| IntroductionChronic obstructive pulmonary disease(COPD)is a common and frequently occurring disease that seriously endangers human health.Over the years,although a lot of attention and investment have been paid to the clinical and basic research of COPD,the effects are far from satisfactory.The reasons involve many aspects,among which,COPD is a highly heterogeneous disease,and its precise diagnosis method is still needed to be investigated and its pathogenesis is not clear,and precise individualized treatment and prevention cannot be carried out are important reasons.The GOLD-2021(Global initiative for chronic Obstructive Lung Disease)defines COPD as:"COPD is a common,preventable and treatable disease that is characterized by persistent respiratory symptoms and airflow limitation that is due to airway and/or alveolar abnormalities usually caused by significant exposure to noxious particles or gases." GOLD has undergone annual updates and several major revisions since its first release in 2001.However,the statement that COPD is characterized by "persistent respiratory symptoms and airflow limitation" has remained unchanged.Pulmonary function test is a prerequisite for the diagnosis of COPD,with FEV1/FVC<70%after inhalation of bronchodilators as the criterion of airflow limitation.As can be seen from the definition of COPD,the pathophysiological basis of the pathophysiological changes in airflow limitation is"airway and/or alveolar abnormalities".The pathological change of COPD including repeated airway inflammation-repair-remodeling and lung parenchyma destruction-emphysema.Airway disease and emphysema together lead to airflow limitation marked by a decrease in FEV1 and FEV1/FVC,which forms the basis of the heterogeneity of COPD.Although a decrease in FEV1/FVC can determine the presence of airflow limitation,it is difficult to obtain more information from a single FEV1/FVC in the identification of underlying causes such as different pathological changes.Second,emphysema is a pathological diagnosis,spirometry parameter alone cannot reflect the actual pathological changes.This study based on the pathological and physiological changes of "airflow limitation"in COPD.On the one hand,we investigate the value of non-invasive and repeatable impulse oscillometry(IOS)in the assessment of airflow limitation heterogeneity,in an attempt to provide help for accurate diagnosis of COPD.On the other hand,we aimed to investigate the role of bone morphogenetic protein 7(BMP7)in airway disease of COPD.It is divided into the Part one and part two.Part ? The value of impulse oscillometry in assessment of airflow limitation heterogeneity in COPDBackgroundIn the field of heterogeneity of COPD,the heterogeneity of airflow limitation has always been paid much attention.Airflow limitation is the critical problem in the diagnosis and evaluation of COPD,and its pathological basis is airway diseasestructural remodeling-airway stenosis and lung tissue destruction-emphysema formation.The weight of airway disease and emphysema varies greatly among different COPD individuals,which is an important component of the heterogeneity of COPD.In the real world,airway disease dominant and emphysema-dominant phenotypes of COPD patients show different clinical characteristics.The study of Celli showed that emphysema-dominant phenotype COPD patients had a faster rate of decline in lung function,BMI and body components,with more acute exacerbations and a higher mortality rate.The assessment of the heterogeneity of COPD airflow limitation is not only helpful to deepen the understanding of the disease and predict the disease progression,but also helpful to carry out individualized treatment and improve the effectiveness of COPD treatment.For example,airway disease-dominant COPD patients respond well to ICS,while emphysema-dominant COPD patients often benefit more from non-pharmacological treatments such as pulmonary rehabilitation.At present,FEV1/FVC and FEV1%,which are widely used in clinical practice,can only evaluate the severity of airflow limitation,but cannot assess the heterogeneity of airflow limitation.They are unable to distinguish the weight of emphysema and airway disease in airflow limitation.Although quantitative CT is widely used to evaluate the heterogeneity of airflow limitation at present,its extensive clinical application is limited due to its need for professional