The Role And Mechanism Of Matrix Protein Tenascin-C Mediate The Effect Of TGF-? In COPD Induced By Biomass Fuel Smoke In Rat

BackgroundChronic obstructive pulmonary disease(COPD),is a common respiratory disease with a high prevalence and a wide range of hazards,which seriously threaten global human health.Increasing attention focus on the effect of the indoor air pollution(especially biomass fuel fumes)as the other key risk factor on the development of COPD.As a heterogeneous disease,COPD manifest the complex and diversity of pathological changes and pathogenesis.The difference of the pathological changes and clinical manifestation between the biomass fuel associated COPD and tobacco smoke induced COPD,and the marked difference is that the damage in lung of biomass fuel induced COPD take placed in the small airway,which remodeling after the recurrent injury and repair,especially in the early stage of COPD.Airway remodeling,resulting from small airway fibrosis,is the feature of early pathological changes in COPD,and is also the main site of airflow limitation.In addition to the contraction character,the function of the proliferation and the secretion of airway smooth muscle cells play a key role in the process of airway remodeling.And the disorder of extracellular matrix(ECM)is an important part of small airway fibrosis.As a member of ECM protein molecule,the expression of the matrix protein TNC increases abnormally in small airway in patients with COPD.But the detail mechanism in the development of COPD still not clear.The report on the ECM deposition in the airway remodeling of COPD caused by biomass fuel was less.Whether the profibrotic factor TGF-?participated in the airway remodeling in biomass fuel induced COPD remains to be further explored.This study aimed at to explore the effect of biofuels on the airway remodeling of COPD,especially the deposition of ECM.And from the following aspects:the biofuel smoke-induced rat model of COPD and BRPM2.5 stimulated the cultured human bronchial smooth muscle cells(HBSMC)in vitro to detect the protein expression of ECM molecule and the proliferation and the inflammation.In addition,to further investigate on the role of deposition matrix protein TNC in the of proliferation,the expression of ECM protein and the status of inflammation of HBSMC induced by TGF-?₁,contributed to airway remodeling in the development of COPD,and then provides a theoretical basis for understanding the involvement of biofuels in the small airway remodeling of COPD.Part I:Increased expression of TNC and TGF-?₁ in COPD of Rat exposed to biomass fuel.Objective biomass fuel is one of the risk factors for chronic obstructive pulmonary disease,the main pathological changes include chronic airway inflammation and small airway remodeling in chronic obstructive pulmonary disease;and ECM deposition was contributed to small airway fibrosis.This section focuses on the abnormal changes of lung morphology,the conditions of pulmonary and systemic inflammation,the deposition of ECM and the expression of TGF-?₁ in rats were exposed to chronic biomass fuel.Methods Twenty-four male SD rats were randomly divided into two groups to receive clean air(CON)and biomass fuel(BMF)exposure for 1 month and 6 months respectively.Rat lung tissue and serum were collected.HE staining was used to observe the changes of lung tissue structure and airway remodeling.The expression of cytokines in lung tissue and serum was detected by Luminex multi-cytokine kit.The ECM protein deposition and the expression of TGF-?₁in lung and airway was observed by immunohistochemistry.The protein expression of TNC and TGF-?₁in serum was detected by ELISA.Results 1.Morphological changes in the lungs of rats:After 6 months of biomass fuel exposure,the mean linear intercept of alveoli(MLI)and the thickness of small airway wall was significantly increased in BMF group compared with the values in clean air group in rats(MLI:76.38±9.16?m vs 50.95±1.31?m,P<0.05;the thickness of small airway:18.45±1.43?m²/?m vs 15.24±1.31?m²/?m,P<0.05);and the?-SMA protein expression of the smooth muscle layer and the percentage of the depth of the smooth muscle layer accounted for the airway thickness(WA%)was markedly increased in BMF group than the values in clean air group in rats(?