| BackgroundChronic obstructive pulmonary disease(COPD)is a systemic disease that seriously endangers human health,mainly involving the respiratory system,causing a huge social,economic and health burden worldwide.As a developing country,China has a more severe COPD disease situation,with high morbidity and mortality rates.At present,the pathogenesis of COPD is unknown,causing difficulties in prevention and treatment.COPD is characterized by irreversible airflow limitation,which is related to the abnormal response of airways and lung tissues exposed to cigarette smoke and other harmful gases or particulate matter.Smoking is the most important risk factor for COPD.The main lesion of COPD is the small airway with an inner diameter of less than 2mm.During the onset of the disease,inflammation and fibrosis of the small airway will cause the airway wall to thicken and narrow the lumen.In addition,excessive mucus secretion will eventually lead to airway occlusion and emphysema is formed.Small airway fibrosis plays a key role in the occurrence of COPD airway occlusion,but its specific mechanism is not clear.In recent years,studies have shown that epithelial-mesenchymal transition(EMT)plays an important role in the process of small airway fibrosis.Under the action of many factors,epithelial cells lose polarity,undergo morphological and functional changes,and transform into mesenchymal cell phenotypes.EMT plays an irreplaceable function in various physiological and pathological events such'as embryonic development,damage repair,fibrosis formation,tumor invasion and metastasis,etc.In recent years,researchers have found evidence of the existence of airway EMT in COPD,and believed that it was related to small airway fibrosis and airway occlusion,but the researches were not deep enough,especially regarding the predisposing factors and molecular mechanisms of airway EMT.Current researches show that cigarette smoke exposure(smoking)is a clear predisposing factor for airway EMT,but the components of cigarette smoke are extremely complex,and after smoking is stopped,EMT can continue to progress,indicating that in addition to the exogenous factor of cigarette smoke exposure,there are still unknown endogenous factors driving EMT.On the other hand,the molecular mechanism of EMT varies with cell types and microenvironments,although TGF-?/Smads,Wnt/?-catenin,uPAR/PI3K,Hedgehog and other signal pathways may be involved in EMT,only TGF-? and uPAR-related pathways are clear in COPD.Whether other signal transduction pathways are involved is still poorly understood.The airway epithelium is the innate immune barrier of the respiratory system.It is first exposed to harmful substances such as cigarette smoke and triggers an inflammatory response,which initiates the pathogenesis of COPD.In addition to physical defenses such as epithelial integrity and mucociliary clearance system,epithelial cells can secrete a variety of biologically active substances including endogenous antimicrobial peptides(AMPs),which participate in the inflammation injury and repair.Endogenous AMPs include two families of defensins and Cathelicidin.The Cathelicidin family has only one member in humans,hCAP18(its active fragment is LL-37),and in mice,there is also only one member,CRAMP(Cathelin-related antimicrobial peptide).The distribution of LL-37 in human tissues is mainly concentrated in cells in direct contact with the external environment such as epithelial cells in the respiratory tract,digestive tract,and urinary tract,as well as inflammatory cells such as neutrophils and macrophages.In addition to antimicrobial effects such as anti-bacteria,fungi and viruses,LL-37 also has many functions such as chemotaxis,immune regulation and immune stimulation,regulation of apoptosis,and injury healing.Previous studies have shown that the synthesis and secretion of LL-37 in the airways of COPD patients are increased,and high levels of LL-37 can be detected in small airway epithelial cells,induced sputum,and bronchoalveolar lavage fluid.These highly expressed LL-37 can participate in the pathogenesis of COPD by inducing airway inflammation,mucus hypersecretion and the formation of emphysema.In this process,the activation of airway epithelial cells occupies an important position.Studies in vitro have shown that both cigarette smoke extract(CSE)and LL-37 can activate airway epithelial cells to promote the release of inflammatory factors such as IL-8 and biologically active substances such as mucus protein MUC5AC.The activation process involves transactivation of epidermal growth factor receptor(EGFR).Specifically,LL-37 first activates TNF-? converting enzyme(TACE)on the surface of airway epithelial cells,and then shears the membrane-anchored proTGF-? extracellular domain to induce soluble TGF-a release,as a ligand to bind to EGFR and activate EGFR and downstream signaling pathways,that is,LL-37 has the potential to activate TACE/TGF-?