The Toxicological Effect And Molecular Mechanism Of NO₂ On Epithelial Cell Metastasis

With the development of industrial production and the increase of vehicle population as well as energy use all over the world,air pollution is becoming more and more serious,which directly endangers human health.As one of the air pollutants,nitrogen dioxide has attracted much attention.At the same time,with the change of people's living environment and lifestyle,the incidence and mortality of cancer are becoming higher and higher.At present,many epidemiological studies at home and abroad have shown that there is a strong correlation between NO₂ exposure and the incidence of lung cancer,but its mechanism is not very clear.Therefore,it has become a hot study to explore the mechanism of NO₂ and the pathogenesis of lung cancer.Recently,the activation of the epithelial-to-mesenchymal transition?EMT?program and the degradation of basement membranes and stromal extracellular matrix?ECM?have been implicated as crucial steps in lung tumor metastasis.In this study,we investigate the effects of NO₂ and its derivatives exposure on migration and invasion of lung epithelial cells as well as its possible molecular mechanism.Firstly,in order to investigate the effects of NO₂ exposure on metastasis of lung epithelial cells and the potential protection of monoacylglycerol lipase?MAGL?inhibition on the effects,we set up an NO₂ exposure model of mice and the mice were exposed to different concentrations of NO₂?0,2.5 and 5mg/m³?.The expression of EMT marker protein E-cadherin and?-SMA in lungs were detected by Western blot;and the content of nitrate and nitrite as well as the expression of prostaglandin E2 were detected by ELISA.These results showed that NO₂ exposure elevated the contents of nitrate and nitrite,and activated EMT by significantly increasing the expression of?-SMA and decreasing the expression of E-cadherin in lungs.MAGL inhibition by injecting JZL184 decreased the content of PGE2,and inhibited E-cadherin decrease and?-SMA elevation in lungs caused by NO₂ exposure.It was implied that NO₂ exposure caused EMT process,and MAGL inhibition prevented the effects by regulating PGE2 release.Secondly,in order to investigate the effects of NO₂ derivatives exposure on metastasis of human lung epithelial cells A549 and the potential protection of reactive oxygen species?ROS?inhibition on the effects,the A549 cells were exposed to different concentrations of NO₂ derivatives?0?10?30?100 and300?M?.The ability of invasion and migration of A549 cells were detected by Transwell.The expression of EMT marker protein E-cadherin and Fibronectin?Fibronectin?as well as ECM degradation marker matrix metalloproteinase-2?MMP-2?and tissue inhibitor of metalloproteinase-2?TIMP-2?of A549 cells were detected by RT-PCR.And the content of ROS was detected by DCF.These results showed that NO₂ derivatives exposure can cause human lung epithelial cells and normal human lung epithelial invasion and metastasis,and increasing the expression of Fibronectin and MMP-2 and decreasing the expression of E-cadherin and TIMP-2.Meanwhile,the production of ROS is increased significantly;ROS inhibition by adding NAC inhibited E-cadherin and TIMP-2 decrease and Fibronectin and MMP-2 elevation of A549 caused by NO₂ derivatives exposure.It was implied that NO₂ derivatives exposure caused EMT and ECM degradation process,and ROS inhibition can prevent the effects.In conclusion,this study established the NO₂ and its derivatives exposure can induce lung epithelial cell invasion and migration,demonstrated the protective effects of MAGL inhibition in NO₂-induced EMT process and revealed ROS participated NO₂ derivatives-induced EMT and ECM degradation process,which will provide an experimental basis for study on the molecular mechanism of tumor metastasis.