Investigate The Mechanism Of Allyl Isothiocyanate Upregulation MRP1 Expression In Pulmonary Bronchial Epithelial Of Nrf2 Knockout Mice Based On The Notch1/Hes1 Signaling Pathway

Objective: Establishing two COPD models of wild-type(Nrf2(+ / +))mice and Nrf2knockout(Nrf2(-/-))mice to study the correlation of Nrf2,MRP1 and Notch1 in the lung bronchial epithelium of COPD mice.The expression of Notch1 and MRP1 in wild-type mice and Nrf2 knockout mice were compared and analyzed.Meanwhile,Allyl isorhodanate(AITC)was used to investigate the expression of Notch1 and MRP1 in wild-type mice and Nrf2 knockout mice,and to analyze the correlation between Notch1 and MRP1 expression.The purpose of the study was to determine whether Nrf2/ARE signaling pathway had an effect on the expression of MRP1 and Notch1.In the state of COPD,it was verified whether Nrf2 was involved in the effect of AITC on the expression of MRP1 and Notch1 protein in lung bronchial tissue of mice,thereby providing a theoretical basis for the treatment of COPD.Methods: To establish the COPD model by passive smoking was used of wild-type mice and COPD model of Nrf2 knockout mice.After the model was successfully established,30 Nrf2(+ / +)mice and 30 Nrf2(-/-)mice were randomly divided into Nrf2(+ / +)COPD group,Nrf2(+ / +)AITC group,Nrf2(+ / +)NAC positive control group,Nrf2(-/-)COPD group,Nrf2(-/-)AITC group,Nrf2(-/-)NAC positive control group,10 Nrf2(+ / +)mice and 10 Nrf2(-/-)mice were divided into Nrf2(+ /+)normal groups and Nrf2(-/-)normal group.After of continuous intragastric administration of AITC for 15 days,pulmonary function tests and HE stainingwere used to detect the lung function parameters and pathological changes.The protein expression of Notch1,Nrf2 and MRP1 protein in lung tissue was analyzed by immunohistochemistry.The protein expression of Nrf2,HO-1,Notch1,Hes1 and MRP1 in the bronchial epithelium of each group was determined by Western blot.Results: 1.In the Nrf2(+ / +)COPD model group mice and the Nrf2(-/-)COPD model group,the PEF,FEV0.3/FVC%,Cdyn and PIF were significantly decreased(P <0.01).Compared with the Nrf2(+ / +)COPD model group,lung function of the Nrf2(-/-)COPD model group comprises FEV0.3/FVC%,PEF,PIF,and Cdyn were significantly decreased(P < 0.01 or P < 0.05).Compared with Nrf2(+ / +)AITC group and NAC group,the indexes of FEV0.3/FVC%,PIF,PEF,Cdyn of Nrf2(-/-)AITC group and NAC group were significantly lower(P < 0.01).The results of HE pathology showed that compared with the normal control group,the bronchus structure of the Nrf2(+ / +)and Nrf2(-/-)COPD model mice showed incomplete bronchial airway smooth muscle thickening with increased goblet cells.Inflammatory cells infiltrate the epithelium of the lung surface,showing a large area of alveolar wall rupture,and the vesicles are thinning,and even fused together to form large bullae.In the Nrf2(+ / +)and Nrf2(-/-)COPD model groups,the bronchial structure was incompletely alleviated after allyl isothiocyanate treatment.However,the symptoms of the Nrf2(-/-)treatment group were only slightly improved compared to the Nrf2(+ /+)treatment group.2.Immunohistochemistry results showed that compared with the normal Nrf2(+ / +)normal group,the expression of Notch1 and MRP1 protein in the Nrf2(-/-)normal group was significantly decreased(P < 0.01);The expression of Notch1 and MRP1 in Nrf2(-/-)COPD model group was lower than that in Nrf2(+ / +)COPD model group(P < 0.01 or P < 0.05).Nrf2(-/-)AITC-administered group and NAC-positive control group compared with Nrf2(+ / +)AITC group and NAC positive control group,the expression of MRP1 and Notch1 protein were also significantly decreased(P <0.01).3.Western blot results showed that compared with the Nrf2(+ / +)normal control group,the expression of MRP1 and Notch1 protein in lung tissue of Nrf2(-/-)normal control group was significantly lower(P < 0.01).Compared with the Nrf2(+ / +)COPD group,the expression of MRP1 and Notch1 protein of Nrf2(-/-)COPD group was significantly decreased(P < 0.01);Nrf2(+ / +)AITC administered group and NAC positive control group were compared with the Nrf2(+ / +)COPD model group,the expression of MRP1 and Notch1 protein expression were also increased significantly(P < 0.01).4.Pearson correlation analysis by SPSS 17.0 software showed that compared with Nrf2(+ / +)COPD model group,the expression of Notch1,MRP1,Hes1 protein in the lung bronchial tissue of Nrf2(-/-)COPD model group was significantly down-regulated(P< 0.01),suggesting that Nrf2 may participate in the regulation of Notch1 and MRP1 protein expression.Conclusion: In the COPD state,Nrf2 can upregulate the expression of Notch1 and MRP1.Nrf2-ARE signaling pathway can be involved in AITC up-regulation in COPD mice,it plays an important role in the treatment of COPD.