-SMA protein expression:2.38±0.15 vs 1.64±0.17,P<0.05;WA%:11.77±1.18%vs 8.34±0.99%,P<0.05);2.Inflammation levels of lung and serum in rats:the levels of chemokines MCP-1,MIP-1?,RANTES,inflammatory factors IL-1?,IL-1?,IL-6,IL-17A and IFN-?were significantly upregulated in lung tissue and the levels of chemokines G-CSF,MCP-1,MIP-1?,RANTES;inflammatory factors IL-1?,IL-2,IL-6,IL-13,IL-17A and IFN-?,VEGF in lung serum in rats exposed to biomass fuel for 1 month and 6 months compared with the value in the clean air group,respectively;3.The expression of TGF-?₁in the serum and lung of rat:a.Compared with the clean air group,the expression of TGF-?₁significantly increased in rat serum in BMF groups both 1 month and 6 months by ELISA.(1 Mon:49.53±2.67pg/m L vs 64.01±4.55 pg/m L,P<0.05;6 Mon:49.42±2.07 pg/m L vs 60.01±3.68 pg/m L,P<0.05);b.Compared with the clean air group,the expression of TGF-?₁significantly up-regulated in rat lung tissue in BMF groups at 6 months by ELISA.(1 Mon:23.75±1.99pg/mg vs 29.83±2.13 pg/mg,P>0.05;6 Mon:21.16±2.69 pg/mg vs 31.11±2.13 pg/mg,P<0.05);c.Compared with the clean air group,the expression of TGF-?₁significantly up-regulated in rat lung tissue in BMF groups at 6 months by IHC.(10.19±0.75 vs13.21±0.84,P<0.05);4.The expression of ECM protein in rat lung:the high expression of ECM proteins,including glycoprotein TNC and FN,proteoglycans VCAN and collagen I and collagen IV in lungs and small airway in rats were exposed to biomass fuel for 6 months relative to control group(TNC:6.27±0.79 vs 3.26±0.55;VCAN:3.01±0.68 vs 3.24±0.54;FN:2.35±0.34 vs 0.93±0.25;Collagen I:9.18±1.16 vs 4.12±0.61;Collagen IV:13.11±1.51 vs 8.55±1.02;P<0.05),and no difference of the expression of collagen III(Collagen III:10.23±1.49 vs 9.01±1.36;P>0.05);5.The correlation analysis between TGF-?₁ expression and ECM proteins expression in rat lungs:the protein expression of TGF-?₁ was significantly related to glycoproteins TNC(r=0.725),FN(r=0.788),proteoglycan VCAN(r=0.711)and Collagen I(r=0.805),Collagen IV(r=0.830),(P<0.05);there is no correlation between TGF-?₁ and Collagen III(P>0.05).Summary The increased of emphysema and airway remodeling in lungs and small airway after biomass fuel chronic exposure for 6 months,and accompanied by the changes of the conditions of lung inflammation and systemic inflammation.Increased smooth muscle cell maybe play a key role in the process of remodeling and inflammation in small airway;the increased expression of TGF-?₁ and the deposition of ECM protein of TNC,VCAN,FN,Collagen I and Collagen IV molecules around the small airways in the lung in the exposed rats,with the most significant difference in the abundance of the matrix protein TNC.And the elevated protein expression of TGF-?₁was significantly correlated with increased expression of ECM protein.Part II:Effect and mechanism of BRPM2.5 on inflammation in HBSMCObjective Chronic airway inflammation is a main pathological change in COPD.The function of smooth muscle cells secreting inflammatory cytokines has involved in the pathological process in the development of COPD.To discuss the effect and of the role of TGF-?pathway in the secretion of HBSMC by BRPM2.5.Methods HBSMC were stimulated at different time-points by different concentrations of BRPM2.5,and pretreated with TGF-?neutralizing antibody,the inhibitor of ALK5and the inhibitors of MAPK pathway(ERK/,respectively)P38)and AKt pathway before incubation with BRPM2.5,respectively.The cell activity was determined by the CCK8,and the m RNA and protein expression of IL6 and IL8 were detected by real-time Q-PCR and ELISA,respectively.Results1.BRPM2.5 induced the increased of IL6 and IL8 m RNA and protein expression in a time dependent and a concentration dependent manner in HBSMC:the expression of IL8 m RNA and protein in cells treated with BRPM2.5 in dose-dependent manner were higher than the DMSO groups(P<0.05),and were increased in 4?g/m L BRPM2.5incubation at indicated time compared with DMSO group(P<0.05);the m RNA and protein expression of IL6 were increased in HBSMC treated with 4?g/m L BRPM2.5 at48h or 72h,compared to without BRPM2.5 incubation(P<0.05);2.The TGF-?neutralizing antibody inhibit BRPM2.5 induced the secretion of IL8:compared with incubated with BRPM2.5 groups,the IL6 and IL8 m RNA and protein expression in cells treated with 1?g/m L TGF-?before incubated with BRPM2.5 were no difference(P>0.05);however the IL8 m RNA and protein expression in the groups of BRPM2.5 treatment after pretreated with 10?g/m L TGF-?were decreased(P<0.05),and no change in IL6 expression.3.The inhibitor of ALK5,ERK1/2,p38 and Akt inhibit the m RNA and protein expression of IL6 and IL8 in HBSMC:compared with cells incubated with BRPM2.5,the IL6 m RNA and protein expression in cells incubated with TGF-?