software,complex process,high cost and radiation risk.In addition,although chest CT has advantages in the assessment of pulmonary structural lesions,it does not fully reflect patients' respiratory physiological changes.For example,some studies have shown that although some people have emphysema on chest CT,they do not have respiratory symptoms and airflow limitation.Therefore,it is of important clinical value to explore a non-invasive,well-reproducible and effective method for evaluating the heterogeneity of airflow limitation without additional medical contamination.Impulse oscillometry(IOS)is a simple and non-invasive method to detect the distribution characteristics of various respiratory impedance in different parts of the respiratory system and different phases.It has the advantages of simple operation,low cost,good repeatability and low requirements for patient cooperation.The IOS has a good specificity for the measurement of respiratory impedance,and can distinguish the location,severity and respiratory dynamics characteristics of the airway obstruction,so it can bring more in-depth information on the reason of airflow limitation.If IOS parameters on inspiratory and expiratory phases were measured at the same time,these parameters may be used to determine the sources and variations of various resistance and reactance during breath in COPD patients,as well as the differences among different individuals and disease states,thus may contributing to the assessment of heterogeneity of airflow limitation.This study was aimed to investigate the value of IOS in evaluating the heterogeneity of airflow limitation in COPD.Materials and methodsThis is a case-control study.All the participants underwent basic information collection,IOS examination,PFTs and inspiratory chest CT scans.1.All the participants were recruited from July 2017 to November 2019 from Qilu Hospital of Shandong University,aged from 40 to 85.1)COPD patients:patients with stable COPD who met the GOLD 2016 diagnostic criteria were recruited.Acute exacerbation within 6 weeks and those with other severe respiratory diseases were excluded.2)Normal controls:the inclusion criteria were healthy people with normal PFTs without smoking history,respiratory symptoms and chronic respiratory disease.Patients with severe diseases of other systems were excluded.2.PFTs1)Spirometry parameters:FEV1,FEV1%,FVC,FVC%,FEV1/FVC;2)IOS parameters:? The whole-breath IOS parameters including:Z5:total respiratory impedance;R5:total airway resistance;R20:central airway resistance;X5:peripheral elastic reactance;Fres:resonance frequency.? The within-breath change of IOS parameters(respiratory phase impedance minus inspiratory phase impedance):?R5;?R20;?R5-R20;?X5;?fres.3.Chest quantitative CT examination:Emphysema index%LAA-950 were calculated using Airway Inspector software.4.The IOS parameters(including the whole-breath and within-breath change of IOS parameters)of COPD patients and the controls were compared.The correlation between IOS parameters and quantitative CT emphysema indexes were analyzed;Using%LAA-950,COPD patients were divided into emphysema-dominant and nonemphysema-dominant groups.ROC curve was used to explore the diagnostic efficacy of IOS parameters for COPD patients with emphysema-dominant phenotype.Results1.A total of 181 COPD patients and 106 controls were recruited in the study.There were no significant differences in age,sex ratio and BMI between the two groups(P>0.05).2.Comparison of IOS parameters between COPD patients and controls:there were significant differences in the whole-breath IOS impedance indexes(including R5,R20,R5-R20,X5 and Fres)between COPD patients and controls(P<0.05).There was no significant difference in ?R20 between COPD group and control group(0.11±0.34 VS 0.078±0.46 cmH2O/L/s,P=0.537),but there were significant differences in ?R5,?R5R20,?X5 and ?Fres between the two groups(P<0.05).3.Comparison of IOS parameters between emphysema-dominant and non-emphysemadominant COPD patients:in the whole-breath IOS parameters,only X5 showed significant difference between emphysema-dominant and non-emphysema dominant COPD patients(-3.33± 1.86 VS-2.49± 1.23 cmH2O/L/s,P=0.001),while R5,R20,R5R20 and Fres showed no significant differences between the two groups(P>0.05).For the Within-breath difference of IOS parameters,there were significant differences in?X5(-3.55±2.82 VS-1.61 ±2.78 