The Expression Of RECK In Patients With COPD And Its Function On Neutrophils

Object:Chronic obstructive pulmonary disease（COPD）is a chronic inflammatory lung disease with high prevalence rate,which brings heavy economic burden on society and seriously threatens human health.Chronic airway inflammation is the major pathogenesis of COPD.Environmental pollutants and cigarette smoke can induce neutrophil infiltration and the release of matrix metalloproteinase-9（MMP-9）,that will finally cause airway remodeling and lung parenchyma injury.Reversion inducing cysteine rich protein with kazal motifs protein（RECK）is a new inhibitor of matrix metalloproteinases.However,the role of RECK in COPD has not been studied.The purpose of the study is to explore the expression of RECK in patients with COPD and the effect on neutrophil function,so as to provide a new scientific basis for the prevention and treatment of COPD.Methods:Fifty COPD patients and fifty healthy controls were recruited.The expression and localization of RECK protein in lung tissue were detected by immunohistochemical staining.The levels of RECK in plasma and sputum supernatant and the inflammatory factors（IL-6,IL-8）in plasma were detected by ELISA.Pearson correlation analysis was used to analyze the correlation between plasma RECK and pulmonary function（FEV₁%predicted）or the inflammatory factors（IL-6 and IL-8）.In vitro,cigarette smoke extract（CSE）was used to stimulate BEAS-2B,a human bronchial epithelial cell line.The expression of RECK protein in the supernatant was detected by ELISA.And RT-PCR was used to detect the levels of IL-6,IL-8 and RECK m RNA.Finally,human peripheral blood neutrophils were isolated by Ficoll-Hypaque and stimulated by CSE combined with rh RECK（human recombinant RECK protein）.The apoptosis and migration of neutrophils were detected by flow cytometry and Transwell assay.Besides,the expression of IL-6,IL-8 and MMP-9 m RNA were detected by RT-PCR.The levels of IL-6 and IL-8 in cell culture supernatant were detected by ELISA.The expression of MMP-9 protein was detected by western blotting.Results:1.The expression and localization of RECK in human lung tissue:RECK was mainly expressed in airway epithelium,and the expression of RECK in airway of COPD patients was significantly lower than that of healthy controls.2.The expression of RECK in plasma and sputum supernatant of COPD patients and its correlation with pulmonary function and inflammatory factors:the concentration of RECK in sputum supernatant of COPD patients was significantly lower than that of healthy controls（0.66±0.59 vs 3.36±3.03 ng/ml,P<0.001）,the concentration of RECK in plasma of COPD patients was also lower than that of healthy controls（1.18±0.59 vs.1.66±0.79ng/ml,P<0.001）.The level of plasma RECK in COPD patients was positively collected to FEV1%pred（r=0.458,P<0.001）,but it had negative correlation between the expression of IL-6 and IL-8（r=-0.386,-0.437,P<0.01）.3.Effect of CSE stimulation on the expressions of RECK,IL-6 and IL-8 in BEAS-2B cells:The levels of IL-6 and IL-8 m RNA in the“CSE group”were significantly higher than that in the control group（P<0.01,0.001）.But RECK m RNA and protein levels in the CSE group were lower than that in the control group（P<0.05,0.001）.4.Effect of recombinant human RECK protein on neutrophil apoptosis,migration and inflammation induced by CSE:The apoptosis rate of neutrophil was higher,while the“CSE group”was controlled with the“control group”.But there was no statistical difference in neutrophil apoptosis rate of“rh RECK pre-stimulated group”and“CSE group”.The number of migrated neutrophils in“CSE group”was significantly higher than that in“control group”,while the number of migrated neutrophils in“CSE+30μg/ml RECK”and“CSE+60μg/ml RECK”groups was significantly lower than that in“CSE group”（P<0.01,P<0.001）,suggesting that rh RECK reduced CSE-induced neutrophil migration.The expressions of IL-6 and IL-8 in neutrophils and culture supernatants in“CSE group”were significantly higher than those in“control group”（P<0.01,P<0.05）.And compared with the“CSE group”,the expressions of IL-6 and IL-8 in neutrophils and culture supernatants were both decreased in“CSE+RECK group”（P<0.05）,suggesting that RECK inhibited the increase of IL-6 and IL-8 in neutrophils induced by CSE.The MMP-9 m RNA and protein levels of neutrophils in“CSE group”were significantly increased,compared with the“control group”（P<0.001,P<0.01）,and the expression of MMP-9 in neutrophils in“CSE+RECK”group was significantly lower than that in“CSE group”（P<0.01）,suggesting that RECK inhibits CSE-induced MMP-9 production in neutrophils.Conclusions:1.The expressions of RECK in COPD patients（lung tissue,plasma,sputum）and CSE-stimulated BEAS-2B are decreased,and the expression of RECK protein in plasma of COPD patients is correlated with pulmonary function and inflammation.2.rhRECK can inhibit CSE-induced neutrophilic inflammation and neutrophil migration,which may be related to its regulation of MMP-9.