基于Notch通路研究化痰降气胶囊含药血清对人支气管上皮细胞MRP1的影响  

Huatan Jiangqi Capsule Containing Serum Up-regulates MRP1 via Notch Pathway in Human Bronchial Epithelial Cells

作　　者：李妮妮 蒋成君 郭艳 汪电雷 LI Nini;JIANG Chengjun;GUO Yan;WANG Dianlei(Graduate School,Anhui University of Chinese Medicine,Hefei 230012 Anhui,China;Laboratory of Drug Metabolism and Pharmacokinetics,Anhui University of Chinese Medicine,Hefei 230012 Anhui,China)

机构地区：[1]安徽中医药大学研究生院,安徽合肥230031 [2]安徽中医药大学药物代谢动力学研究室,安徽合肥230031

出　　处：《中药新药与临床药理》2019年第12期1442-1448,共7页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：国家自然科学基金项目（81473536）

摘　　要：目的基于Notch通路探讨化痰降气胶囊含药血清对人支气管上皮细胞(16HBE)中多药耐药相关蛋白1(MRP1)表达和外排功能的影响。方法根据血清药理学的方法制备化痰降气胶囊含药血清和空白血清,然后将含药血清分为5%、10%、15%、20%4个浓度对16HBE细胞进行干预,CCK-8法检测细胞增殖率筛选出最佳作用浓度;细胞分为空白血清组、香烟烟雾提取物(CSE)+空白血清组、CSE+含药血清组、化痰降气胶囊含药血清组、Notch通路抑制剂DAPT+空白血清组、DAPT+含药血清组,用Western Blot法检测各组Notch1、Hes1、MRP1蛋白表达;以5-羧基二乙酸荧光素(5-CFDA)为底物,采用流式细胞术检测细胞内荧光强度的变化评价MRP1的外排功能;免疫荧光法检测Notch1的胞内段(NICD)核移位活性。结果化痰降气胶囊含药血清的最佳作用浓度为15%。与空白血清组比较,含药血清组Notch1、Hes1、MRP1蛋白表达呈浓度依赖性升高(P<0.05,P<0.01),MRP1转运体的外排能力明显提高(P<0.01),细胞核内NICD的荧光强度有所增强;而CSE+空白血清组蛋白表达水平明显降低(P<0.01)。与CSE+空白血清组比较,CSE+化痰降气胶囊含药血清组的Notch1、Hes1、MRP1蛋白表达增加(P<0.05,P<0.01);与化痰降气胶囊含药血清组比较,DAPT+化痰降气胶囊含药血清组的Notch1、Hes1、MRP1蛋白表达水平均降低(P<0.05,P<0.01),MRP1的外排能力也降低(P<0.01),胞质胞核荧光强度均减弱。结论化痰降气胶囊含药血清可能通过Notch通路上调16HBE细胞中MRP1的表达和外排功能,这可能是其治疗慢性阻塞性肺疾病(COPD)的作用机制之一。Objective The aim of this study was to investigate the effect and mechanism of Huatan Jiangqi capsule containing serum on the expression and function of MRP1 based on the Notch signaling pathway through in vitro experiments.Methods According to the method of serum pharmacology,the drug containing serum and blank serum were prepared,and then 5%,10%,15%and 20%drug containing serum were used to intervene with human bronchial epithelial(16 HBE)cells.CCK-8 kit was used to detect the proliferation of 16 HBE cells for the optimal concentration.Cells were divided into blank serum control group,Huatan Jiangqi capsule containing serum group,cigarette smoke extract(CSE)+blank serum group,CSE+drug containing serum group,DAPT+blank serum group,DAPT+Huatan Jiangqi capsule containing serum group.Western Blot was used to detect protein expression levels of Notch1,Hes1 and MRP1.Using fluorescein 5-carboxydiacetic acid(5-CFDA)as substrate,the change of intracellular fluorescence intensity in 16 HBE cells was detected by flow cytometry,which can reflect the efflux function of MRP1.The NICD nuclear translocation activity was detected by immunofluorescence.Results The optimal concentration of drug containing serum was 15%.Compared with the blank control group,the expression levels of Notch1,Hes1 and MRP1 protein were significantly up-regulated in Huatan Jiangqi capsule containing serum group(P<0.05,P<0.01),and the increase was concentration-dependent.The efflux capacity of MRP1 transporter was significantly improved(P<0.01)and the fluorescence intensity of NICD in the nucleus was enhanced.While the protein expression levels in CSE+blank serum group were significantly reduced(P<0.01).Compared with the CSE+blank serum group,protein expression levels of Notch1,Hes1 and MRP1 were all increased in the CSE+drug containing serum group(P<0.05,P<0.01).Compared with the Huatan Jiangqi capsule containing serum groups,the expression levels of Notch1,Hes1 and MRP1 protein in DAPT+Huatan Jiangqi capsule containing serum group were all decreased(P<

关 键 词：化痰降气胶囊含药血清 慢性阻塞性肺疾病 多药耐药相关蛋白1 Notch信号通路 人支气管上皮细胞 

分 类 号：R285.5[医药卫生—中药学]