cmH2O/L/s,P<0.001)and ?Fres(4.97±5.70 VS 3.03±5.06 Hz,P=0.0162)between the two groups,but no significant differences were shown in ?R5,?R20 and ?R5-R20(P>0.05).4.Correlation between IOS parameters and emphysema index:correlation analysis showed that%LAA-950 was significantly correlated with ?X5(r=0.545,P<0.001),X5(r=-0.229,P=0.002)and ?Fres(r=0.200,P=0.009),among which ?X5 showed the best correlation.5.The ROC curve analysis of ?X5,X5 and ?Fres in the diagnosis of emphysema predominant COPD:the area under the ROC curve(AUC)of ?X5 in judging emphysema-predominant COPD patients was 0.751(P<0.05),which was significantly higher than that of X5 and ?Fres.The sensitivity,specificity,positive predictive value and negative predictive value of ?X5 ?-2.66 cmH2O/L/s were 62.65%,93.62%,89.66%and 73.95%,respectively.Conclusion of Part ?The within-breath difference of X5(?X5)is an effective parameter for diagnosing emphysema-dominant phenotype of COPD,which can be used to evaluate the heterogeneity of airflow limitation in COPD.Part ? The role of BMP7 in airway disease in COPDBackgroundThe airway disease of chronic obstructive pulmonary disease(COPD)is the cause of persistent symptoms of COPD and the main pathological and anatomical basis of airflow limitation.Airway disease in COPD involves both large and small airways.Recurrent airway inflammatory damage and abnormal repairing lead to increasing collagen deposition,scar tissue formation,and airway structural remodeling,resulting in airway narrowing,loss of airway counts and airflow obstruction.The mechanisms of continuous airway inflammation,mucus hypersecretion,squamous epithelial metaplasia,and structural remodeling in COPD are far from being fully elucidated.It is generally recognized the pathogenesis of COPD is mainly a pathological process in which the respiratory tract is exposed to cigarette smoke,organic dust or atmospheric pollution,followed by the inflammatory response and damages,and airway-lung tissue remodeling is initiated while the inflammatory response is amplified and sustained.As the first barrier,the epithelial tissue of the respiratory tract is inevitably and unavoidably exposed to a variety of harmful stimuli causing damage,at the same time,a variety of factors such as chemokines,activating stimulating factors,growth factors and so on,can be produced in response to stimuli.Due to the abnormally high inflammatory response to stimuli,these factors are likely to be abnormally expressed in COPD patients and lead to a series of pathological changes.The study on the influence of these abnormal expressed factors in the bronchial epithelia of COPD patients may help to elucidate the pathogenesis of COPD and seek new prevention strategies.In our previous work,we found that the level of Bone Morphogenetic Protein-7(BMP7)in induced sputum was significantly higher in stable COPD patients than that in nonCOPD patients,and further immunohistochemistry results of lung tissue from COPD patients showed dramatically higher bronchial epithelial BMP7 expression than that of non-COPD patients.BMPs are cytokines that belong to the TGF-? superfamily which plays an important role in bone formation in vitro.More than 20 members of BMPs have been identified and BMP7 is a molecule that has attracted much attention.Recent studies have confirmed that the BMP family play a key role in osteogenesis,proliferation,differentiation and apoptosis.BMP7 is expressed in multiple tissues,such as Thymus,bone marrow,heart,kidney,lung and prostate.The function and mechanism of BMP7 in chronic pulmonary disease and pulmonary injury repair are not yet fully elucidated.Previous studies shown that the expression of BMP7 in airway epithelium of smokers is increased,and BMP7 may participate in the repair process of airway epithelium in mice.Our question is whether highly expressed BMP7 in airway is related to airway disease in COPD patients.If so,what role did BMP7 play?Our study involves the following three parts:1.The correlation between BMP7 expression in bronchial epithelial and clinical characteristics in COPD patients;2.The role of BMP7 in proliferation and differentiation of bronchial epithelial cells;3.The role and mechanisms of BMP7 in apoptosis in bronchial epithelial cells.Chapter 1 The BMP7 expression in bronchial epithelia of COPD and its correlation with clinical characteristicsObjectivesWe aimed to study the