(Bu-Fei Yi-Shen Formula, BYF)组、益气滋肾(Yi-Qi Zi-Shen Formula, YZF)组、ERK1/2抑制剂(PD98059)组、补肺健脾联合抑制剂(BJF+PD98059)组、补肺益肾联合抑制剂(BYF+PD98059)组和益气滋肾联合抑制剂(YZF+PD98059)组。第1~8周采用香烟烟雾暴露联合细菌反复感染的方法建立COPD大鼠模型,9~16周Control组和COPD组给予生理盐水每只2 mL,BJF组、BYF组和YZF组分别给予对应的调补肺肾三方灌胃,第16周PD98059组、BJF+PD98059组、BYF+PD98059组和YZF+PD98059组给予PD98059腹腔注射7 d。16周结束后取材,检测大鼠肺功能、肺组织形态、肺组织水含量、BALF中炎性细胞计数和血清炎性因子水平。AB-PAS染色和免疫组化法分别检测杯状细胞比例和Muc5AC、Muc5B表达。PCR检测ERK1、ERK2、ENaC、CFTR、AQP5 mRNA表达。Western Blot测定肺组织中ERK1/2、P-ERK1/2蛋白表达。结果 与Control组相比,COPD组大鼠TV、MV、FVC、FEV_(0.1)及FEV_(0.1)/FVC均显著下降(P<0.01);肺病理显示肺泡紊乱,肺泡壁大量断裂,气管壁严重皱缩增厚,周围有大量炎性细胞浸润;肺组织水含量明显升高(P<0.01);BALF中巨噬细胞比例显著降低(P<0.01),中性粒细胞和淋巴细胞比例显著升高(P<0.01);血清炎性因子TNF-α、IL-1β水平显著增高(P<0.05,P<0.01);气道上皮杯状细胞比例和Muc5AC、Muc5B表达水平均显著升高(P<0.01);肺组织ERK1、ERK2、ENaC mRNA表达量均明显升高(P<0.01),CFTR和AQP5 mRNA的表达均明显降低(P<0.01);肺组织P-ERK1/2, ERK1/2表达明显升高(P<0.01);与COPD组相比,各治疗组能不同程度改善上述指标变化(P<0.05,P<0.01),调补肺肾三方联合PD98059组治疗效果优于单一治疗组(P<0.05,P<0.01)。结�Objective To investigate the roles of three Tiao-Bu Fei-Shen Traditional Chinese Medicine(TCM)therapies in improving airway mucus hypersecretion in rats with stable chronic obstructive pulmonary disease(COPD).Methods Ninety rats were divided randomly into nine groups:control(Control)group,model(COPD)group,Bu-Fei Jian-Pi Formula(BJF)group,Bu-Fei Yi-Shen Formula(BYF)group,Yi-Qi Zi-Shen Formula(YZF)group,ERK1/2 inhibitor(PD98059)group,Bu-Fei Jian-Pi combined with inhibitor(BJF+PD98059)group,Bu-Fei Yi-Shen combined with inhibitor(BYF+PD98059)group,and Yi-Qi Zi-Shen combined with inhibitor(YZF+PD98059)group.A rat model of COPD was established by exposing rats to cigarette smoke followed by repeated bacterial infection from weeks 1~8.From weeks 9~16,rats in the control and COPD groups were given 2 mL normal saline,rats in the BJF,BYF,and YZF groups were given the three Tiao-Bu Fei-Shen formulas by gavage,and rats in the PD98059,BJF+PD98059,BYF+PD98059,and YZF+PD98059 groups were given PD98059 by intraperitoneal injection for 7 days at the 16th week.Lung function tests were conducted after 16 weeks and lung tissue morphology,lung water content,inflammatory cell count in bronchoalveolar lavage fluid,and serum levels of inflammatory factors were also assessed.Goblet cell proportion was determined by Alcian blue-periodic acid-Schiff staining,and Muc5AC and Muc5B expression levels were detected by immunohistochemistry.mRNA expression levels of ERK1,ERK2,ENaC,CFTR,and AQP5 were detected by polymerase chain reaction and protein expression levels of ERK1/2 and P-ERK1/2 in lung tissue were determined by Western Blot.Results TV,MV,FVC,FEV 0.1,FEV 0.1/FVC were significantly decreased(P<0.01)in COPD rats compared with those in the control group.Lung pathology revealed alveolar disorder,massive fracture of the alveolar wall,and severe shrinkage/thickening of the airway wall accompanied by extensive infiltration of inflammatory cells.Lung tissue water content was significantly increased in COPD rats(P<0.01),while the proportion of m

关 键 词：慢性阻塞性肺疾病 气道黏液高分泌 调补肺肾三法 ERK1/2信号通路 

分 类 号：Q95-33[生物学—动物学]