Investigate The Mechanism Of Allyl Isothiocyanate Upregulation MRP1 Expression In Pulmonary Bronchial Epithelial Of COPD Based On The DJ-1/NRF2

Objective:In this study,we investigated the effects of DJ-1/Nrf2 on the expression of MRP1 protein in COPD disease,and explored the important protective effects of DJ-1/Nrf2 system on lung tissue.Furthermore,we verified whether AITC can up-regulate the expression of MRP1 protein by maintaining the stability of DJ-1 protein and activating Nrf2 pathway to inhibit the development of COPD induced by cigarette smoke stimulation.Methods:In vivo,COPD models of wild-type（WT）mice and Nrf2 knockout（-/-）mice were established by passive smoking.The effects of Nrf2 gene knockout on cigarette smoke（CS）-induced COPD development were assessed by detecting lung function and HE staining.And the expressions of Nrf2 and MRP1,DJ-1 in lung bronchial epithelium of COPD mice were determined by immunohistochemistry and Western blot.Subsequently,we compared the expression of MRP1 and DJ-1 in WT and Nrf2^-/-control groups to investigate the effect of Nrf2 knockout on their expression.At the same time,we investigated the effect of Allyl isorhodanate（AITC）on the expression of DJ-1,Nrf2and MRP1 in lung bronchial epithelium of WT and Nrf2^-/-model mice,and explored whether AITC up-regulated the expression of MRP1 in lung tissue of COPD mic was related to DJ-1/Nrf2.In vitro,cigarette smoke extract（CSE）was used to stimulate human bronchial epithelial cells 16HBE.Western blot and Real time RT-PCR were used to detect the expression of DJ-1,Nrf2,HO-1 and MRP1 after CSE stimulation and AITC pre-protection.Then,the expression of DJ-1 in 16 HBE cells was silenced by siRNA,and the effect of DJ-1 expression level on CSE-stimulated protein degradation and AITC-induced protein expression was examined.Results:1.Pulmonary function and HE staining results showed that Nrf2 gene deletion aggravated COPD caused by cigarette smoke stimulation and inhibited the pharmacological effects of AITC.2.Compared with the WT control group,the expression of DJ-1,Nrf2,HO-1 and MRP1 was significantly decreased in the WT model group（P<0.05）.After administration of AITC,the expression of each protein was significantly increased compared with the WT model group.（P<0.05）.3.Nrf2 gene deletion inhibited the expression of MRP1 protein in lung tissue of Nrf2^-/-control group（P<0.05）and attenuated the effect of AITC on up-regulating MRP1expression in COPD group.4.5%CSE stimulation could reduce the expression of DJ-1,Nrf2,HO-1 and MRP1proteins in 16HBE cells in a time-dependent manner.And AITC（10μM～40μM）could increase DJ-1,Nrf2,HO-1,MRP1 protein expression in 16HBE cells in a concentration-dependent manner,and inhibit proteins degradation induced by CSE stimulation.5.Silencing the expression of DJ-1 in 16HBE cells did not affect the expression of Nrf2,HO-1,MRP1 m RNA and protein under normal conditions,but accelerated CSE-induced proteins degradation and significantly attenuated the effect of AITC-induced Nrf2,MRP1 m RNA and protein expression.Conclusion:Down-regulation of MRP1 expression in lung tissue of COPD mice may be caused by inactivation of Nrf2 pathway and degradation of DJ-1 protein.And AITC up-regulates MRP1 expression by activating Nrf2 signaling pathway.In addition,the expression level of DJ-1 significantly affected the role of AITC in inducing MRP1protein expression through Nrf2 pathway.In summary,AITC may increase the stability of Nrf2 and up-regulate the expression of MRP1 by inhibiting the degradation of DJ-1protein,which may play an important role in the treatment of COPD.