| Objective:Chronic obstructive pulmonary disease（COPD）is a life-threatening disease.Its morbidity and mortality are on the rise globally.The main characteristics of COPD are chronic airway inflammation,irreversible airflow limitation,and accelerated decline in lung function.The pathogenesis and corresponding pathophysiological changes of COPD are very complex,and a better understanding of the underlying mechanisms is urgently required to investigate important targets for prevention and treatment.In recent years,cysteine-rich angiogenic inducer 61（Cyr61）has gradually become a research hotspot in various fields due to its multiple biological effects.However,the studies on Cyr61 in COPD are very rare.Airway remodeling is the core pathological change of COPD and is closely related to persistent airflow limitation.The purpose of this research is to study the expression and significance of Cyr61 in serum,induced sputum and lung tissue of COPD patients,explore its role in airway remodeling of COPD by cell research in vitro,and explore whether Cyr61monoclonal antibody has potential therapeutic effect in COPD rat model.Materials and Methods:In clinical research,a cross-sectional study design was adopted.33 patients who underwent surgery for benign pulmonary diseases in the Department of Thoracic Surgery of Chengdu First People’s Hospital were recruited and divided into the COPD group and the non-COPD group,and relevant clinical data were collected.ELISA was used to detect the levels of Cyr61 in serum and induced sputum of the two groups.The peripheral lung tissue that was more than 5 cm away from the lesion was collected.q RT-PCR and immunohistochemistry were used to detect the expression of Cyr61.The correlations of the expression of Cyr61 in serum,induced sputum and lung tissue with smoking index,FEV1%pred and CAT score were analyzed.In vitro cell research,16 human bronchial epithelial cells（16HBE）were treated with cigarette smoke extract（CSE）to establish an in vitro exposure model.Firstly,16HBE were divided into the control group,model group,Cyr61-1 group（+5ng/m L Cyr61）,Cyr61-2 group（+50ng/m L Cyr61）and Cyr61-3 group（+100ng/m L Cyr61）.ELISA,Western Blot and q RT-PCR were adopted for the detection of the expression of IL-8,MCP-1,TGF-β1,MMP-9 and E-cadherin in each group at 1h,12h and 24h respectively.Secondly,the CSE-stimulated 16HBE were divided into the model group and the experimental group,and the empty vector and Cyr61si RNA plasmid were implanted respectively.After successful transfection,the expression of IL-8,MCP-1,TGF-β1,MMP-9 and E-cadherin in the two groups was detected by ELISA,Western Blot and q RT-PCR.The apoptosis rate was detected by flow cytometry.Finally,the model group,blocking group 1 and blocking group 2 were set up.Wnt/β-catenin signaling pathway inhibitor XAV939 and P38MAPK signaling pathway inhibitor SB202190 were added into blocking group 1 and blocking group2 respectively to observe their effects on the expression of inflammatory cytokines and EMT-related factors induced by Cyr61.In animal research,SD rats were assigned to the control group,model group,treatment group and placebo group randomly.The treatment group was given an intraperitoneal injection of specific Cyr61 monoclonal antibody,and the placebo group was given a placebo intraperitoneal injection.The lung tissues of four groups were stained with HE and Masson.The degree of airway inflammation,pulmonary alveolar destruction,small airway smooth muscle thickness and airway wall thickness were observed under a microscope.WAt/Pbm、WAm/Pbm and N/Pbm were used for quantitative analysis.The counting statistics of the total number of cells and cell classification in BALF were completed.The levels of Cyr61,IL-8,MCP-1,TGF-β1,MMP-9 and E-cadherin in the BALF supernatant and serum of the4 groups of rats were detected by ELISA.Western Blot and q RT-PCR were adopted for the detection of the expression of Cyr61,IL-8,MCP-1,TGF-β1,MMP-9 and E-cadherin in lung tissue.Results:1.The clinical research results are as follows:1.1 Both the COPD group and the non-COPD group had a similar baseline level,and there was no statistically significant difference in gender,age,smoker proportion and smoking