Study On The Effect Of Soluble Receptor For Advanced Glycation End-products In Cigarette Smoke-induced COPD In Mice

Background:In recent years,attention has been paid to the role of chronic inflammation in the development of Chronic Obstructive Pulmonary Disease（COPD）.Smoking is the most important predisposing factor of COPD.Studies have suggested that Cigarette Smoke（CS）can promote the activation of various inflammatory signals in lung through AGE-RAGE/s RAGE axis,thus mediating the continuous airway inflammation and emphysema of COPD.However,studies on s RAGE levels in smokers have mixed results,and how CS affects circulating s RAGE levels in COPD patients remains unclear.Objective:The purpose of this study was to investigate the role of s RAGE in the pathogenesis of COPD induced by cigarette smoke exposure.（1）To dynamically observe the changes of s RAGE levels in bronchoalveolar lavage fluid of mice from early exposure to cigarette smoke to the development of COPD,and to provide theoretical basis for the feasibility of s RAGE as a biomarker for COPD diagnosis;（2）The lung function,degree of emphysema and inflammatory response in each group were detected,and the potential relationship between s RAGE and pulmonary function decline,emphysema and inflammatory response in the mouse model of cigarette smoke exposure was evaluated.Methods:（1）48 6-weeks-old C57BL/6J male mice were randomly divided into smoke-exposed group（SE group）and control group.Mice in the SE group were placed in a self-made glass smoking box and lit 10 cigarettes for 1h each time,twice a day.The mice in the SE group were exposed to CS at 3,7,15,30,60,90 and 120 days,with 6 mice in each group.The mice in control group were exposed to fresh room air at the same time.Mice were weighed for the last time 24 hours after the last smoking,anesthetized with sodium pentobarbital,the lung function of the mice was measured,and bronchoalveolar lavage was performed.The supernatant was frozen at-80℃after centrifugation for use.The cell precipitates were resuspended in PBS,discarded,stained,and the cells（including neutrophils,macrophages and lymphocytes）were sorted and counted under the microscope.The lung tissues of mice were collected,and the pathological changes of lung tissues in each group were observed by HE staining.The morphological quantitative analysis of lung tissues was performed to evaluate the degree of lung tissue damage（emphysema degree）.（2）Enzyme-Linked Immunosorbent Assay（ELISA）was used to detect the levels of inflammatory factors in BALF supernatant of each group.These cytokines included Interleukin-1β,Interleukin-6 and Tumor Necrosis Factor-α.ELISA was used to measure the level of s RAGE in BALF supernatant.（3）Pearson correlation analysis was used to study the correlation between s RAGE and the degree of reduced lung function and pulmonary inflammation in the COPD model group.Results:（1）After 90 days of cigarette smoke exposure,FEV_0.1,FEV_0.1/FVC and PEF of mice were significantly lower than those of control group,Rn was significantly increased;A large number of alveolar dilation and fusion,alveolar wall structure disorder and inflammatory cell infiltration were observed in lung tissues.The MLI,PAA were significantly higher than those in the control group,and the MAN was significantly reduced.The number of macrophages,neutrophils and lymphocytes in BALF was significantly higher than that in the control group,and the expressions of inflammatory mediators IL-1β,IL-6 and TNF-αwere significantly increased.All the differences were statistically significant（P<0.05）.These results suggest that a mouse model of COPD was successfully established after 90 days of CS exposure.（2）Compared with control group,s RAGE increased significantly in the early stage of CS exposure（days 7-15）,and the number of macrophages and the levels of inflammatory factors in BALF also showed a transient upward trend（P<0.05）.The expression of s RAGE gradually decreased during the time course of CS exposure,and decreased significantly after the formation of COPD compared with the control（P<0.01）.（3）In the 120-day CE group,the s RAGE level was positively correlated with FEV_0.1（r=0.955,P=0.003）,FEV_0.1/FVC（r=0.969,P=0.002）.and it was negatively correlated with the number of neutrophils（r=-0.948,P=0.004）and IL-1β（r=-0.869,P=0.025）.Conclusion:In the process of the occurrence and development of chronic obstructive pulmonary disease induced by cigarette smoke exposure,the level of s RAGE in bronchoalveolar lavage fluid showed a dynamic change of first increase and then decrease.The expression of s RAGE increased in the early stage of smoke exposure and played a transient pro-inflammatory role.With long-term exposure to cigarette smoke,the progression of emphysema,the inflammatory response is gradually aggravated in lung,and the expression of s RAGE is significantly decreased,and its reduction degree is closely related to the degree of reduced lung function and inflammation（the severity of COPD）.