/EGFR signaling pathways in airway epithelial cells.Studies in tumor cells have found that the TACE/TGF-?/EGFR pathway may be a potential molecular mechanism of EMT.For example,in prostate cancer and liver cancer cells,EMT mediated by the TACE/TGF-?/EGFR pathway can promote tumor cell invasion and metastasis.But in other cell types,such as airway epithelial cells,whether the TACE/TGF-?/EGFR pathway can mediate EMT is still unknown.In view of(1)the small airway epithelium of COPD patients highly expresses LL-37,(2)LL-37 can activate airway epithelial cells through the TACE/TGF-?/EGFR pathway,and(3)the TACE/TGF-?/EGFR pathway may have The potential for mediating EMT,(4)COPD patients have smoking-related small airway EMT,it can be speculated that during the pathogenesis of COPD,the high expression of LL-37 in airway epithelial cells may mediate the EMT in small airways caused by cigarette smoke exposure through the TACE/TGF-?/EGFR pathway,promoteing small airway fibrosis and COPD progression.In order to verify this hypothesis,this study explored the mediating role of LL-37 in the small airway EMT of COPD caused by tobacco smoke and the signal pathways that may be involved in the three levels of human histopathology,in vitro cell experiments and animal models.Objective1.To observe the relationship between Cathelicidin and EMT in the small airway epithelium of COPD patients;2.To study the molecular mechanisms underlying the induction of EMT by Cathelicidin in vitro;3.To testify the effects of Cathelicidin on EMT and potential mechanisms in vivo.Methods1.Human Histopathology1.1 Inclusion of research objects and data collectionAdult patients who underwent lobectomy due to lung tumors were recruited and divided into three groups based on preoperative lung function tests:normal lung function non-smoker group(normal non-smoker group),normal lung function smoking group(normal smoking group)and COPD group,The number of cases were 10,9 and 9 respectively.Before surgery,record gender,age,smoking history(pack years),forced expiratory volume in one second(FEV1)and FEV1/FVC(FEV1/forced vital capacity).The diagnosis of COPD is based on GOLD(2017)criteria.1.2 Lung tissue specimen collection and stainingTo collect the lung lobes tissues of the three groups of study subjects who have undergone surgery,and take the materials 5cm beyond the edge of the tumor lesion.Masson staining and immunohistochemical staining were performed after serial sections to compare the morphological changes of the small airway in the three groups of pathological specimens,as well as the expression of LL-37 and EMT markers E-cadherin and vimentin in the small airway epithelium.2.In vitro experiments2.1 In vitro culture of human bronchial epithelial cell line NCI-H2922.2 Intervention grouping and examination2.2.1 In order to detect the effect of cigarette smoke on the expression of LL-37 in airway epithelial cells,the cultured cells were divided into the following intervention groups:(1)normal control group;(2)CSE stimulation group.The expression of LL-37 was examined by immunofluorescence.2.2.2 In order to detect the effect of LL-37 siRNA on airway epithelial cells,the cultured cells were divided into the following intervention groups:(1)normal control group;(2)LL-37 siRNA interference group;(3)control siRNA treatment group.The expression of LL-37 was examined by immunofluorescence.2.2.3 In order to detect the effects of LL-37 on airway epithelial EMT,the cultured cells were grouped according to the following interventions:(1)normal control group;(2)exogenous LL-37 synthetic peptide stimulation group.The expression of EMT markers E-cadherin and vimentin were examined