| BackgroundChronic obstructive pulmonary disease(COPD)is a common chronic respiratory disease,which could cause systemic organs throughout the body.The clinical characteristics are mainly focused on the limited by airflow and accompanied with progressive decline lung function.The morbidity,disability and fatality rate of COPD was associated with serious harmful impact on the quality of life due to the COPD with longer course of disease.Meanwhile,COPD was caused a heavy social and economic burden in society and families.Currently,COPD is a common disease endangers the health of individuals respiratory system,and the direct and indirect mortality caused by COPD contributed the fourth leading cause of death worldwide.Besides,the mortality rate of COPD will increase gradually in 2010 due to the proportion of smokers was increased.The prevalence rate of COPD nearly 6.2%in China if the age of subjects greater than 30 years.There are fist leading cause of death in rural areas and fourth leading cause of death in urban areas for respiratory diseases.Currently treatment strategies were not significantly improvement the progression of COPD,and limited the airflow reversal.However,the appropriate treatment strategies could improve COPD patients endurance and quality of life.As the concept of precision medical emerge,this new disease management model could combined internal biological information and external clinical manifestations,the implement personalization is expected to clinically to achieve the purpose of accurate treatment in the future COPD treatment.Therefore,the expression of new biomarkers in COPD patients should be exploredPrevious studies have already illustrated the occurrence,development and prognosis of COPD patients are affected by a variety of serum markers.These serum markers including C-reaction protein(CRP),procalcitonin(PCT),Nampt,tumour necrosis factor-?(TNF-?),endothelin(ET),interleukin(IL-6),adiponectin(ADP),uric acid(UA),fibrinogen,growth differentiation factor-15(GDF-15),clara cell protein 16(CC16),eosnophils,copeptin,serun amyloid A(SAA),and YKL-40.However,the expression levels of matrix metalloprote inases-9(MMPs-9)and macrophage stimulating protein(MSP)in various phenotype of COPD and related impact factors remains unclear.MMPs is the main enzyme,which could regulates the degradation of extracellular matrix.The expression levels of MMPs were significantly correlated with the restoration of airway inflammation.MMPs can be detected in lung tissue,bronchoalveolar lavage and induced sputum in COPD patients,while the increased level of MMPs was not affect the activity of MMPs.Further,the expression level of MMPs in the bronchoalveolar lavage of smokers was significantly higher than non-smokers,and MMPs expression level is inverse correlated with airflow obstruction.Previous studies have already demonstrated that MMP-9 is involved in the destruction,remodeling and degradation of alveolar wall matrix in airway structures Besides,MMP-9 is also involved in dediating cytokines and other protease activities,which were correlated with various inflammatory factors and play an important role in the progression of emphysema.Thus infer than,MMP-9 may be associated with chronic inflammatory response in COPD,airway repair and remodeling and emphysema formation.Similarly,MSP as immunomodulatory activity of glycoprotein which mainly synthesized in liver,it lays in the specific receptor tyrosine kinase after combined RON,which could stimulate the alveolar macrophages to produce oxygen free radical and cytokine,and regulate cell differentiation,migration and the matrix Therefore,there is a close related between MSP and the progression of COPD,and affect lung function.ObjectiveIn this study,the expression levels of MMP-9 and MSP in COPD group and corresponding control group were evaluated.Besides,the expression levels of MMP-9 and MSP in patients with different types of COPD were also compared.Finally,potential impact factors were explored,which could affect the expression levels of MMP-9 and MSP in patients with phenotype COPD.Materials and MethodsSample sourceFourty-seven patients with COPD who were admitted to the department of respiratory medicine in Yancheng hospital of southeast university from December 2015 to January 2018 were selected as observation group.Meanwhile,we select 30 patients from physical examination center in our hospital as the control group.The inclusion criteria are listed as follows:(1)the diagnostic criteria of COPD was compliance with the revised guidelines for COPD of the Chinese medical association:FEV1/FVC less than 0.7 after inhalation bronchodilators,or patients with continuous flow limitation;(2)age of patients greater than 40.0 years;(3)patients and family should signed informed consent.The inclusion criteria in control group are:(1)age of patients greater than 40.0 years;(2)subjects without history of smoking;(3)The results of health check indicated normal lung function and normal body functions;(4)patients and family should signed informed consent.The exclusion criteria are presented as follows:(1)subjects not fulfill the inclusion criteria;(2)subjects