The Role Of UCHL1 In Smoking-induced Airway Epithelial Damage In COPD

Objective:The expression of UCHL1 in smoking and COPD states was analyzed by bioinformatics,and mice and BE AS-2B cells were stimulated with cigarette smoke and its extracts to construct an exposure model to explore the role of UCHL1 in the process of cigarette smoke-induced airway epithelial damage changes,effects and related mechanisms.Methods:（1）Bioinformatics analysis screened the non-smoking group,smoking group,and COPD group in the GSE5058 data set with progressive expression genes,and re-validated the differentially expressed genes in the GSE76925 data,than selected UCHL1 for the next analysis.（2）Search the GeneCards database and GTEx database for an overview of the expression of UCHL1 in various tissues and organs and the expression in various types of cells in lung tissue.Pearson correlation analysis screened the GSE5058 data set in the non-smoking group,smoking group,and COPD group.The 200 genes with the highest correlation between expression progressive genes and UCHL1 expression were analyzed by GO function enrichment.（3）Establishment COPD model mice,16 male clean-grade C57BL/6J mice were randomly divided into control group and smoking group.The mice in the control group breathed air freely,and the mice in the cigarette group were exposed to cigarette smoke for half an hour each time,twice a day,5 days a week,for 24 consecutive weeks,record mouse body weight on time.Lung tissues of mice were collected for HE staining and immunohistochemistry,and bronchoalveolar lavage fluid was used for ELISA detection.（4）Establishment cell exposure model:Beas-2B cells were divided into control group and CSE group,with 3 samples in each group.The CSE group was added with complete medium containing 2%CSE,and the control group was given complete medium for 8 weeks.The effects of CSE treatment on the expression of UCHL1,GPX1/2,SOD1,Trx,E-cadherin,a-SMA and Vimentin were detected by WB,the levels of ROS,total SOD and total GPX were detected by the kit,and the changes of cell migration ability were detected by cell scratch assay.（5）BEAS-2B cells were transfected with lentivirus to establish three stable transfection cell lines,LV-UCHL1 overexpressing UCHL1,LV-UCHL1-RNAi inhibiting UCHL1 expression,and empty control LV-Control.The transfection efficiency was verified by qPCR and Western blot,MTT assay were used to detect cell proliferation.The following groups were constructed:LV-Control,LV-UCHL1,LV-UCHL1-RNAi,LV-Control+CSE,LVUCHL1+CSE,LV-UCHL1-RNAi+CSE,treated for 8 weeks,and the ROS,total SOD and The total GPX level were detected by kits,transwell experiments were used to observe the cell migration.（6）Set up UCHL1 overexpression groups:LV-Control,LV-UCHL1,LV-Control+CSE,LV-UCHL1+CSE;UCHL1 inhibition groups:LV-Control,LV-UCHL1-RNAi,LVControl+CSE,LV-UCHL1-RNAi+CSE;treated for 8 weeks.The protein expression levels of UCHL1,GPX1/2,E-cadherin,α-SMA and Vimentin in the above grouped cells were detected by Western blot.Results:（1）The expression of UCHL1 in the small airway epithelium of non-smoking controls,smokers and COPD patients was increased.The expression of UCHL1 in lung tissue of COPD patients was higher than that of non-COPD smokers and was not significantly affected by age and gender.（2）UCHL1 was expressed in alveolar type I epithelium,alveolar type II epithelium,alveolar macrophages and ciliated cells.Analysis of GSE5058 showed that among the nonsmoking controls,smokers,and COPD patients with increased expression in small airway epithelium,the GO function enrichment analysis of the top 200 genes correlated with UCHL1 expression mainly focused on redox processes and oxidation.Reductase activity,etc.（3）HE staining of mouse lung tissue:The alveolar spaces of mice exposed to smoke increased,the alveolar septum was partially ruptured,the alveolar fusion and the formation of emphysema occurred.Smoke exposure increases alveolar mean linear intercept（MLI）（p<0.01）.Smoke exposure increased IL-6 levels in bronchoalveolar lavage fluid and lung tissue significantly（p<0.01）.Immunohistochemical results:The expression of UCHL1 and α-SMA in the lung tissue of smoking group increased,while the expression of GPX1/2,SOD1,Trx and E-cadherin decreased（p<0.05）.（4）Long-term treatment with CSE can increase the level of ROS in BEAS-2B cells,decrease the levels of total SOD,and make BEAS-2B cells migrate Enhanced（p<0.05）.CSE treatment increased the expression levels of UCHL1,GPX1/2,α-SMA and Vimentin,and inhibited the expression levels of SOD1,Trx and E-cadherin.（p<0.05）.（5）Overexpression of UCHL1 accelerated the proliferation of BEAS-2B cells,whereas inhibition of UCHL1 expression decreased the rate of cell proliferation（p<0.05）.In the case of CSE treatment,overexpression of UCHL1 increased the levels of total GPX and total SOD compared with the empty vector group,and the level of ROS decreased;inhibiting the expression of UCHL1 decreased the levels of total GPX and total SOD,and increased the level of ROS（p<0.05）.Transwell migration experiments showed that overexpression of UCHL1 could inhibit the increase in migration ability induced by CSE stimulation,while inhibiting the expression of UCHL1 further enhanced the migration ability of cells stimulated by CSE（p<0.05）.（6）Overexpression of UCHL1 further increased GPX1/2 levels under CSE stimulation,whereas inhibition of UCHL1 expression significantly limited GPX 1/2 levels under CSE stimulation（p<0.05）.Overexpression of UCHL1 suppressed the decline of E-cadherin and the raise of Vimentin and α-SMA induced by CSE stimulation,while the inhibition of UCHL1 expression enhanced the decrease of E-cadherin expression and the increase of α-SMA and Vimentin expression induced by CSE stimulation（p<0.05）.Conclusion:In the process of smoking-induced COPD,oxidative stress occurs in the airway epithelium,the levels of inflammatory factors increase,and the airway epithelial cells undergo epithelial-mesenchymal transition,which is accompanied by an increase in the expression of UCHL1.UCHL1 can negatively regulate the ROS level of BEAS-2B cells stimulated by CSE.It may be that UCHL1 positively regulates the expression of GPX1/2 and affects the antioxidant capacity of bronchial epithelial cells.At the same time,UCHL1 can also regulate the epithelial-mesenchyme translation of bronchial epithelial cells stimulated by CSE.UCHL1 has the potential to be a diagnostic molecular marker and therapeutic target for COPD.