| BackgroundBronchial asthma is a chronic airway inflammatory disease characterized by airway hyperresponsiveness,airway inflammation and airway remodeling.Current treatments for airway inflammation and airway hyperresponsiveness are effective in relieving and preventing symptoms in most patients,but some patients who experience persistent symptoms and progressive decline in lung function are described as irreversible or refractory asthma.Airway remodeling is one of the main features of asthma.This remodeling process is a key feature of severe asthma and is believed to play a key role in refractory or persistent asthma.Therefore,the research on the mechanism of airway remodeling and the development of biological agents for various components of airway remodeling are particularly necessary for asthma,especially refractory asthma.At present,animal models,vitro studies and some clinical studies have been put forward about the airway remodeling of the cellular and molecular pathways involved in the existing knowledge,which promotes the aim of airway remodeling of various components for the development of biological agents,in order to limit the airway remodeling in patients with asthma,to cure bronchial asthma to the one step closer.Connective tissue growth factor(CTGF)is an secreted peptide rich in cysteine.The expression of CTGF was significantly increased in the lung of ova-induced mice,and CTGF was positively correlated with MMP-9、TIMP-1 and smooth muscle layer thickness,suggesting that the up-regulation of CTGF was correlated with MMP-9,and CTGF might be involved in the pathogenesis of airway remodeling in asthma.Further studies have found that CTGF in plasma may be a potential biomarker for stable asthma when assessing the degree of persistent airway obstruction,and CTGF may be associated with airway remodeling in asthma.Follistatin-Like 1(Fstll)is an extracellular matrix glycoprotein belonging to the family of follicular statin proteins,which is a secretory protein.It plays an important role in embryo development,organ formation,tumor,inflammatory response and myocardial protection,and can improve endothelial function,promote vascular regeneration,and inhibit the proliferation and migration of vascular smooth muscle cells.Abnormal proliferation and migration of airway smooth muscle cells play an important role in airway remodeling in asthma and lead to airway hyperresponsiveness.Fstl1 has been shown to play an important role in promoting the proliferation and migration of human airway smooth muscle cells.In the lungs of patients with severe asthma,Fstll is highly expressed by macrophages,and Fstll knockout can inhibit smooth muscle cell migration,suggesting that Fstll may represent a new therapeutic target for airway remodeling in bronchial asthma.Disconnected(disco)interaction protein homologue 2 A(DIP2A)is a kind of cytoplasmic proteins.Immunocoprecipitation revealed the direct physical interaction between FSTL1 and DIP2A,which mediated the downstream action of FSTL1 as a receptor of FSTL1.Transforming growth factor beta(TGF-β)is a multifunctional protein that can affect the growth,differentiation,apoptosis and immune regulation of various cells.The level of TGF-β is increased in the airways of asthmatic patients.Many of the effects of TGF-β are mediated by CTGF and CTGF is one of the key fibrogenic factors related to TGF-β.TGF-β is the main central media of tissue fibrosis,Fstl1 is an important fibrinogen in the process of pulmonary tissue fibrosis,mainly through the TGF-βsignaling pathway to promote fibrosis.TGF-β can induce the formation of Fstll in lung fibroblasts and up-regulate the expression of Fstl1 in mouse pulmonary fibrosis.Many effects of TGF-β are mediated by CTGF.Previous studies have shown that both FSTL1 and CTGF can promote airway remodeling,but the specific mechanism of the action is still unclear.In this study,CTGF and FSTL1 were used as entry points to clarify the specific mechanism of airway remodeling and to provide clinical treatment ideas for the early prevention and treatment of asthma patients with fixed airflow limitation.ObjectivesTo explore whether CTGF promotes the occurrence of airway remodeling in asthma by affecting the FSTL1/DIP2A signaling pathway.Methods1.To clarify the role of CTGF and FSTL1 in remodeling allergic airway disease model in mice induced by OVA.1.1 female BALB/C mice at 6-8 weeks were given OVA to induce allergic airway disease model in mice.The allergic airway disease model was induced by intraperitoneal injection and local challenge by nasal drip inhalation.1.2 After the successful establishment of the model,the airway responsiveness of mice in each group was measured,and the airway resistance after methacholine atomization inhalation at different concentrations was recorded.1.3 After lung function monitoring,the bronchoalveolar lavage fluid,blood and lung tissue samples of mice in the asthma model group and the control group were collected.1.4 The bronchoalveolar lavage fluid of mice in each group was counted and classified.1.5 The concentration of IgE in peripheral blood of mice in each group was determined.1.6 HE staining was used to observe the characteristics of airway remodeling,including exfoliation of epithelial cells,increase of smooth muscle bundles and thickening of basement membrane.1.7 Masson staining was used to observe the