expression of BMP7 in lung tissue and the relationship of BMP7 expression with smoking history,airway numbers,airway wall thickness and FEV1.We also detected the levels of BMP7,IL-6,IL8 and other cytokines in induced sputum of COPD patients and examine the relationship between them.Materials and methods1.Histopathology study1)Study population:Patients who underwent lobectomy for lung lesions at Qilu Hospital of Shandong University between 2017.6 and 2020.11 were recruited,including COPD patients,smoking controls and non-smoking controls.Lung tissue specimens that were more than 5 cm away from the lesion margin were collected.The localization and expression of BMP7 in bronchial epithelial were detected by immunohistochemistry.2)Demographic,clinical and imaging data collection:PFTs parameters included FEV1,FVC,FEV1/FVC.Quantitative CT parameters:airway parameters:number of airways in the right upper lung(grade 4 to 9 bronchial counts),square root of airway wall area with a circumference of 10 mm(Pi10);(?)emphysema index(%LAA-950)3)The correlation of the BMP7 expression levels in bronchial epithelial and quantitative chest CT airway parameters,emphysema index and FEV1.2.Induced sputum studyWe collected induced sputum of COPD patients and normal controls and detected the expression of BMP-7,BMP-4,IL-6,IL-8/CXCL8,IL-1?,CCL18,and MMP-12 using Luminex liquid phase microarray technology.1)Participants:Patients who visited our hospital from June 2017 to November 2019 were divided into COPD Group and control group.Patients with stable COPD meeting the diagnostic criteria of GOLD2016 were included.Subjects aged 40-80 years who had no respiratory symptoms,no chronic respiratory diseases,and normal pulmonary ventilation function tests were recruited as controls.The sputum specimens were induced by hypertonic saline atomization for 10 min,and supernatant was obtained.2)Detection of BMP7 levels in induced sputum supernatant:Using Luminex liquid phase microarray technology,The levels of 7 cytokines in induced sputum supernatant were detected:BMP-7,BMP-4,IL-6,IL-8,IL-1?,CCL18,and MMP-12.3)The correlation between BMP7 and IL-1?,IL-6,IL8 and FEV1%in induced sputum supernatant was analyzed.Results1.Histopathology study1)There was no significant difference in the age of subjects among the 3 groups.2)Localization of BMP7 in bronchial epithelial:BMP7 was expressed in all layers of bronchial epithelial in large airways;in small airways,BMP7 was expressed in all layers of small bronchial epithelial.BMP7 was also expressed in epithelial cells of large airways mucus glands.3)BMP7 expression in bronchial epithelial of COPD:The bronchial epithelial BMP7 expression in COPD patients was significantly higher than smoking controls(P<0.001)and non-smoking controls(P<0.001),and the bronchial epithelial BMP7 expression in smoking controls was higher than non-smoking controls(P<0.001).4)Correlation between bronchial epithelial BMP7 expression and quantitative CT index:bronchial epithelial BMP7 expression was negatively correlated with right lung grade 4-9 bronchial count(r=-0.454,P=0.0173),positively correlated with airway wall thickness index Pi-10(r=0.374,P<0.001),and positively correlated with emphysema index%LAA-950(r=0.355,P<0.001).5)Bronchial epithelial BMP7 expression was negatively correlated with FEV1.0%(r=0.401,P<0.001).2.Induced sputum study1)There was no significant difference in age,sex ratio and BMI between COPD group and normal group(P>0.05).2)BMP7 expression in induced sputum of COPD group:the level of BMP7 in induced sputum of COPD patients was significantly higher than controls(355.38±287.64 VS 213.56±85.73 pg/mL,P=0.013).3)The levels of IL-6,IL-8,IL-1? in induced sputum of COPD patients were significantly higher than control group(P<0.05).4)The level of BMP7 in induced sputum of COPD patients was significantly related with levels of IL-6(r=0.461,P<0.001),IL-8(r=0.306,P=0.005)and IL-1?