The Mechanism Of Notch1/Hes1 Pathway Regulating MRP1 Expression In Human Bronchial Epithelial Cells And The Intervention Of Allyl Isothiocyanate

Objective:In this study,we investigated the changes of multidrug resistance-associated protein 1（MRP1）expression and function in human bronchial epithelial cells（16HBE）stimulated by cigarette smoke extract（CSE）based on Notch1/Hes1 signaling pathway.On this basis,whether Notch1/Hes1 signaling pathway is involved in the upregulation of MRP1 expression by allyl isothiocyanate（AITC）was further investigated.This study was designed not only to clarify the mechanism of Notch1/Hes1 signaling pathway in the influence of CSE stimulation and AITC intervention on MRP1 expression,but also to provide a theoretical basis and a new thought for the prevention and treatment of chronic obstructive pulmonary disease（COPD）.Methods:MTT method was used to screen out the appropriate concentration of CSE acting on 16HBE cells.The protein expression levels of Notch1,Hes1 and MRP1 in16HBE cells after CSE stimulation and AITC intervention were determined by Western blot.Further,cells were incubated with Notch signaling pathway inhibitor DAPT to observe its effect on proliferation of 16HBE cells and expression of NICD,Hes1,Hey1and MRP1 proteins.Silencing the expression of Notch1 gene in 16HBE cells by siRNA technology,and then exposed to CSE stimulation or AITC pretreatment,the expression of Notch1,Hes1 and MRP1 m RNA and protein were detected by Western blot and RT-PCR.Using 5-CFDA as substrate,flow cytometry was used to detect the effect of incubation with AITC on the efflux function of MRP1 on the 16HBE cells transfected with Notch1 siRNA.Electrophoresis mobility variability analysis（EMSA）detected specific binding of 16HBE nuclear protein extract and probe containning NICD/CBF-1 complex.Results:1.Cigarette smoke extraction device was prepared by injection syringe and Y-shaped latex tube,the average OD₃₂₀value of 100%CSE was 0.525,5%CSE was applied to 16HBE cells for 24 h,and the cell survival rate was larger than 80%,which was suitable for subsequent experiments;2.The expression of Notch1,Hes1 and MRP1 was decreased in a time-dependent manner by stimulating 16HBE cells with 5%CSE for 12,24 and 36 h（P<0.05,P<0.01）.The expression of Notch1 protein was positively correlated with that of MRP1protein（P<0.01,r=0.752）;AITC（10μM、20μM、40μM）pretreatment could reverse the CSE-induced degradation of Notch1,Hes1,and MRP1,and significantly improve the protein expression level（P<0.01）.There was a significant positive correlation between Notch1 protein and MRP1 protein expression（P<0.01,r=0.927）;3.DAPT inhibited the activity of 16HBE cells at different concentrations,and IC₅₀at 24 h and 48 h was 100.79±6.41μM and 92.37±5.01μM,respectively.The IC₅₀of DAPT+AITC group was 107.51±7.76μM.DAPT（10μM、20μM、50μM）were incubated in 16HBE cells for 24 h,the expressions of Notch1,Hes1,Hey1 and MRP1in 16HBE cells were significantly decreased（P<0.05,P<0.01）.Compared with the DAPT group,the expression levels of Notch1,Hey1 and MRP1 in the DAPT+AITC group were significantly increased（P<0.05）,while compared with the AITC group（1.57 times）,the ability of AITC to up-regulate the expression of MRP1 was significantly decreased（1.29 times）（P<0.01）;4.Notch1 siRNA significantly decreased the m RNA and protein expression levels of Notch1,Hes1 and MRP1 in 16HBE cells in the basal statue（P<0.05,P<0.01）.After silencing the expression of Notch1 in 16HBE cells by siRNA,then stimulated with 5%CSE,Notch1 siRNA accelerated the CSE-induced m RNA and protein degradation of Notch1,Hes1 and MRP1（P<0.05,P<0.01）.Notch1 siRNA weakened the up-regulation of m RNA and protein expression of Notch1,Hes1 and MRP1 induced by AITC pre-incubation（P<0.05,P<0.01）;5.Transfection with Notch1 siRNA weakened the efflux transport activity of MRP1 induced by AITC.Compared with the fluorescent control group group,AITC significantly increased the efflux transport capacity of MRP1（P<0.01）,and compared with the AITC+control siRNA group,the efflux transport capacity of MRP1 in AITC+Notch1 siRNA group was significantly decreased（P<0.05）;6.EMSA assay demonstrated that NICD,the Notch1 intracellular segment,in16HBE cells could specifically bind to specific binding sites in the promoter region of MRP1 gene.Conclusion:The down-regulation of MRP1 expression induced by 5%CSE stimulation of 16HBE cells could be due to the inactivation of Notch1/Hes1 signaling pathway,and the silencing of Notch1 gene expression by siRNA can further aggravate the down-regulation of MRP1 expression.AITC could up-regulate the expression and function of MRP1 by activating the Notch1 signaling pathway.DAPT inhibits the Notch signaling pathway or siRNA silencing the Notch1 gene significantly weakens the induction effect of AITC on MRP1.In 16HBE cells,Notch1 intracellular segment NICD binding to MRP1 promoter region fragments,and regulate MRP1 expression.