expression of collagen fibers in airway mucosa of mice1.8 The expression of CTGF,FSTL1 and the expression of a-smooth muscle actin(a-SMA),fibronectin and collagen I were determined by immunohistochemistry.1.9 The expression of CTGF,FSTL1 and DIP2A,as well as remodeling related proteins were determined by Western blot.1.10 The expressions of CTGF,FSTL1 and its receptor DIP2A and remodeling related markers mRNA were detected by real-time quantitative polymerase chain reaction(qRT-PCR).2.The effect of CTGF on the FSTL1/DIP2A signaling pathway was observed at the cellular level.2.1 Human lung fibroblasts were cultured in vitro,and the expressions of DIP2A,a-SMA,fibronectin and collagen Ⅰ were detected after the stimulation of FSTL1 recombinant protein2.2 Human lung fibroblasts were cultured in vitro,and the expressions of FSTL1、DIP2A,a-SMA,fibronectin and collagen Ⅰ were detected after the stimulation of CTGF recombinant protein.2.3 Human lung fibroblasts were cultured in vitro,transfected with FSTL1 siRNA and treated with CTGF recombinant protein,and the expressions of FSTL1,DIP2A,a-SMA,fibronectin and collagen Ⅰ were detected2.4 Human lung fibroblasts were cultured in vitro,transfected with DIP2A siRNA,and treated with CTGF recombinant protein,and the expressions of FSTL1,DIP2A,a-SMA,fibronectin and collagen Ⅰ were detected.2.5 Human lung fibroblasts were cultured in vitro and transfected with DIP2A siRNA.After being stimulated by FSTL1 recombinant protein,the expressions of CTGF,a-SMA,fibronectin and type Ⅰ collagen were detected.3.Effects of intervention of CTGF expression on FSTL1 and airway remodeling in asthmatic mice in vivo.3.1 female BALB/C mice of 8 weeks old were randomly divided into WT group,WT+OVA group,WT+OVA+Dex group and WT+OVA+CTGF group.The OVA asthma mouse model was established in WT+OVA group by the method described in the first part.WT+OVA+ Dex group was injected intraperitoneally with dexamethasone.WT+OVA+CTGF was intranasally dripped with CTGF recombinant protein.Both of them were on the basis of WT+OVA group model preparation.3.2 After the successful establishment of the model,the airway responsiveness of mice in each group was measured,and the airway resistance after methacholine atomization inhalation at different concentrations was recorded.3.3 After pulmonary function monitoring,the mice were killed and the lung tissue samples of each group were collected.3.4 HE staining was used to observe the characteristics of airway remodeling in each group,including exfoliation of epithelial cells,increase of smooth muscle bundles and thickening of basement membrane.3.5 The expression of FSTL1 and remodeling related markers a-SMA,fibronectin and type I collagen were detected by immunohistochemistry.3.6 The expression of FSTL1 and remodeling related proteins were determined by Western blot.3.7 The expressions of FSTL1 and remodeling related markers mRNA were detected by real-time quantitative polymerase chain reaction(qRT-PCR).Results1.Airway remodeling in asthmatic mice was increased,accompanied by increased expression of CTGF and FSTL1.1.1 The airway responsiveness of mice with allergic airway disease induced by OVA in the model group was higher than that in the control group.1.2 Compared with the control group,the number of total cells,neutrophils and eosinophils in the bronchoalveolar lavage fluid(BALF)of OVA-induced mice increased.1.3 The level of plasma IgE in OVA-induced asthmatic mice was significantly higher than that in the control group.1.4 HE staining showed that airway remodeling in the asthmatic mouse model group was characterized by epithelial cell shedding,increased smooth muscle tracts and thickened basement membrane1.5 Masson staining showed a significant increase in collagen fibers in the airway tissues of the asthmatic mouse model group1.6 Immunohistochemistry showed high expression of CTGF and FSTL1 in the airway mucosa of asthmatic mouse model,accompanied by high expression of remodeling related proteins a-SMA,fibronectin and collagen Ⅰ1.7 Western blot showed high expression of CTGF,FSTL1 and its receptor DIP2A in lung tissues of asthmatic mice,accompanied by high expression of remodeling related proteins a-SMA,fibronectin and collagen Ⅰ1.8 qRT-PCR showed high expression of CTGF,FSTL1 and their receptor DIP2A mRNA in the lung tissue of asthmatic mice,accompanied by high expression of remodeling-related proteins a-SMA,fibronectin and type Ⅰ collagen mRNA2.CTGF promotes airway remodeling through the FSTL1/DIP2A signaling pathway.2.1 Western blot showed that human lung fibroblasts stimulated by recombinant FSTL1 protein had high expression of its receptor DIP2A protein,accompanied by high expression of remodeling-related proteins.Compared with the control group,the expression of DIP2A protein of FSTL1 receptor and the expression of remodeling-related proteins in FSTL1 recombinant protein group were significantly higher than those in control group(p<0.05).2.2 Western blot showed that FSTL1 and its receptor DIP2A protein were highly expressed in human lung fibroblasts stimulated by CTGF recombinant protein,accompanied by high expression of remodeling-related proteins.Compared with the control group,the expression of FSTL1 and its receptor DIP2A protein and remodeling-related proteins in the CTGF recombinant protein group were significantly higher than those in the