The Role And Mechanism Of Chemokine Receptor 1(CCR1) In Smoking-related COPD Airway Inflammation

Chronic obstructive pulmonary disease is a chronic airway inflammatory disease characterized by irreversible airflow limitation.Patients with COPD are admitted to hospital for multiple acute exacerbations every year,which brings economic burden to the country and patients,and repeated acute exacerbations can also lead to Deterioration of lung function makes patients prone to shortness of breath and unable to go out or even staying in bed for a long period of time.This aggravates the quality of life,and even some patients experience depression and suicidal tendencies.Smoking-induced airway inflammation is the main reason for the acute exacerbation of COPD,but the mechanism of how smoking causes airway inflammation in COPD has not been fully elucidated.Chemokines can bind to receptors on the surface of inflammatory cells such as macrophages and neutrophils,thereby recruiting the adhesion and homing of inflammatory cells,inducing their activation and amplifying the inflammatory response.CCR1 is a CC chemokine receptor,which has the functions of mediating leukocyte migration,Ca2+ mobilization,increased extracellular acidification rate,and CD11b expression.CCR1 is involved in the process of lung inflammation,tumor,cardiovascular disease and other diseases,but CCR1 smoking COPD little studied aspects of airway inflammation,the subject of hope through clinical research,cell and experimental animal model studies,discovered and confirmed CCR1 involved in COPD airway inflammation process,and to clarify its mechanisms,designed to COPD diagnosis,and Evaluation of treatment and efficacy,providing new molecular markers and potential therapeutic targets.Objective:To investigate the role and mechanism of chemokine receptor 1(CCR1)in airway inflammation in smoking-related COPD.Method:1.clinical samples:collection COPD bronchial mucosa biopsies and peripheral blood of patients and controls,bronchial mucosa was observed by immunohistochemical methods and immunofluorescence CCR1 differential expression,RT-the PCR method for detecting peripheral blood CCR1 of mRNA expression levels with ELISA detection in blood levels of inflammatory cytokines in peripheral blood method,and peripheral blood CCR1 of mRNA expression and correlation with pulmonary function and inflammatory factors.2.In vitro cell experiment:prepare cigarette smoke extract(CSE),select the appropriate CSE concentration(5%)by CCK-8 method.siRNA transfection method and CCR1 antagonist BX471 pretreatment method were used to construct lowexpressing CCR1 mouse alveolar macrophage MH-S cell line,and plasmid transfection method to construct high-expressing CCR1 mouse alveolar macrophage MH-S cell line.CSE stimulated mouse alveolar macrophages(MH-S)of different groups for 24h.The experiment is divided into 8 groups:① normal control group,② empty plasmid group,③ CCR1 overexpression group,④ CCR1 siRNA NC interference group,⑤CCR1 siRNA interference group,⑥CCR1 overexpression group+CSE group,⑦CCR+siRNA interference group+CSE group,⑧ CCR1 antagonist BX471+CSE group.Detect the mRNA and protein expression of mouse alveolar macrophages CCR1 and signaling pathway JAK2,STAT3,NFκB by RT-PCR and WB methods,and detect the chemokine macrophage inflammatory protein in the cell supernatant by ELISA-1(MIP-1 β)and the expression levels of inflammatory factors IL-6 and TNF-α.3.COPD mouse model research:using cigarette smoking and air tube instillation of LPS method to establish a tobacco-induced COPD mouse model.The experiment is divided into 5 groups:①normal mice+normal saline(100ul),②COPD mice+empty lentivirus(5×10^8TU/ml,100ul),③COPD mice+overexpressing CCR1 lentivirus(5×10^8TU/ml,100ul),④ COPD mice+shRNA-CCR1 lentivirus(5×10^8TU/ml,100ul),⑤ COPD mice+intravenous injection of CCR1 antagonist BX 471(30mg/kg).Bronchial mucosal tissues and alveolar lavage fluid of each group of mice were taken,HE staining method was used to observe the pathological changes of bronchial mucosa,immunofluorescence method was used to observe the expression of CCR1 in bronchial mucosa,TUNEL method was used to detect cell apoptosis in bronchial mucosa,RTPCR and WB was used to detect the mRNA and protein expression of CCR1,JAK2,STAT3 