with serious major cardiovascular events,liver and kidney failure,or disturbance of consciousness;(3)history of malignant tumors within history;and(4)informed consent was not signed.Phenotype COPD,MMP-9,and MSP expression levels measurementsEight-one COPD patients were divided into emphysema phenotype(A group),bronchiectasis phenotype(B group),and other(C group)according to the guideline in Czech published in 2013.After this,we divided the included patients into smoker group and non-smoker groups.MMP-9 and MSP kits were employed to measure the expression levels of MMP-9 and MSP by using Guangzhou rainbow the biological technology co.,LTD.Data collection and potential impact factorsThe items collected are listed as follows:gender,age,body mass index,history of disease,urban population or not,exposure to biofuel or not,smoking status,pulmonary artery pressure,oxygen partial pressure,partial pressure of carbon dioxide,brain natriuretic peptide,hemameba,neutrophilic granulocyte percentage,eosinophil,eosinophil percentage,sputum culture,history of cardiovascular disease,hypertension,diabetes mellitus,FEV1,FEV1%,FVC,and FEV1/FVC.Statistical ana lysisThe normality and homogeneity of variance were tested before the analysis.Mean and deviation was used assess continuous data which fulfilled normal distribution,and the median and interquartile was used for assess continuous data,which not fulfill normal distribution.t test,ANOVA,and non-parametric tests were used calculated the baseline characteristics among various groups.Chi-square test was employed to test categories data.The t test and non-parametric tests were used to compare the MMP-9 and MSP expression levels in observational and control groups.ANOVA test was used to compare the MMP-9 and MSP expression levels in various phenotype of COPD,and multiple test was used LSD test.Spearman method and scatter plot were used to measure the expression levels of MMP-9 and MSP in observational and control group Multiple linear regression analysis was also conducted.All reported P values are 2-sided,and P values<0.05 were considered statistically significant for all analyses Statistical analyses were performed using SPSS 19.0 softwareResultsExpression levels of MMP-9 and MSP in observational and control groupThere were 47 samples in observational group,and 30 samples in control group The expression level of MMP-9 in observational group was(323.94±270.06 ng/ml),which was significantly greater than control group(217.91±134.24 ng/ml)(P=0.0402)Further,the expression level of MSP in observational group(158235.1±76887.45 ng/ml)was higher than control group(38787.05±35668.51 ng/ml)(P<0.0001)Baseline characteristics in types A,B,and C COPDThere were 81 patients included in this study,which contained 48 patients with type A COPD,25 patients with type B COPD,and 8 patients with type C COPD.There were significant differences among these 3 types COPD group for gender(P=0.0022),body mass index(P=0.0013),smoking status(P<0.0001),eosinophil(P=0.0002),and eosinophil percentage(P=0.0001)Expression levels of MMP-9 and MSP in A,B,C types of COPDThere are 48 patients with type A COPD,the expression levels of MMP-9 and MSP were(320.41±268.29 ng/ml)and(155820.70±77882.66 ng/ml),respectively.In 25 type B COPD patients,the expression levels of MMP-9 and MSP were(323.02±354.40 ng/ml)and(184360.45±93059.75 ng/ml),respectively.Finally,in 8 patients with other type COPD,the expression levels of MMP-9 and MSP were(299.96±157.35 ng/ml)and(252622.17±123718.30 ng/ml),respectively.We noted no significant differences among 3 types COPD for the expression level of MMP-9(P=0.980).However,there are significant differences of expression level of MSP among these three groups.The results of pairwise comparison suggested the expression level of MSP in type A COPD was significantly lower than type C COPD patients.No other significant differences were observedThe expression levels of MMP-9 and MSP in types A,B,C COPD in smoker and non-smoker groupA total of 38 smoker patients were included,which involved 10 patients with type A COPD,23 patients with type B COPD,and 5 patients with type C COPD.The results of expression levels of MMP-9 in types A,B,C COPD were 316.19±305.62 ng/ml,262.86±206.52 ng/ml,and 350.79±185.02 ng/ml,respectively.We noted the expression level of MMP-9 were not associated with statistically significant among these 3 types COPD(P>0.05).The results of expression levels of MSP in types A,B,C COPD were 152658.88±73370.61 ng/ml,180554.19±95961.49 ng/ml,and 284880.86±134831.11 ng/ml,respectively.There was significant difference among 3 type COPD for the expression level of MSP(P<0.05).The results of pairwise comparison suggested the expression level of MSP in type A(P=0.017)and type B(P=0.035)COPD were lower than type C COPDA total of 43 non-smoker patients were included,which involved 38 patients with type A COPD,2 patients with type B COPD,and 3 patients with type C COPD.The results of expression levels of MMP-9 in types A,B,C COPD were 321.52±262.13 ng/ml,1014.76±1017.81 ng/ml,and 215.25±31.16 ng/ml,respectively.We noted the expression level of MMP-9 were associated with