control group(p<0.05).2.3 After human lung fibroblasts were transfected with FSTL1 siRNA and treated with CTGF recombinant protein,Western blot showed that the expressions of FSTL1,DIP2A and remodeling-related proteins in human lung fibroblasts in the negative control group stimulated by CTGF recombinant protein were significantly higher than those in the negative control group without CTGF stimulation.The expressions of FSTL1,DIP2A and remodeling related proteins in FSTL1 siRNA transfected human lung fibroblasts stimulated by CTGF recombinant protein were significantly lower than those in the negative control group stimulated by CTGF recombinant protein.The expressions of FSTL1,DIP2A and remodeling related proteins in human lung fibroblasts transfected with CTGF recombinant protein were not significantly different from those in human lung fibroblasts transfected with FSTL1 siRNA without CTGF recombinant protein stimulation.(P>0.05).It is suggested that the expression of FSTL1,receptor DIP2A and remodeling markers in pulmonary fibroblasts stimulated by recombinant CTGF protein increased.After blocking FSTL1,the expressions of FSTL1,receptor DIP2A and remodeling markers were affected and did not increase.2.4 After human lung fibroblasts were transfected with DIP2A siRNA and treated with CTGF recombinant protein,Western blot showed that the expressions of FSTL1,DIP2A and remodeling-related proteins in human lung fibroblasts in the negative control group stimulated by CTGF recombinant protein were significantly higher than those in the negative control group without CTGF stimulation.The expressions of DIP2A and remodeling related proteins in DIP2A siRNA transfected human lung fibroblasts stimulated by CTGF recombinant protein were significantly lower than those in the negative control group stimulated by CTGF recombinant protein,while the expression of FSTL1 was almost unchanged.The expression of DIP2A and remodeling related proteins in human lung fibroblasts transfected with DIP2A siRNA stimulated by recombinant CTGF protein were not significantly different from those in human lung fibroblasts transfected with DIP2A siRNA without CTGF recombinant protein(p>0.05),while the expression of FSTL1 was significantly increased in human lung fibroblasts transfected with DIP2A siRNA2.5 Human lung fibroblasts were treated with FSTL1 recombinant protein after transfection of DIP2A siRNA,Western blot showed that the expression of remodeling-related proteins in human lung fibroblasts stimulated by FSTL1 recombinant protein was significantly higher than that in the negative control group without FSTL1 stimulation,while the expression of CTGF was basically unchanged.The expression of remodeling-related proteins in DIP2A siRNA-transfected human lung fibroblasts stimulated by recombinant FSTL1 protein was significantly lower than that in negative control group stimulated by FSTL1 recombinant protein,while the expression of CTGF remained unchanged.The expression of remodeling-related proteins in DIP2A siRNA-transfected human lung fibroblasts stimulated by recombinant FSTL1 protein was not significantly different from that in DIP2A siRNA-transfected human lung fibroblasts without FSTL1 recombinant protein stimulation(p>0.05),while the expression of CTGF was almost unchanged in human lung fibroblasts transfected with DIP2A siRNA.3.FSTL1 and airway remodeling in asthmatic mice were affected by in vivo after intervention of CTGF expression.3.1 After aerosol inhalation of methacholine,the airway resistance of WT+OVA group was higher than that of WT group,the airway resistance of WT+OVA+Dex group was lower than that of WT+OVA group,and the airway resistance of WT+OVA+CTGF group was higher than that of WT+OVA group.3.2 HE staining showed that the WT+OVA model group showed airway remodeling such as exfoliation of epithelial cells,increase of smooth muscle bundles and thickening of basement membrane.The airway inflammation and remodeling in WT+OVA+Dex group were less than those in WT+OVA group.The airway inflammation and remodeling in WT+OVA+CTGF group were more serious than those in WT+OVA group3.3 Immunohistochemistry showed that the expression of FSTL1,Collagen Ⅰ,Fibronectin and a-SMA in WT+OVA group was higher than that in WT group,the expression of FSTL1,Collagen Ⅰ,Fibronectin and a-SMA in WT+OVA+Dex group was lower than that in WT+OVA group,and the expression of FSTL1,Collagen Ⅰ,Fibronectin and a-SMA in WT+OVA+CTGF group was higher than that in WT+OVA group.3.4 Western blot showed that the expression of FSTL1,Collagen Ⅰ,Fibronectin and a-SMA in WT+OVA group was higher than that in WT group,the expression of FSTL1,Collagen I,Fibronectin and a-SMA in WT+OVA+Dex group was lower than that in WT+OVA group,and the expression of FSTL1,Collagen I,Fibronectin and a-SMA in WT+OVA+CTGF group was higher than that in WT+OVA group.3.5 qRT-PCR showed that the expression of FSTL1,Collagen Ⅰ,Fibronectin and a-SMA in WT+OVA group was higher than that in WT group,the expression of FSTL1,Collagen I,Fibronectin and a-SMA in WT+OVA+Dex group was lower than that in WT+OVA group,and the expression of FSTL1,Collagen Ⅰ,Fibronectin and a-SMA in WT+OVA+CTGF group was higher than that in WT+OVA group.ConclusionCTGF promotes airway remodeling in asthma by affecting FSTL1/DIP2A signaling pathway. |
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