补肺益肾方通过调控Notch信号通路改善香烟烟雾提取物诱导气道上皮细胞黏液高分泌  

Bufei Yishen formula attenuates cigarette smoke extract-induced airway mucus hypersecretion by regulating Notch signaling pathway

作　　者：梅晓峰 任周新[2] 余海滨[3] Mei Xiaofeng;Ren Zhouxin;Yu Haibin(First Clinical Medical College of Henan University of Traditional Chinese Medicine,Zhengzhou 450046,Henan,China;Henan University of Traditional Chinese Medicine,Collaborative Innovation Center for Chinese Medicine and Respiratory Diseases Co-constructed by Henan Province&Education Ministry of P.R.China,Zhengzhou 450046,Henan,China;Department of Scientific Research,the First Affiliated Hospital of Henan University of Traditional Chinese Medicine,Zhengzhou 450000,Henan,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,呼吸疾病中医药防治省部共建协同创新中心,郑州450046 [3]河南中医药大学第一附属医院科研部,郑州450000

出　　处：《中华危重病急救医学》2022年第7期704-709,共6页Chinese Critical Care Medicine

基　　金：国家自然科学基金(81473650);河南省中医药科学研究项目(2016ZY2006)。

摘　　要：目的基于Notch信号通路探讨补肺益肾方(BYF)抑制香烟烟雾提取物(CSE)诱导气道上皮细胞黏液高分泌的机制。方法体外培养人气道上皮细胞株16HBE, 取对数生长期细胞用于实验。①干预条件筛选实验:将16HBE细胞分组, 分别采用噻唑蓝(MTT)比色法和酶联免疫吸附试验(ELISA)检测不同浓度CSE(2.5%、5%、10%、20%、40%)、不同浓度BYF含药血清(5%、10%、20%、40%)及不同浓度Notch信号通路阻断剂DAPT(5、10、20、40 μmol/L)对细胞活性与分泌黏蛋白5AC(MUC5AC)水平的影响, 并设空白对照组, 筛选出制备CSE诱导细胞黏液高分泌模型及BYF、DAPT干预的最佳条件。②干预实验:将16HBE细胞分为4组。空白对照组不给予任何处理;CSE模型组采用10% CSE诱导16HBE细胞24 h制备黏液高分泌模型;CSE+BYF组和CSE+DAPT组在加入10% CSE的同时分别给予10% BYF或20 μmol/L DAPT干预24 h。采用实时荧光定量聚合酶链反应(qPCR)检测细胞中MUC5AC、Notch3和多毛分裂增强子1(HES1)的mRNA表达;采用蛋白质免疫印迹试验(Western blotting)检测细胞中Notch3和HES1的蛋白表达。结果①干预条件筛选实验结果:与空白对照组比较, 10% CSE诱导24 h是既不影响细胞活性又可增加MUC5AC分泌的制备细胞黏液高分泌模型的最佳条件;而10% BYF和20 μmol/L DAPT为最佳干预条件。②干预实验结果:与空白对照组比较, CSE模型组细胞中MUC5AC、Notch3、HES1的mRNA表达及Notch3、HES1的蛋白表达均显著升高, 说明CSE可能通过促进Notch3、HES1信号活化, 诱导16HBE细胞分泌黏蛋白;与CSE模型组比较, BYF和DAPT均可显著下调细胞中MUC5AC、Notch3、HES1的mRNA及蛋白表达〔MUC5AC mRNA(2^(-ΔΔCT)):1.03±0.13、0.96±0.05比1.35±0.07, Notch3 mRNA(2^(-ΔΔCT)):1.10±0.14、1.10±0.02比1.31±0.15, HES1 mRNA(2^(-ΔΔCT)):1.26±0.10、1.14±0.15比1.45±0.08, Notch3蛋白(Notch3/GAPDH):0.10±0.03、0.16±0.03比0.31±0.09, HES1蛋白(HES1/GAPDH):0.37±0.06、0.34±0.08比Objective To explore the mechanism of Bufei Yishen formula(BYF)on attenuating cigarette smoke extract(CSE)-induced airway mucus hypersecretion by regulating Notch signaling pathway.Methods The human airway epithelial cell 16HBE was cultured in vitro,and the cells in logarithmic growth phase were used for the experiments.①Intervention condition screening experiment:the 16HBE cells were grouped,methylthiazolyldiphenyl-tetrazolium(MTT)method and enzyme-linked immunosorbent assay(ELISA)were used to detect the effects of different concentrations of CSE(2.5%,5%,10%,20%,40%),different concentrations of BYF drug-containing serum(5%,10%,20%,40%),and different concentrations of Notch signal pathway blocker DAPT(5,10,20,40μmol/L)on cell activity and secretion of mucin 5AC(MUC5AC)levels.In addition,a blank control group was set up to screen out the best conditions for preparing CSE-induced cell mucus hypersecretion model and BYF and DAPT intervention.②Intervention experiment:the 16HBE cells were divided into four groups.The blank control group was not given any treatment;the 16HBE cells were induced by 10%CSE for 24 hours to prepare mucus hypersecretion model in the CSE model group;the cells in the CSE+BYF group and CSE+DAPT group were given 10%BYF or 20μmol/L DAPT,respectively,for intervention at the same time for 24 hours.Real-time fluorescent quantitative polymerase chain reaction(qPCR)was used to detect the mRNA expressions of MUC5AC,Notch3 and hairy and enhancer of split 1(HES1)in the cells.Western blotting was used to detect the protein expressions of Notch3 and HES1 in the cells.Results①Results of the screening experiment of intervention conditions:compared with the blank control group,10%CSE induction for 24 hours was the best condition for establishing cell mucus hypersecretion model that neither affected cell viability nor increased the secretion of MUC5AC;while 10%BYF and 20μmol/L DAPT was the optimal intervention condition.②Intervention experiment results:compared with the blank control group,the mRNA

关 键 词：补肺益肾方 NOTCH信号通路 香烟烟雾提取物 气道上皮细胞 黏液高分泌 

分 类 号：R285[医药卫生—中药学]