statistically significant among these 3 types COPD(P<0.05).The results of pairwise comparison suggested the expression level of MMP-9 between type A and type B COPD was associated with statistically significant(P=0.003).Further,significant difference for the expression level of MMP-9 between type B and type C COPD(P=0.006).The results of expression levels of MSP in types A,B,C COPD were 156652.76±79951.47 ng/ml,228132.39±32989.28 ng/ml,and 198857.70±101367.76 ng/ml,respectively.There was no significant difference among 3 type COPD for the expression level of MSP(P>0.05)The impact factors on MMP-9 and MSP expression levels and its correlated with lung functionThere were 81 patients included in this study,which contained 48 patients with type A COPD,25 patients with type B COPD,and 8 patients with type C COPD.We noted body mass index(P=0.015),and hemameba(P=0.008)were significantly associated with the expression level of MMP-9 in\type A COPD.Further,smoking status(P=0.001),exposure to biofuel(P=0.040)were significantly associated with the expression level of MMP-9 in type B COPD.In addition,history of disease(P=0.017)was significantly associated with the expression level of MMP-9 in type C COPDWe noted history of disease(P=0.035)was significantly associated with the expression level of MSP in type A COPD.Further,no pre-defined factors could affect the expression level of MSP in type B COPD.In addition,history of disease(P=0.001),hemameba(P=0.004),and pulmonary artery pressure(P=0.025)were significantly associated with the expression level of MSP in type C COPDFinally,we noted the expression level of MMP-9 was inverse correlated with FEV1(P=0.029),FEV1(%)(P=0.022),and FVC(P=0.043).However,the expression level of MMP-9 was not correlated with FEV1/FVC(P=0.088).Further,the expression level of MSP in COPD patients were not correlated with the elvels of FEV1(P=0.476),FEV1(%)(P=0.903),FVC(P=0.894),and FEV1/FVC(P=0.249)ConclusionsThe summary results are presented as follows:1)The expression levels of MMP-9 and MSP in COPD group were significantly higher than control group.2)There were significant differences for eosinophil,and eosinophil percentage among 3 types COPD.3)There were no significant differences for the expression level of MMP-9 among 3 types COPD,while the expression level of MSP among 3 types COPD.4)There was no significant difference for the expression level of MMP-9 among 3 types COPD in smoker,while the expression level of MSP among 3 types COPD in smoker was associated with statistically significant.Conversely,there was significant difference for the expression level of MMP-9 among 3 types COPD in non-smoker,while the expression level of MSP among 3 types COPD in smoker was not associated with statistically significantly.5)BMI and hemameba were significantly correlated with the expression level of MMP-9 in patients with type A COPD;smoking status,exposure to biofuel were significantly correlated with the expression level of MMP-9 in patients with type B COPD;history of disease was significantly correlated with the expression level of MMP-9 in patients with type C COPD.Further,history of disease was significantly correlated with the expression level of MSP in patients with type A COPD;all of inclusion factors were not associated with statistically significant for the expression level of MSP;history of disease,hemameba,and pulmonary artery pressure were significantly associated with the expression level of MSP in type C COPD.Finally,the expression level of MMP-9 was significantly correlated with the levels of FEV1,FEV1(%),and FVCBackgroundChronic obstructive pulmonary disease(COPD)is a common chronic respiratory disease,which could cause systemic organs throughout the body.The clinical characteristics are mainly focused on the limited by airflow and accompanied with progressive decline lung function.The morbidity,disability and fatality rate of COPD was associated with serious harmful impact on the quality of life due to the COPD with longer course of disease.Meanwhile,COPD was caused a heavy social and economic burden in society and families.Currently,COPD is a common disease endangers the health of individuals respiratory system,and the direct and indirect mortality caused by COPD contributed the fourth leading cause of death worldwideIt is generally believed that the main pathogenesis of COPD includes chronic inflammation,oxidative/antioxidant imbalance,protease/antiprotease imbalance and so on.MMPs are the major enzymes that regulate the degradation and synthesis of extracellular matrix(ECM).There is a significant correlation between the expression level of MMPs and the repair and remodeling of airway inflammation.MMP-9 is a member of MMPs family,whose main function is to degrade extracellular matrix MMP9 is mainly expressed in macrophages,neutrophils and vascular endothelial cells,and is involved in airway structural damage,airway inflammation,airway remodeling,mediation of cytokines and other protease activities.It plays an important role in the occurrence and development of COPD.Therefore,inhibiting the expression and activity of MMP9 can provide a new research direction for