index（P>0.05）,which was comparable.1.2 The levels of Cyr61 in the serum and induced sputum in the COPD group were higher than those in the non-COPD group（P<0.001）.1.3 Compared with the non-COPD group,Cyr61m RNA was highly expressed in the lung tissue of the COPD group（P<0.01）.1.4 Immunohistochemical images showed that Cyr61 was expressed in both groups,mainly in epithelial cells and some inflammatory cells such as alveolar macrophages,and the staining in the COPD group was more intensive than that in the non-COPD group.The semi-quantitative results of immunohistochemistry showed that the expression of Cyr61 in the lungs of the COPD group was higher than that of the non-COPD group（P=0.0013）.1.5 In COPD group,the expression of Cyr61 in serum,induced sputum and lung was negatively correlated with FEV1%pred（r=-0.684,P<0.001;r=-0.858,P<0.001;r=-0.794,P<0.001）,while positively correlated with CAT score（r=0.694,P<0.001;r=0.821,P<0.001;r=0.805,P<0.001）and smoking index（r=0.756,P<0.001;r=0.773,P<0.001;r=0.722,P=0.002）.2.The results of cell studies are as follows:2.1 Compared with the control group,the secretion of IL-8,MCP-1,TGF-β1 and MMP-9 in cell culture supernatant of CSE-stimulated model group was increased,while the secretion of E-cadherin was decreased（P<0.05）.And the protein levels and m RNA transcription levels of cytokines IL-8,MCP-1,TGF-β1 and MMP-9 in the model group cells were increased,while the protein level and m RNA transcription level of E-cadherin were decreased（P<0.05）.2.2 Compared with the model group,the low,medium and high dose of Cyr61stimulation could significantly increase the levels of cytokines IL-8,MCP-1,TGF-β1 and MMP-9 in the cell culture supernatant,while the secretion of E-cadherin was decreased（P<0.05）.The Cyr61 stimulation could also significantly increase the protein level and m RNA transcription level of IL-8,MCP-1,TGF-β1and MMP-9 in each group cells,and reduce the protein and m RNA transcription level of E-cadherin（P<0.05）,and the higher the concentration of Cyr61 and the longer the time,the more significant the effect（P<0.05）.2.3 Compared with the model group,the secretion of cytokines IL-8,MCP-1,TGF-β1 and MMP-9 in cell culture supernatant was decreased in the Cyr61si RNA plasmid transfected group,while the secretion of E-cadherin was increased（P<0.01）.And the protein levels and m RNA transcription levels of the cytokines IL-8,MCP-1,TGF-β1 and MMP-9 in the experimental group cells were decreased,while the E-cadherin protein levels and m RNA transcription levels were increased（P<0.05）.The apoptosis rate of 16HBE was decreased in the experimental group（P<0.001）.2.4 Compared with the model group,the level of IL-8 and TGF-β1 was reduced being pretreated with Wnt/β-catenin inhibitor XAV939 in the blocking group 1（P<0.05）,while there was no significant change after pretreatment with P38MAPK signaling pathway inhibitor SB202190 in the blocking group 2（P>0.05）.3.The results of animal experiments are as follows:3.1 HE staining and Masson staining resultsThe HE stained and Masson stained pathological sections were observed under a microscope.In the control group,the rats had complete airway epithelium,neatly lined epithelial cilia,no inflammatory cell infiltration around the airway,complete alveolar structures with normal size,no thickening of the wall and smooth muscle layer,and no lumen stenosis.In the model group and the placebo group,the rats had similar airway lesions,including incomplete airway epithelium,thickened airway wall and smooth muscle layer,obvious collagen proliferation,a great quantity of inflammatory cells gathering around the airway,pulmonary alveolar structure disorder,alveolar wall rupture,alveolar cavity expansion and fusion and pulmonary bullae formation.In the treatment group,all the lesions of the lungs mentioned above were significantly reduced compared with the model group.3.2 Comparison of morphological parametersCompared with the control group,the model group had significantly increased WAt/Pbm,WAm/Pbm and N/Pbm（P<0.001）.Compared with the model group,the rats in the treatment group had significantly decreased WAt/Pbm,WAm/Pbm and N/Pbm（P<0.001）.There was no statistical difference between the placebo