Study On The Down-regulation Mechanism Of MRP1 And Drug Intervention In A549 Cell Inflammation Model Induced By Cigarette Smoke Extract Combined With LPS

Chronic obstructive pulmonary disease(COPD)is an inflammatory disease that cannot be completely reversed and is characterized by airway obstruction and abnormal inflammatory responses.Multidrug resistance associated protein 1(MRP1)exudes a variety of toxic exogenous substances and certain pro-inflammatory molecules,and the up-regulation of MRP1 can further reduce pulmonary inflammation and damage.DJ-1(PARK7)is a multifunctional protein that protects neurons from oxidative stress and stabilizes the transcription factor nuclear factor erythrocyte2-associated factor 2(Nrf2).Wnt/β-catenin signalling is involved in embryonic development,autophagy,and human disease pathogenesis,in which Wnt3 a is resistant to COPD and emphysema.Tiotropium bromide(TB)and odaterol(O)are currently the inhaled drugs of choice for the treatment of COPD,and in addition to their own bronchiectasis,they have clearly demonstrated anti-inflammatory effects.However,it remains unclear whether DJ-1 regulates Nrf2-mediated MRP1 expression and lung antioxidant defense through activation of Wnt3a/β-catenin signalling pathway,as well as the anti-inflammatory mechanisms of TB and O in pulmonary inflammation.AimsAlveolar epithelial cells(A549)were exposed to cigarette smoke extract(CSE)and lipopolysaccharide(LPS)to establish an inflammatory cell model and explore the effects of CSE and LPS on MRP1 protein expression,the regulatory mechanism of MRP1 protein expression changes in the inflammatory state and the intervention effects of TB and O on inflammation.Methods1.MTT assay was used to detect the effects of CSE,LPS,TB and O on the activity of A549 cells,and combined with literature reference to determine the appropriate administration concentration.2.Western blot was used to detect the expression of major markers in A549 cells stimulated by CSE alone,LPS alone or combined use of CSE and LPS.3.Western blot and quantitative real-time PCR were used to detect the expression of major markers in A549 cells stimulated by different concentrations of CSE combined with LPS.4.Western blot was used to detect the expression of major markers after overexpression or silencing of Wnt3 a in A549 cells under inflammatory state.5.Western blot was used to detect the expression of major markers after Wnt3 a and Nrf2 were,respectively,overexpressed and silenced in A549 cells under inflammatory state.6.Western blot and immunofluorescence staining were used to detect the expression of major markers after overexpression or silencing of DJ-1 in A549 cells under inflammatory state.7.Western blot was used to detect the expression of major markers after DJ-1 and Wnt3 A were,respectively,overexpressed and silenced in A549 cells under inflammatory state.8.Western blot was used to detect the expression of major markers in A549 cells stimulated by TB alone,O alone or combined TB and O under inflammatory state.ResultsCSE alone,LPS alone and CSE combined with LPS stimulated A549 cells,all of which could induce down-regulation of the protein expressions of p-Nrf2,Nrf2 and MRP1 and up-regulation of the protein expressions of TNF-α and IL-6,and the combination of CSE and LPS has a stronger effect.CSE combined with LPS stimulated A549 cells,the expressions of DJ-1,Wnt3 a,β-catenin,p-Nrf2,Nrf2,MRP1,and HO-1 were down-regulated while the expressions of TNF-α,IL-6,IL-18 and IL-1βwere up-regulated,and with the increase of CSE concentration,the protein expressions of DJ-1,Wnt3 a,Nrf2 and MRP1 gradually decreased in a dose-dependent manner.After exposing A549 cells to CSE and LPS,Nrf2 protein expressions were significantly decreased,while there was no change in Nrf2 m RNA levels.In A549 cells exposed to CSE and LPS,overexpression of DJ-1 and Wnt3 a can activate Nrf2 signal,increase MRP1 and HO-1 protein levels,and reduce IL-6 protein levels.Overexpression of Wnt3 a can also increase the expressions of p-GSK3β and decrease the expressions of Axin1 and GSK3β.The knockdown of DJ-1 and Wnt3 a is the opposite.In addition,overexpression of DJ-1 and knockdown of DJ-1 increased and decreased the protein levels of Wnt3 a and β-catenin,respectively.The lack of Nrf2 and Wnt3 a reduced the protective effects of Wnt3 a and DJ-1 on A549 cells,respectively.However,the protein levels of DJ-1 and Wnt3 a did not change due to the deletion of Wnt3 a and Nrf2.In A549 cells exposed to CSE and LPS,we found that both TB and O can up-regulate the protein levels of p-Nrf2,Nrf2 and MRP1,and down-regulate the protein levels of IL-6,TNF-α and IL1β.After showing a stronger effect.ConclusionsIn conclusion,in A549 cells treated with CSE and LPS,DJ-1 regulates Nrf2-mediated MRP1 expression and antioxidant defense,at least in part,by activating the Wnt3a/β-catenin signalling pathway.In the treatment of inflammation,Tiotropium and Odaterol may partly play an anti-inflammatory effect by up-regulating the expressions of Nrf2 and MRP1.These findings may provide potential therapeutic targets for COPD intervention.