The Role And Mechanism Of Slit2-Robo Signaling Pathway On The Formation Of Cigarette Smoke-Induced Emphysema

PART ?Objective:To explore the differentially expressed genes(DEGs)between severe chronic obstructive pulmonary disease(COPD)patients and healthy smokers to reveal the potential mechanism underpinning the development of COPD.Methods: The m RNA microarray datasets including lung tissue samples from both severe COPD patients and healthy smokers without carcinoma were screened by GEO online database.Subsequently,the DEGs in each dataset were screened by the GEO2 R online analysis and then were integrated using the Robust Rank Aggregation method.Protein-protein interaction(PPI)networks of integrated DEGs were constructed using the STRING online database.GO and KEGG pathway of the DEGs were performed by a DAVID online tool.Results:(1)Three microarray datasets(GSE1650,GSE38974 and GSE76925),including 139 cases of severe COPD and 52 cases of normal smokers,were integrated to screen DEGs using bioinformatics methods.314 DEGs(119upregulated and 195 downregulated)were identified in three databases.(2)20 hub genes were identified by PPI networks analysis,and these genes mostly were associated with B cell signaling pathway,inflammatory response and alveolar destruction.(3)GO and KEGG pathway analyses suggested that the inflammatory response,extracellular matrix disassembly,immune response,the apoptotic signaling pathway,ubiquitination and the Roundabout signaling pathway all together were involved in the development of COPD.(4)In GSE38974 dataset,the genes Slit2 and Robo2 were decreased in patients with COPD and these decreases were significantly negatively correlated with the GOLD stages of COPD.The downstream genes Rac2 and Cdc42 were increased in patients with COPD and were significantly positively correlated with the GOLD stages of COPD.Conclusion: Slit2-Robo signaling pathway may be involved in the pathogenesis of COPD.PART ?Objective:To investigate the expression of Slit2-Robo signaling pathway in cigarette smoke(CS)-induced emphysema mice model and explore the relationship between Slit2 and neutrophils in the airway.Methods: Sixteen male C57BL/6J mice aged 6-8 weeks were randomly divided into the air-exposure(AIR)group and the CS-exposure group.The mice were exposed to chronic cigarette smoke or air for 24 weeks.The right lung was pathologically stained with HE and the alveolar mean linear intercept(Lm)was calculated.Immunohistochemical staining of Slit2 was conducted.The m RNA expression of Slit2,Robo1,Robo2,Rac2 and Cdc42 in lung were conducted by the RT-PCR,and the protein expression of Slit2,Robo1 and Robo2 were examined by Western blot.The neutrophils expression in the bronchoalveolar lavage fluid(BALF)were detected by flow cytometry.Results: The Lm in CS-exposed mice was significantly higher than that in AIRexposed mice.Slit2 was mainly expressed in bronchial epithelial cells,the m RNA and protein expressions of Slit2,Robo1 and Robo2 in the lung of CS-exposed mice were both significantly lower than that in the AIR-exposed mice,however there was on statistically difference in the m RNA expressions of both Rac2 and Cdc42 in the lung of mice in two group.The proportion of neutrophils in BALF was significantly higher in CS-exposed mice than that in AIR-exposed mice,the expression of Slit2 was significantly negative correlated with Lm and the proportion of neutrophils in BALF.Conclusion: The Slit2-Robo signaling pathway is down-regulated in the CSinduced emphysema mice,and the expression of Slit2 is negatively correlated with both the emphysema severity and the neutrophils in the airway.The Slit2- Robo signaling pathway is involved in the development of CS-induced emphysema.PART ?Objective:To investigate the direct effect of cigarette smoke on the expression of Slit2 in human bronchial epithelial cells and whether Slit2 can mediate the migration of neutrophils induced by cigarette smoke extract(CSE)or not.Methods: In four GEO databases(GSE7895,GSE20250,GSE37147 and GSE97010),bronchial epithelial cells were collected via fiberoptic bronchoscopy from different individuals including non-smoker,current smoker,former-smoker and COPD patients.RNA from these samples was profiled on microarrays.The comparisons of the m RNA expression of Slit2 were conducted in each database.Human bronchial epithelial cells BEAS-2B were divided into control group and CSE intervention group.The survival rate of bronchial epithelial cells at different CSE intervention concentrations was determined by CCK8 assay.After the intervention with CSE at different concentrations,the protein expressions of Slit2 in each group were detected by Western blot.Neutrophils from peripheral blood of healthy people were divided into four groups:(1)control group(2)CSE intervention group(3)Slit2 intervention group(4)CSE+Slit2 intervention group.The migration rate of neutrophils in each group were detected by Transwell assay.Results: In vivo,the m RNA expression of Slit2 in human bronchial epithelial cells was both significantly decrease in COPD patients and smokers compared with healthy people,what's more,it could be down-regulated even with an exposure to cigarette smoke for 24 hours.The concentration of CSE less than 0.3% showed no obvious cytotoxicity in the bronchial epithelial cells.Compared with the control group,the expression of Slit2 was significantly decreased in bronchial epithelial cells simulated with 0.15%CSE for 24 hours.The migration rate of neutrophils was significantly increased when stimulated with CSE,which could be attenuated by the treatment of Slit2.Conclusion: Cigarette smoke can directly inhibit the Slit2 expression in bronchial epithelial cells.Slit2 can inhibit the migration of neutrophils induced by CSE.PART ?Objective:To investigate the role of exogenous Slit2 on the infiltration of neutrophils in the airway and chronic pulmonary inflammation in CS-induced emphysema mice model.Methods: Thirty-two male C57BL/6J mice aged 6-8 weeks were randomly divided into the air-exposure(AIR)group,the CS-exposure group,the low dose Slit2 administrated(SLIT2-S)group and the high dose Slit2 administrated(SLIT2-L)group.The mice in the CS,SLIT2-S and SLIT2-L groups were exposed to chronic cigarette smoke for 24 weeks.Low dose(1?g/kg/d)or high dose(5?g/kg/d)of recombinant Slit2-N were injected into the peritoneal cavity of mice from the end of the 12 th week to the end of the 24 th week.The mice in the AIR group were exposed to air for 24 weeks.The expression of NETs-DNA in BALF was detected by Picogreen,the expressions of MPO and NE in BALF were detected by ELISA.The total cells in the BALF were calculated manually,and the expression of neutrophils in the BALF,lung and spleen were detected by flow cytometry.The right lung was pathologically stained with HE and the alveolar mean linear intercept(Lm)was calculated.The ratio of Th1 and Tc1 cells in the lung and spleen were determined by flow cytometry.Results: The total cells,NETs-DNA formation and the expressions of MPO and NE in BALF,Lm,the proportion of neutrophils in BALF,lung and spleen,and the Th1 and Tc1 cells in lung and spleen,were all increased in CS group compared with AIR group,however,the Slit2-S group and Slit2-L group were lower than those in CS group.The proportion of neutrophils in the lung and spleen of the Slit2-L group was comparable to the AIR group.Conclusion: Exogenous Slit2 could not only inhibit the infiltration of neutrophils and the formation of NETs in the airway of CS-induced emphysema mice,but also attenuate the CS-induced emphysema.Slit2 could serve as a potential target for the treatment of CS-associated emphysema.