by immunofluorescence.2.2.4 In order to detect the signal pathway of LL-37-induced EMT,the cultured cells were divided into the following groups according to the interventions:(1)normal control group;(2)exogenous LL-37 synthetic peptide stimulation group;(3)EGFR inhibitor(AG1478)pretreatment+exogenous LL-37 synthetic peptide stimulation group;(4)TGF-? neutralization antibody(TGF-? Abs)pretreatment+exogenous LL-37 synthetic peptide stimulation group;(5)TACE inhibitor(TAPI-1)pretreatment+exogenous LL-37 synthetic peptide stimulation group.The expression of EMT markers E-cadherin and vimentin were examined by immunofluorescence.2.2.5 In order to detect the effects of CSE on airway epithelial EMT,the cultured cells were grouped according to the following interventions:(1)normal control group;(2)CSE stimulation group.The expression of EMT markers E-cadherin and vimentin were examined by immunofluorescence.2.2.6 In order to detect the signal pathway of CSE-induced EMT,the cultured cells were divided into the following groups according to the interventions:(1)normal control group;(2)CSE stimulation group;(3)EGFR inhibitor(AG1478)pretreatment+CSE stimulation group;(4)TGF-? neutralization antibody(TGF-? Abs)pretreatment+CSE stimulation group;(5)TACE inhibitor(TAPI-1)pretreatment+CSE stimulation group.The expression of EMT markers E-cadherin and vimentin were examined by immunofluorescence.2.2.7 In order to investigate the role of LL-37 in CSE-induced EMT,the cultured cells were divided into the following groups:(1)normal control group;(2)CSE stimulation group;(3)LL-37 siRNA pretreatment+CSE stimulation group.The expression of EMT markers E-cadherin and vimentin were examined by immunofluorescence.3.Animal experiments3.1 Grouping of experimental miceThirty 6-week-old BALB/c mice were randomly divided into 6 groups,5 in each group.According to the different intervention measures,they were:normal control group(Control),COPD group(COPD),COPD+lentiviral vector negative control group(COPD+NC),COPD+CRAMP down-regulation group(COPD+CRAMP-),CRAMP up-regulation group(CRAMP+),COPD+CRAMP up-regulation group(COPA+CRAMP+).The normal control group did not apply any intervention measures;the COPD group used the cigarette smoke exposure method to establish a COPD mouse model,and the modeling time was 8 weeks;the up-regulation and down-regulation of CRAMP were transfected with corresponding lentiviral vectors to transfect mouse airway epithelial cells to change the expression level of CRAMP.3.2 Lentiviral vector transfection to miceThe lentiviral vector was used to enter the mouse airway to transfect the airway epithelial cells by the nose drop method to achieve the up-regulation or down-regulation of CRAMP expression.During the exposure to cigarette smoke,the first dose of lentiviral vector was given intranasally on the first day,then once every 10 days,and the last dose on the 50th day,for a total of 6 doses,with each dose of 108 TU.3.3 Fluorescent quantitative PCRAfter the intervention,the lung tissues of each group of mice were homogenized and then fluorescent quantitative PCR was performed to detect the changes in CRAMP mRNA levels to verify the effect of lentiviral transfection.3.4 Mouse lung tissue stainingHE staining,Masson staining and immunofluorescence staining were performed on mouse lung tissue,and the airway morphology changes of each group of specimens were compared,and the expression and distribution of CRAMP,E-cadherin,and vimentin were compared.3.5 Western blot detection of signal pathway proteinsAfter homogenizing the lung tissue of each group of mice,Western blot was performed to evaluate the protein expression levels of TACE,TGF-? and EGFR.4.Statistical analysisData statistical analysis adopts SPSS 17.0 software.Normally distributed data with uniform variances are compared between groups by analysis of variance;non-normally distributed data between groups are compared by non-parametric testing methods such as Kruskcal-Wallis H test or Mann-Whitney U test.P<0.05 was considered statistically significant.Results1.The pathology of human airway1.1 Comparison of general information and lung function parametersThe COPD group and the normal smoking group were older than the normal non-smoking group(both P<0.01);the smoking index difference between the normal