(r=0.246,P=0.027).5)BMP7 level in induced sputum of COPD patients was negatively correlated with FEV1%(r=-0.242,P=0.029).Summary1.BMP7 expression in bronchial epithelial of COPD patients was significantly higher than smoking controls and non-smoking controls.2.The expression of BMP7 in bronchial epithelial of COPD was negatively correlated with the airway counts measured by quantitative CT,positively correlated with airway wall thickness,and negatively correlated with FEV1.3.BMP7 expression in induced sputum of COPD patients was higher than controls and was related with the levels of IL-6,IL-8 and IL-1? in induced sputum.Chapter 2:The role of BMP7 in proliferation and differentiation of human bronchial epithelial cellsObjectivesChapter 1 showed that BMP7 was abnormally high expressed in airways of COPD patients and was related with the number of class 4-9 bronchioles and Pi-10.Previous studies showed that BMP7 played a key role in regulating proliferation,differentiation and apoptosis.We next investigated the effect of BMP7 on proliferation and differentiation of bronchial epithelial cells.The expression of KRT5 and p63(symbols of basal cells)was detected by immunofluorescence staining.Materials and methods1.Culture and identification of human primary bronchial epithelial cells(HPBECs):Non-smokers with normal pulmonary ventilation for bronchoscopy were selected,and brushed cells were cultured in vitro followed by isolation and purification of human primary bronchial epithelial cells.2.Effects of BMP7 on the proliferation of HPBECs:Experimental groups:(1)rhBMP7(100ng/ml)treated group;(2)rhBMP7(10Ong/ml)combined with BMP pathway inhibitor LDN193189(50nM)treated group;(3)blank control group.Treat time:48 h.EdU assay was performed to detect cell proliferation ability.3.Effects of BMP7 on the differentiation of HPBECs:1)Effects of BMP7 on the differentiation of monolayer cultured HPBECs Experimental groups:?rhBMP7 gradient concentration treatment group:0,10,20,40,80 ng/ml treated cells for 72h;?rhBMP7(40ng/ml)treated group,rhBMP7(40ng/ml)combined with BMP pathway inhibitor LDN193189(50nM)treated group,control group.the treatment time was 72h.Western Blot was used to detect involucrin and FOXJ1.2)Effects of BMP7 on cell differentiation in the airway model of air-liquid interface culture(ALI):? ALI model:Cells were cultured in the upper chamber of Transwell and exposed to air.The HPBECs differentiated into human pseudostratified ciliated columnar epithelium in vitro and established airway model.? The expression of involucrin and FOXJ1 was detected by Western Blot and immunofluorescence in ALI model.Results1.Culture and identification of HPBECs:Immunofluorescence staining showed that more than 95%of HPBECs expressed both KRT5 and p63,with KRT5 expressed in the cytoplasm and cell membrane as well as P63 expressed in the nucleus.2.Effects of BMP7 on proliferation of HPBECs:The proportion of EdU-positive cells was significantly reduced in rhBMP7 group compared with control group,while the proportion of EdU-positive cells was significantly increased inrhBMP7+LDN193189 group compared with rhBMP7 group.3.Effects of BMP7 on the differentiation of HPBECs1)Effects of BMP7 on the differentiation of monolayer cultured HPBECs:There was no significant difference in involucrin and FOXJ1 expressions between the 10 ng/ml group and the control group(P>0.05),whereas involucrin expressions were significantly higher and the expression of FOXJ1 were significantly lower in the 20,40 and 80 ng/ml groups compared to the control group(P<0.05).Compared to the rhBMP7(40 ng/ml)treated group,the expression of involucrin was significantly reduced and the expression of FOXJ1 was significantly increased in the group treated with both rhBMP7(40 ng/ml)and the BMP pathway inhibitor LDN193189(50 nM)(P<0.05).2)Effects of BMP7 on cell differentiation in ALI:HPBECs were cultured for 4 weeks,and multilayer cell formation and the appearance of cilia were observed.WB and IF results showed that involucrin expression was higher in the rhBMP7(40ng/ml)treated group than that in the control group,and FOXJ1 expression was lower than that in the control group(P<0.05).Summary1.BMP7 can inhibit the proliferation of human bronchial epithelial cells.2.BMP7 can promote squamous metaplasia of human bronchial epithelial cells,and inhibit ciliated cell differentiation.Chapter 3:The role and mechanism of BMP7 in apoptosis in bronchial epithelial cells.ObjectivesWe demonstrated that BMP7 regulated the proliferation and differentiation the bronchial epithelial.This negative effect of BMP7 on the biological function of bronchial epithelium may further lead to epithelial cell reorganization,causing an imbalanced state of cell proliferation/death.Abnormal apoptosis was a vital part of the airway remodeling,so we hypothesized that BMP7 might be involved in the regulation of bronchial