group and the model group（P>0.05）.3.3 Comparison of total number and classification of cells in BALFThe total number of cells,neutrophils,macrophages and lymphocytes in the BALF of the model group were increased,which was significantly different from that of the control group（P<0.001）.Compared with the model group,the total number of cells,neutrophils,macrophages and lymphocytes in the BALF of the treatment group were reduced（P<0.01）.There was no statistical difference between the placebo group and the model group（P>0.05）.3.4 Comparison of the levels of cytokines in peripheral blood and BALFCompared with the rats in the control group,the levels of Cyr61,IL-8,MCP-1,TGF-β1 and MMP-9 in the peripheral blood and BALF of rats in the model group were significantly increased,while the levels of E-cadherin were decreased（P<0.001）.Compared with the model group,the levels of Cyr61,IL-8,MCP-1,TGF-β1and MMP-9 in the peripheral blood and BALF of rats in the treatment group were significantly decreased,while the levels of E-cadherin were increased（P<0.001）.There was no statistical difference between the placebo group and the model group（P>0.05）.3.5 Comparison of the expression of cytokines in the lungCompared with the control group,the protein and m RNA levels of Cyr61,IL-8,MCP-1,TGF-β1 and MMP-9 of lung tissue in the model group were greatly increased,while the protein and m RNA levels of E-cadherin were decreased significantly（P<0.01）.Compared with model group,the protein and m RNA levels of Cyr61,IL-8,MCP-1,TGF-β1 and MMP-9 of lung tissue in treatment group were decreased significantly,while the protein and m RNA levels of E-cadherin were increased（P<0.05）.There was no statistical difference between the placebo group and the model group（P>0.05）.3.6 Correlation analysisThe Cyr61 concentration in BALF of the model group was positively correlated with the concentrations of IL-8（r=0.846,P<0.01）,MCP-1（r=0.739,P<0.05）,TGF-β1（r=0.809,P<0.01）and MMP-9（r=0.659,P<0.05）in BALF,and negatively correlated with the concentration of E-cadherin（r=-0.756,P<0.05）in BALF.The Cyr61 concentration in BALF of the model group was positively correlated with the total cell number of BALF（r=0.823,P<0.01）.The protein levels of Cyr61 in lung tissue was positively correlated with those of IL-8（r=0.803,P<0.01）,MCP-1（r=0.722,P<0.05）,TGF-β1（r=0.829,P<0.01）and MMP-9（r=0.704,P<0.05）in lung tissue,and negatively correlated with that of E-cadherin（r=-0.716,P<0.05）in lung tissue.And the protein levels of Cyr61 in lung tissue was positively correlated with WAt/Pbm（r=0.724,P<0.05）,WAm/Pbmm（r=0.820,P<0.01）and N/Pbm（r=0.688,P<0.05）.Conclusion:1.Cyr61 was mainly expressed in airway epithelial cells.Compared with the non-COPD group,the expression of Cyr61 in serum,induced sputum and lung tissue in COPD group was greatly increasded,and it was positively correlated with smoking index and CAT score,and negatively correlated with FEV1%pred,suggesting that Cyr61 is involved in the pathogenesis of COPD and closely related to the severity of the disease.2.Exogenous Cyr61 promoted 16HBE to up-regulate the expression of IL-8,MCP-1,TGF-β1 and MMP-9,while the expression of E-cadherin was down-regulated,and it was concentration-and time-dependent.Knocking down the expression of Cyr61with Cyr61si RNA could weaken its function and inhibit the apoptosis of 16HBE cells.It suggested that Cyr61 could promote the secretion of inflammatory factors,epithelial mesenchymal transformation and apoptosis,and might play an important role in airway remodeling of COPD.The mechanism of action might be partly related to Wnt/β-catenin signaling pathway.3.The results of animal experiments showed that the expression of Cyr61,IL-8,MCP-1,TGF-β1 and MMP-9 in BALF,serum and lung tissue in treatment group treated with Cyr61 monoclonal antibody was lower than that in the model group,and the expression of E-cadherin was increased.The degree of airway inflammation,emphysema and airway wall thickening in treatment group was reduced compared with that in the model group.It suggested that Cyr61 monoclonal antibody has the potential to reduce airway inflammation and airway remodeling in COPD. |
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