the prevention and treatment of COPDObjectiveIn this study,the selective inhibitor of MMP9,SB-3CT,was used to interfere with cigarette smoking-COPD model in rats.The effects of selective inhibition of MMP9 on inflammatory response,oxidative stress response and MSP(MST-1)/RON expression in the progression of COPD were observed,which provided a basis for clinical prevention and treatment of COPDMaterials and MethodsAnimals grouping and interventionThe rats were randomly divided into 3 groups,8 in each group,including control group,model group and treatment group.The control group did not do any treatment The model group established COPD model by cigarette smoking.The treatment group was given SB-3CT(50mg/kg)intervention on the basis of COPD modelLung tissue detectionHematoxylin-Eosin staining was used to observe the lung tissue of rats in each group.Immunohistochemical method was used to detect the expression of MSP,MMP9 and iNOS in lung tissueAlveolar macrophage detectionThe density and morphology of alveolar macrophages were observed by Gimsa staining and Diff rapid staining.And the content of nitric oxide and the activity of nitric oxide synthase were detected by biochemical methods.The mRNA transcription and protein expression of RON,MMP9 and iNOS in cells were further studied by Realtime PCR and Western blot respectivelyBronchoalveolar lavage fluid detectionThe concentrations of MSP(MST-1),TNF-alpha and IL-1beta in alveolar lavage fluid were determined by enzyme-linked immunoassay(ELISA)ResultsLung tissue detectionSB-3CT intervention alleviated the destruction of alveolar structure in the COPD model and reduced the infiltration of inflammatory cells.The results of immunohistochemistry showed that the expression of MSP protein in the model group was significantly higher than that in the treatment group(Wilcoxon rank sum test,P=0.0273);the expression of MMP 9 protein in the model group was significantly higher than that in the treatment group(Wilcoxon rank sum test,P=0.0273);and the expression of iNOS protein in the model group was significantly higher than that in the treatment group(Wilcoxon rank sum test,P=0.0273).Rank sum test,P=0.0273)Alveolar macrophage detectionGimsa staining and Diff-rapid staining showed that there were more cells in the alveolar lavage fluid of the model group.NO and iNOS biochemical markers were detected in alveolar macrophages of rats in each group.NO measurement results showed that the model group was 85.62±18.49?M,significantly higher than the treatment group 42.99±10.23?M(P<0.001).iNOS activity test showed that the result of model group was 51.82±60.86,which was significantly higher than that of treatment group 178.09±48.16(P<0.001)In the expression of RON,MMP9 and iNOS protein in alveolar macrophages,the expression of RON protein in model group 13757.14(12577.35-17899.01)was significantly higher than that in treatment group 10107.55(8622.321-12943.99),P=0.0209.The expression of MMP9 protein in model group 2610.06(11345.19-17287.92)was also significantly higher than that in treatment group 8453.893(7392.847-11808.9),P=0.0274.The expression of iNOS protein in model group 8058.529(6541.528-12138.83)was significantly higher than that in treatment group 4663.286(3730.725-6754.671),P=0.0274The mRNA transcription results of RON,MMP9,iNOS and GAPDH in alveolar macrophages of rats were detected by Realtime PCR.RON in the model group was 0.0321182±0.0061654,which was significantly higher than that in the treatment group(0.0136875±0.0033243),P<0.001.The results of MMP9 were 0.0104691±0.0021502 in model group and 0.0044161±0.0017602 in treatment group,and the model group was significantly higher than the treatment group,P<0.001.The results of iNOS were 0.0150488±0.003004 in model group and 0.0051904±0.0010381 in treatment group,and the model group was significantly higher than the treatment group,P<0.001Bronchoalveolar lavage fluid detectionELISA method was used to analyze the concentration of MST1,TNF-?,IL-1? protein in bronchoalveolar lavage fluid of each group.The expression of MST1 in the model group was 0.90±0.21ng/ml,which was significantly higher than that in the treatment group(0.50±0.16ng/ml,P<0.001).The expression of TNF-? in the model group was 56.74±12.58ng/L,which was significantly higher than that in the treatment group(29.02±1083ng/L,P<0.001).The expression of IL-1? in the model group was 394.59±97.48ng/L,which was significantly higher than that in the treatment group(222.89±60.35ng/L,P<0.001)ConclusionsSB-3CT,an inhibitor of MMP-9,could decrease the expression of MMP-9 in COPD model,the transcription and expression of RON in rat alveolar macrophages,and the concentration of MST1 in alveolar fluid.And SB-3CT intervention can relieve the oxidative stress reaction in COPD pathological process,and can reduce the inflammatory mediator content in alveolar fluid.Therefore,SB-3CT may delay the occurrence and development of COPD through the above anti-inflammatory,anti-oxidation effect and inhibition of MST-1/RON protein,and ultimately alleviate the damage of cigarette smoke to alveolar tissue in rats. |
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