Downregulation Of Secretory Lekocyte Proteinase Inhibitor In Chronic Obstructive Lung Disease: The Role Of TGF Beta1/Smads Signaling Pathways

Chronic obstructive pulmonary disease(COPD)is characterized by airflow obstruction,which comprises emphysema and chronic bronchitis/bronchiolitis.While the molecular mechanisms by which small airway obstruction occurs remain unknown.Recently,some studies indicated that an imbalance between neutrophil protease and surrounding antiprotease levels has been shown to be important in the pathogenesis of COPD.Secretory leukocyte proteinase inhibitor(SLPI),known as one of the most important antileukoproteases in airway,is a 12-kDa non-glycosylated,cationic protein that is produced by serous cells of the submucosal bronchial glands,by non-ciliated cells of the bronchial epithelium,and by neutrophils.Its major physiological function is considered to be the inhibition of the destructive capacity of neutrophil elastase(NE),and some data indicated that COPD is related to the reduction of SLPI.Whereas little is known about the regulation of SLPI expression in the lung.Other studies suggest some fibrogenic growth factors may be involved in the remodeling processes of the small airways,iOne of the most potent and extensively studied growth factors is transforming growth factor(TGF)-betal,which induces fibroblast proliferation, increased production of collagen and other extracellular matrix proteins, and decreased collagen degradation.TGF-β₁ is also chemotactic for neutrophil,macrophages and mast cells,and its major intracellular signaling effector is the Smad proteins.It is well known that Smad pathways are central mediators of signals from the receptors for TGF-beta superfamily members to the nucleus.Phosphorylation of receptor-activated Smads(R-Smads)leads to formation of complexes with the common mediator Smad(Co-Smad)(Smad4),which are imported to the nucleus.Nuclear Smad oligomers bind to DNA and associate with transcription factors to regulate expression of target genes. TGF-β₁ is widely localized in the lung.Several studies recently demonstrated that there was a significant expression of TGF-β₁ in airway epithelial cells in subjects with COPD as compared with the control.Low levels of SLPI and high levels of TGF-β₁ were observed in the bronchi and lung tissues of COPD,and recent study indicated that TGF-β₁ is a potent inhibitor of SLPI in a bronchial epithelial cell. However,whether the decreased expression of SLPI in the bronchi and lung tissues of COPD is related to the increased expression of TGF-β₁ is unknown.If so,whether the role of SLPI is mediated through Smads signal pathway needs further investigation.To address the role of TGF-β₁/Smads on the regulation of SLPI in the bronchi and lung tissues of COPD,COPD model was established and the rats were treated with TGF-β₁ monoclonal antibody;the normal human bronchial epithelial cell (NHBE)line was cultured and it was stimulated with TGF-β₁ and siRNA (Smad4).Then,the relationship among the expression of SLPI TGF-β₁ and Smad4 in the bronchi and lung tissues were observed,and the role of TGF-β₁/Smads on the decreased expression of SLPI in NHBE cells was investigated.1.Expression of secretory leukocyte proteinase inhibitor in the bronchi and lung tissues of chronic obstructive pulmonary disease rat models and the regu-lative mechanism by TGF-β₁Rat COPD model was established by intratracheal instillation of lipopoly-saccharide(LPS)twice and exposure to cigarette smoke daily; drug intervention group received TGF-β₁ monoclonal antibody 0.5 mg twice via the tail venous injection.The spirometry was conducted and the pathological changes were observed,the concentrations of SLPI in bronchoalveolar lavage fluid(BALF)was measured by enzyme-linked inmunosorbent assay(ELISA);The expressions of TGF-β₁,Smad4 and SLPI in the bronchi and lung tissues were examined by using immunohistochemistry,and the expressions of TGF-β₁ mRNA,Smad4 mRNA and SLPI mRNA in the bronchi and lung tissues were detected by reverse transcription-polymerase chain reaction(RT-PCR)respectively.Results showed:The PEF,FEV_0.3and FEV_0.3/FVC in COPD model group were significantly lower than those in the control group(all P＜0.01).After treated with TGF-β₁ monoclonal antibody,the PEF,FEV_0.3 and FEV_0.3/FVC in the TGF-β₁ monoclonal antibody intervention group were all significantly improved as compared with the COPD model group (all P＜0.01).Compared with the control group,a portion of the airway epithelium and some cilia had been shed,inflammatory cells infiltrated some airway walls,smooth muscles in the airway walls and small artery walls had proliferated irregularly,air spaces were enlarged in an irregular manner,some alveoli were confluent and bullae were seen in the COPD model group.Compared with the COPD model group,significant improvement was seen in the shedding of airway epithelium and cilia,the proliferation of smooth muscle in airway walls and small artery walls was great alleviated,air spaces were not obviously enlarged in the TGF-β₁ monoclonal antibody intervention group.The concentration of secretion of SLPI in BALF of the COPD model group was significantly lower than that in the control group(P＜0.01).After treated with TGF-β₁ monoclonal antibody,the concentration of secretion of SLPI in BALF of the TGF-β₁ monoclonal antibody intervention group was significantly improved as compared with the COPD model group(all P＜0.01).Both at the mRNA level and the protein level,the expressions of both TGF-β₁ and Smad4 in the COPD model group were higher than those in the control group(all P＜0.01),while the expression of SLPI was lower in the COPD model group than that in the control group(P＜0.01).After treated with TGF-β₁ monoclonal antibody,the expressions of both TGF-β₁ and Smad4 were all significantly decreased in TGF-β₁ monoclonal antibody intervention group both at the mRNA level and the protein level as compared with the COPD model,grouP(all P＜0.01),however,the expression of SLPI was significantly increased in TGF-β₁ monoclonal antibody intervention group both at the mRNA level and the protein level as compared with the COPD model group(all P＜0.01).According to TGF-β₁ and Smad4,the main immune positive cell types were vascular smooth muscle cell, airway epithelial cell,alveolar epithelial cell,pulmonary interstitial cell and macrophage cells;but to SLPI,the main immune positive cell type was only airway epithelial cell.TGF-β₁ was mainly stained in membrane, Smad4 and SLPI were both stained in cytoplasm,but Smad4 was stained in nuclus,too.2.The expression of secretory leukocyte proteinase inhibitor in human bronchial epithelial cell is downregulated by TGF-β₁/Smads pathway.The normal human bronchial epithelial cell(NHBE)was cultured, preincubated with or without siRNA(Smad4),and then stimulated with or without TGF-β₁.The expressions of Smad4 and SLPI were detected by immunocytochemistry,western blot and RT-PCR respectively.Results showed:The expression of SLPI in NHBE was inhibited by TGF-β₁ both at the mRNA level and the protein level(all P＜0.01).The expression of Smad4 in NHBE was successfully inhibited by siRNA (Smad4)both at the mRNA level and the protein level(all P＜0.01),but it was not affected by negative control siRNA(all P＞0.05).At the same time,the results indicated that,after preincubated with siRNA(Smad4) then stimulated with TGF-β₁,the effects of TGF-β₁-inhibited expression of SLPI in NHBE was disengaged by siRNA(Smad4)both at the mRNA level and the protein level(all P＜0.01),but it was not affected by negative control siRNA.(all P＞0.05)The above two parts prompted:the expression of SLPI in the airway of the COPD rat model was significantly decreased,which may be mainly caused by the increased expression of TGF-β₁,and the activation of Smads signal pathway play a crucial role in this process.