and NF-κB in bronchial mucosa,and the expression of chemokine MIP-1β and inflammatory factors IL-6 and TNF-α in alveolar lavage fluid were detected by ELISA.Result:1.35 COPD patients,all have a history of smoking,(42.42±3.18)package years;16 cases in the control group,all have no history of smoking.There was no statistical difference between the two groups in gender and age(P>0.05).The COPD group’s FEV 1/FVC and FEV 1%pred were significantly lower than those in the control group(P<0.01).Immunohistochemistry and immunofluorescence showed that the expression of CCR1 in the bronchial mucosa of COPD patients was significantly increased.The expression level of CCR1 mRNA in the peripheral blood of COPD patients was significantly higher than that of the control group.CCR1 mRNA in peripheral blood is significantly negatively correlated with patients’ FEV1%pred,and significantly positively correlated with IL-8 and TNFα levels.The chemokine MIP-1β,inflammatory factors IL-6,IL-8,and TNF-α were significantly higher than those in the control group.2.The cell experiments,the CSE stimulation 24h after normal NH-S as compared to cell lines with low expression CCR1 in MH-S cell line CCR1 and downstream passage the JAK2,of STAT3,of NF-κB of mRNA expression and protein significantly reduced,cell supernatants chemokine the MIP-1β,inflammatory cytokines of IL-.6,the TNF-reducingα expression,and high expression of CCR1 in MH-S cell line CCR1 and downstream passage the JAK2,of STAT3,of NF-κB of mRNA and protein The expression of chemokines MIP-1β,inflammatory factors IL-6,and TNF-α increased significantly.3.In the mouse model experiment,compared with the normal control group,the lung tissue of the bronchial mucosa of the empty virus COPD model mice showed inflammatory cell infiltration,airway remodeling and emphysema,and the expression of CCR1 and apoptosis were significantly increased.high.Overexpressing CCR1 of COPD mouse model of the bronchial mucosa of lung tissue inflammation and emphysema increased,CCR1 expression and apoptosis significantly increased.In COPD model mice with low CCR1 expression(shRNA-CCR1 transfection and BX471 pretreatment),airway inflammation and emphysema were reduced,and CCR1 expression and apoptosis were also inhibited;CCR1 overexpression COPD model was small bronchial mucosa in the rat lung tissue CCR1,the JAK2,of STAT3 the mRNA and protein expression was significantly increased in CCR1 low expression(shRNA designed CCR1-transfection and BX471 pretreatment)of COPD mouse model of bronchial mucosa CCR1,the JAK2,of STAT3 the mRNA and protein Decrease in expression.In the mouse alveolar lavage fluid,CCR1 overexpression COPD model mouse chemokine MIP-1β,inflammatory factors IL-6,TNF-α expression increased,while in CCR1 low expression(shRNA-CCR1 transfection and BX471 pretreatment Treatment)COPD model mouse chemokine MIP-1β,inflammatory factors IL-6,TNF-α expression decreased.Conclusion:1.chemokine receptor.1(CCR1)related to smoking in COPD patients airway mucosa and peripheral blood expression,CCR1 and smoking-related COPD correlated with negative lung function in patients with peripheral blood of the TNF-[alpha],of IL-.6 and MIP-1β is positively correlated,suggesting that CCR1 is involved in the inflammatory response of the COPD airway.2.After cigarette smoke extract(CSE)stimulates mouse alveolar macrophages MHS,the expression level of CCR1 increases,the secretion of related inflammatory factors and chemokines increases,and the inflammatory response increases.The mechanism may be related to the activation of JAK/STAT The signal pathway is related.3.The experimental study of tobacco-induced COPD mouse model further verified that CCR1 participates in the airway inflammatory reaction process of COPD and plays a pro-inflammatory effect.Inhibiting the expression of CCR1 or using the CCR1 antagonist BX471 can inhibit the airway inflammatory reaction of COPD.In this study,clinical research,research cell experiments and animal models,confirmed the CCR1 in COPD important role in airway inflammation processes play a proinflammatory and clearly its mechanism may be through activation of JAK/STAT related pathways for COPD diagnosis of,Treatment and efficacy evaluation provide new molecular markers and potential therapeutic targets.