smoking group and the COPD group was not significant(P>0.05);the FEV1/FVC of the COPD group was significantly lower than normal smoking group and normal non-smoker group(both P<0.01).1.2 Pathological analysis of human lung tissueThe Masson staining results of the lung tissues of the three groups showed that compared with the normal non-smoking group,the subepithelial blue staining area of the small airway in the normal smoking group and the COPD group increased,indicating that the subepithelial collagen deposition increased and the presence of small airway fibrosis.Immunohistochemistry staining results showed that,compared with the normal non-smoking group,the expressions of LL-37 and vimentin in the small airway epithelium of the normal smoking group and the COPD group were significantly increased(both P<0.01),and the expressions of E-cadherin were significantly decreased(both P<0.01),indicating that the expression of LL-37 in the airway epithelium of smokers(regardless of COPD or not)was accompanied by airway EMT.2.In vitro experiments2.1 CSE induced the expression of LL-37 in airway epithelial cellsAfter stimulation with CSE(5%)for 24 hours,LL-37 immunofluorescence staining was performed on airway epithelial cells.The results showed that the background expression of LL-37 was seen in the epithelial cells of the normal control group,and the expression of LL-37 in the CSE stimulation group was significantly increased,indicating that cigarette smoke exposure could specifically induce the expression of LL-37 in airway epithelial cells.2.2 Effect of LL-37 siRNA transfection on the interference of LL-37 expression in airway epithelial cellsImmunofluorescence staining results showed that the expression of LL-37 in the LL-37 siRNA interference group was significantly lower than that in the normal control group,indicating that LL-37 siRNA could successfully down-regulate the expression of LL-37 in airway epithelial cells,and the interference was effective.2.3 The induction of EMT by LL-37 in airway epithelial cellsAfter stimulating with LL-37 synthetic peptide(5?g/mL)for 24 hours,the fluorescence expression intensity of E-cadherin in airway epithelial cells was significantly lower than that of the normal control group,while the fluorescence expression intensity of vimentin was significantly higher than that of the normal control group,indicating that LL-37 could induce EMT in airway epithelial cells.2.4 LL-37 induced EMT of airway epithelial cells involving the TACE/TGF-?/EGFR signaling pathwayAfter pretreated with TAPI-1(l0?mol/L),TGF-? Abs(1?g/mL)and AG 1478(2?mol/L)for 30 minutes,the epithelial cells were then stimulated with LL-37 synthetic peptide(5?g/mL)for 24 hours.The E-cadherin and vimentin were examined.The results showed that after blocking TACE,TGF-?,and EGFR,both the down-regulation of E-cadherin expression and the up-regulation of vimentin expression induced by LL-37 were inhibited,indicating that the TACE/TGF-?/EGFR signaling pathway played an important role in EMT induced by LL-37 in airway epithelial cells.2.5 The induction of EMT byvCSE in airway epithelial cellsAfter stimulating with CSE(5%)for 24 hours,the fluorescence expression intensity of E-cadherin in airway epithelial cells was significantly lower than that of the normal control group,while the fluorescence expression intensity of vimentin was significantly higher than that of the normal control group,indicating that CSE could induce EMT in airway epithelial cells.2.6 CSE induced EMT of airway epithelial cells involving the TACE/TGF-?/EGFR signaling pathwayAfter pretreated with TAPI-1(10?mol/L),TGF-? Abs(1?g/mL)and AG1478(2?mol/L)for 30 minutes,the epithelial cells were then stimulated with CSE(5%)for 24 hours.The E-cadherin and vimentin were examined.The results showed that after blocking TACE,TGF-?,and EGFR,both the down-regulation of E-cadherin expression and the up-regulation of vimentin expression induced by CSE were inhibited,indicating that the TACE/TGF-?