The Role Of Microsomal Epoxide Hydrolase In Lung Injuryinduced By Cigarette-induced Chronic Obstructive Pulmonary Disease In Mice

Objectives:1.To observe the expression level changes of mEH and Nrf2 from lung tissue of CSE induced COPD model mice.2.To explore the effect of Nrf2 gene level changes on the expression level of mEH in CSE-induced BEAS-2B cells.3.To explore the effect of mEH gene level changes on inflammatory reaction of CSE-induced BEAS-2B cells.Methods1.Changes of mEH and Nrf2 gene expression level in CSE-induced COPD model mice3-5 weeks old C57BL/6 mice were randomly divided into control group and COPD model group.The control group did not do any treatment under the same room temperature environment.For the COPD model group,a 24-hour smoke intervention was performed on C57BL/6 mice using a homemade smoke box.Observing the state of two group mice during modeling.After the modeling was completed,the mice were sacrificed,and the lung function and HE staining were detected.Mice were collected bronchoalveolar lavage fluid,lung tissue and plasma.Using qPCR and Western blot methods to measure the expression levels of mEH and Nrf2 mRNA or protein in lung tissue of mice.The concentration of mEH in bronchoalveolar lavage fluid and plasma was detected by ELISA.2.Effect of transcription factor Nrf2 on the expression of mEH in CSE induced BEAS-2B cellsBEAS-2B cells were cultured in DMEM medium containing 10%FBS in an incubator at 37?,5%CO₂ atmosphere.Measuring the OD₃₄₀of CSE after prepared for testing its stability and consistency.The toxicity of CSE to BEAS-2B cells was detected by CCK-8 method,and the optimal concentration of CSE was determined.The cells were divided into?1?Control group and?2?CSE intervention group.The BEAS-2B cells were treated with 5%CSE for 12H,and the expression levels of mEH and Nrf2 mRNA were detected by Q-PCR and WB methods,respectively.The cells were divided into?1?Control group,?2?Nrf2 siRNA group,?3?CSE intervention group,and?4?Nrf2 siRNA+CSE group.Inhibiting the expression level of Nrf2 mRNA through transfection of siRNA-Nrf2.After abandoning the transfection solution,the 12H of BEAS-2B cells was continued to be treated with 5%CSE.The mRNA and protein expression level of mEH and Nrf2 in BEAS-2B cells were detected by qPCR and WB respectively.3.The effect of mEH on CSE-mediated inflammation in BEAS-2B cellsThe cells were divided into?1?Control group,?2?mEH siRNA group,?3?CSE intervention group,?4?mEH siRNA+CSE group.Inhibiting the expression level of mEH mRNA through transfection of siRNA-mEH.After abandoning the transfection solution,the BAES-2B cells were further treated with 5%CSE.The expression of mEH mRNA in BEAS-2B cells was detected by qPCR.The IL-6 and IL-8 in the supernatant of cells were evaluated by ELISA.Results:1.Changes of mEH and Nrf2 gene expression level in CSE-induced COPD miceA mouse model of chronic obstructive pulmonary disease was successfully established after 24 weeks of intervention with cigarette smoke.The mRNA and protein expressions of mEH and Nrf2 in the COPD model group were significantly higher than those in the control group,P<0.05.The plasma level of mEH in the COPD model group was significantly higher than that in the blank control group,P<0.05.There was no significant difference in the concentration of mEH in bronchoalveolar lavage fluid between the COPD model group and the blank control group?P>0.05?.2.Transcription factor Nrf2 regulates CSE-induced mEH expression in BEAS-2B cells2.1There has no statistical difference in the OD₃₄₀ of CSE prepared at 6 different times,P>0.05,suggesting that the cigarette extract prepared by the method has good consistency and stability.CCK-8 method suggested that CSE increased the concentration of BEAS-2B cells with increasing concentration and prolonged intervention time.The inhibition rate of 5%CSE was significantly increased at 12H and later,and the expression of Nrf2 mRNA was the most significant in the intervention of 12H.2.2The expression of mEH and Nrf2 mRNA and protein in CSE intervention group was significantly higher than that in Control group?P<0.05?.2.3Effect of mEH on CSE-mediated inflammation in BEAS-2B cellsInhibiting the expression of mEH and interventing the BEAS-2B cells with5%CSE for 12H.The results showed that,mEH siRNA+CSE group mEH mRNA expression was significantly down-regulated compared with CSE intervention group?P<0.05?,compared with mEH siRNA group significantly increased?P<0.05?.Compared with the Control group,the levels of IL-6 and IL-8 in the CSE intervention group were significantly higher,P<0.05.Compared with mEH siRNA group and CSE intervention group,IL-6 and IL-8 levels in mEH siRNA+CSE group were significantly increased,P<0.05.Conclusion1.Theexpression of mEH and Nrf2 genes in lung tissue of CSE-induecd COPD model mice were significantly increased,which can cause an up-regulationof mEH concentrationin plasma.2.Transcription factor Nrf2 regulates the expression of mEH in BEAS-2B cells induced by CSE.3.Silencing the mEH gene leads to a significant increase in CSE-induced inflammatory response.