epithelial apoptosis.This part of the study intended to investigate the roles and the mechanism of BMP7 in apoptosis of bronchial epithelial cells.Methods1.BMP7 overexpression lentivirus(OE-BMP7-LV)and lentivirus with empty vector(NC-LV)were used to transfect HPBECs,and BMP7 overexpression HPBECs were constructed by puromycin selection,followed by WB to verify BMP7 overexpression.2.Experimental groups:?BMP7 overexpression group(OE-BMP7-LV)and negative control group(NC-LV);?BMP7 overexpression group(OE-BMP7-LV)and BMP7 overexpression(OE-BMP7-LV)+LDN193189(50 nM)group.3.AnnexinV-FITC/PI double-staining flow cytometry was used to detect apoptosis.4.We used WB to detect the expression of apoptosis-related proteins:Cleaved-caspase3,BCL-2/BAX.5.Mechanisms of BMP7 in regulating apoptosis in bronchial epithelial cells:1)We used proteomics to detect the differentially expressed proteins between the BMP7 overexpression HPBECs and the negative control HPBECs.2)To explore and validate the role of BMP7 in the regulation of bronchial epithelial cell apoptosis by interfering the cellular signaling pathway associated with the differentially expressed protein.Result1.BMP7 pexpression was significantly increased in OE-BMP7-LV transfected group compared to negative control group(NC-LV)(P<0.05).2.Flow cytometry showed that the percentage of early apoptotic cells was significantly higher in BMP7 overexpression group than negative control group(12.1±2.2%VS 5.26 ± 1.16%,P=0.015),and the percentage of early apoptotic cells was significantly lower in BMP7 overexpression+LDN193189(50nM)group than BMP7 overexpression group(9.29±1.55%VS 5.24±0.92%,P=0.0042).3.Compared with the negative control group,BMP7 overexpression HPBECs was significantly decreased in Pro-caspase3 and BCL-2expression and significantly increased in Cleaved-caspase3 and BAX expression,(p<0.05).In BMP7 overexpression group,treatment with LDN193189 significantly reduced Cleavedcaspase3 and BAX expression and promoted BCL-2 expression(p<0.05).4.Exploration of the mechanism of BMP7 regulation of apoptosis in HPBECs:1)A total of 189 proteins were upregulated and 169 proteins were downregulated in BMP7 overexpression group compared with the control group.Of which,44 proteins were associated with signal transduction.Sorted by differential expression fold,MAP2K7(also known as MKK7,mitogen-activated protein kinase kinase 7,which specifically activates the JNK signaling pathway)was the most differentially expressed in the two groups(2.698 fold).2)MKK7 expression and p-JNK2 were significantly higher in BMP7 overexpression group than negative control group(p<0.05).3)Inhibition of the JNK signaling pathway reduced BMP7 overexpression-induced apoptosis in bronchial epithelial cells:? JNK pathway inhibitor SP600125 significantly reduced p-JNK2 in BMP7 overexpression group.?Flow cytology results showed that the percentage of early apoptotic cells was significantly lower in BMP7 overexpression+SP600125 group than BMP7 overexpression group(5.28±0.86%VS 9.74±0.60%,P=0.018).?Cleaved-caspase3 expression in BMP7 overexpression+SP600125 group was lower than BMP7 overexpression group(P<0.05).SummaryBMP7 overexpression can promote apoptosis in human bronchial epithelial cells,through regulating MKK7/JNK2 signaling pathway and activating Caspase3 pathway.Conclusions of Part ?1.BMP7 expression in the bronchial epithelium of COPD was elevated and correlated with quantitative CT airway remodeling index.2.BMP7 can inhibit human bronchial epithelial cells proliferation,promote human bronchial epithelial cell squamous metaplasia and inhibit ciliated cell metaplasia.3.BMP7 overexpression may promote apoptosis in human bronchial epithelial cells through regulating MKK7/JNK2 signaling pathway and activating Caspase3.Conclusions1.The within-breath difference of X5(?X5)is an effective parameter for diagnosing emphysema-dominant phenotype of COPD,which can be used to evaluate the heterogeneity of airflow limitation in COPD.2.The expression of BMP7 in the airway epithelium of COPD is increased.BMP7 can inhibit the proliferation of human bronchial epithelial cells,promote squamous metaplasia,inhibit ciliary metaplasia,and promote the apoptosis of human bronchial epithelial cells through MKK7/JNK2 signaling pathway,which may play a role in the pathogenesis of airway disease in COPD. |
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