/EGFR signaling pathway played an important role in EMT induced by CSE in airway epithelial cells.2.7 The mediating role of LL-37 in CSE-induced EMT in airway epithelial cellsAfter interfered with LL-37 siRNA to down-regulate the expression of LL-37,the epithelial cells were stimulated with CSE(5%)for 24 hours.CSE-induced down-regulation of E-cadherin expression and up-regulation of vimentin expression could be inhibited by LL-37 siRNA,indicating that LL-37 played a mediating role in CSE-induced EMT of airway epithelial cells.3.Animal experiments3.1 Pathological results of mouse lung tissueThe HE staining and Masson staining of mouse lung tissues showed that there were significant subepithelial collagen deposition and airway fibrosis in the COPD group(P<0.05).After the airway was locally down-regulated the expression of CRAMP,collagen deposition and airway fibrosis were significantly reduced(P<0.05).3.2 Fluorescence quantitative PCR resultsThe fluorescence quantitative PCR detection of mouse lung tissues showed that compared with the normal control group,CRAMP mRNA levels in CRAMP+ group mice were significantly increased(P<0.01);compared with COPD group,CRAMP levels in COPD+CRAMP-group mice The mRNA level was significantly reduced(P<0.01),and the CRAMP mRNA level of mice in the COPD+CRAMP+group was significantly increased(P<0.01).It shows that the corresponding lentivirus transfection can successfully up-regulate or down-regulate the expression of airway CRAMP at the transcriptional level.3.3 Results of immunofluorescence staining(1)Compared with the normal control group,the expression of CRAMP in the airway epithelium of the COPD group was significantly increased,the expression of E-cadherin was significantly decreased,and the expression of vimentin was significantly increased(all P<0.01),indicating that cigarette smoke exposure can induce the up-regulation of CRMAP in the tract epithelium and the occurrence of EMT.(2)Compared with the normal control group,the CRAMP expression in the airway epithelium of the CRAMP+ group was significantly increased,the expression of E-cadherin was significantly reduced,and the expression of vimentin was significantly increased(all P<0.01),indicating that the airway epithelial CRAMP was overexpressed and could induce the occurrence of EMT.(3)Compared with the COPD group,the expression of CRAMP in the airway epithelium of the COPD+CRAMP-group was significantly reduced,the expression of E-cadherin was significantly increased,and the expression of vimentin was significantly reduced(all P<0.01),indicating that the CRAMP of the airway epithelium may be involved in the occurrence of EMT induced by exposure to cigarette smoke.(4)Compared with the COPD group,the expression of CRAMP in the airway epithelium of the COPD+CRAMP+group was significantly increased,the expression of E-cadherin was significantly decreased,and the expression of vimentin was significantly increased(all P<0.01),indicating that the airway epithelium was highly expressed CRAMP can act synergistically with cigarette smoke to induce EMT.3.4 Protein expression levels of signal molecules Western blot test results show:(1)Compared with the normal control group,the expression levels of TACE,TGF-?and EGFR in the lung tissues of the COPD group were significantly increased(P<0.05),indicating that cigarette smoke exposure can induce high expression of TACE,TGF-? and EGFR,and activate TACE/TGF-?/EGFR pathway.(2)Compared with the normal control group,the expression levels of TACE,TGF-?and EGFR in the lung tissues of the CRAMP+ group were also significantly increased(P<0.05),indicating that the upregulation of CRAMP expression in the airway epithelium can also induce high expression of TACE,TGF-? and EGFR,activating the TACE/TGF-?/EGFR pathway.(3)Compared with the COPD group,the expression levels of TACE,TGF-? and EGFR in the lung tissues of the COPD+CARMP-group were significantly reduced(P<0.05),indicating that CRAMP mediates the activation of the TACE/TGF-?/EGFR pathway induced by cigarette smoke exposure.(4)Compared with the COPD group,the expression levels of TACE,TGF-? and EGFR in the lung tissues of the COPD+CRAMP+group were significantly increased(P<0.05),indicating that CRAMP,which is highly expressed in the airway epithelium,can act synergistically with cigarette smoke to jointly activate TACE/TGF-?/EGFR pathway.ConclusionsCathelicidin overexpressed in airway epithelial cells could mediate airway EMT induced by cigarette smoke via activating TACE/TGF-?/EGFR signaling pathway to promote the pathogenesis of COPD. |
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