₁ after pretreated with the inhibitor of ALK5,P38 and Akt were down-regulated,respectively(P<0.05);and for the m RNA and protein expression of IL8 in the cells which pretreated with inhibitor of ALK5,ERK1/2 and P38 then incubated with TGF-?₁were less than the TGF-?₁ treatment groups(P<0.05).4.BRPM2.5 promote the release of TGF-?₁ in 16HBE,but not in HBSMC:compared with DMSO groups,the levels of TGF-?₁protein in the supernatant were increased in the treatment of BRPM2.5 groups on 16HBE(P<0.05);but no difference in the protein expression of TGF-?₁ in HBSMC;5.BRPM2.5 decreased m RNA expression of TNC and increased m RNA expression of VCAN in HBSMC,and there is no difference in the m RNA expression of other ECM proteins after BRPM2.5 treatment:compared with control groups,the BRPM2.5treatment downregulated the m RNA expression of TNC,and upregulated the m RNA expression of VCAN in HBSMC(P<0.05).Summary BRPM2.5 stimulate the secretion of IL6 and IL8 through the TGF-?/LAK5pathway and non-canonical pathway(ERK/P38/Akt)in HBSMC.BRPM2.5 promote the release of TGF-?₁ in 16HBE,but not in HBSMC.Part III:TNC participates in the COPD-like function changes of airway smooth muscle induce by TGF-?₁Objective The proliferation and the ECM protein production of the smooth muscle cell involved in the process of the small airway remodeling,which contributes to the airflow limitation in COPD.To examine the effects and mechanisms TGF-?₁ on the proliferation,the secretion of ECM proteins and IL6,IL8 in bronchial smooth muscle cells(HBSMC),and investigate the role and mechanism of matrix protein TNC mediated the proliferation,the expression of other ECM proteins and secretion of IL6and IL8 induced by TGF-?₁ in this section.Methods 1.HBSMCs were stimulated with TGF-?₁ in time and concentration dependent manner,and the real-time quantitative PCR and Western blot were used to determine the m RNA and protein expression of ECM protein TNC,FN,Collagen I and?-SMA in cells,respectively.2.HBSMCs were stimulated with 2ng/m L TGF-?₁ for 48h,the proliferation of HBSMCs were determined by Immunofluorescence and Flow Cytometry;HBSMCs were incubated with TGF-?₁ in time-and concentration-dependent manner,the expression of IL6 and IL8 expression were determined by ELISA.3.The inhibitor of TGF-?receptor ALK5,and the inhibitor of ERK/P38/Akt pathway pretreatment before the stimulation of TGF-?₁,and the real-time quantitative PCR and Immunoblotting to detect the expression of m RNA and protein of ECM proteins include TNC,FN,Collagen I and?-SMA.4.After transfected the si RNA of SMAD2/SMAD3 and TNC,the HBSMC were stimulated with TGF-?₁ for indicated time,and the real-time QPCR and Immunoblotting were used to detect the expression of m RNA and protein of ECM proteins include TNC,FN,Collagen I and?-SMA;and the phosphorylation level of SMAD2 and SMAD3 were determined by western blot after TNC-si RNA transfection in the stimulation of TGF-?₁;the expression of IL6 and IL8 were detected by ELISA.Result1.TGF-?₁ induced the proliferation,the expression of ECM proteins include TNC,FN,Collagen I and?-SMA and the secretion of IL6 and IL8 in HBSMC:Compared with the control group without incubated with TGF-?₁,the increased expression of proliferation,the upregulated of the TNC,FN,Collagen I and?-SMA m RNA and protein expression and the higher secretion of IL6 and IL8 in HBSMCs incubated with TGF-?₁.(P<0.05)2.SMAD2/SMAD3-si RNA inhibit-induced the m RNA and protein expression of?-SMA and ECM proteins include TNC,FN,Collagen I:compared with stimulated with TGF-?₁after transfected with NC-si RNA,the expression of?-SMA and ECM proteins include TNC,FN,Collagen I in the cell incubated with TGF-?₁after transfected with SMAD2-si RNA or SMAD3-si RNA were reduced,respectively(P<0.05).3.The inhibitor of ALK5,ERK1/2,p38 and Akt inhibit the expression of?-SMA and ECM proteins include TNC,FN,Collagen I:compared with cells incubated with TGF-?₁,the TNC m RNA and protein expression in cells that incubated with TGF-?₁ after pretreated with the inhibitor of ALK5,ERK1/2,P38 and Akt were down-regulated,respectively(P<0.05);and for the m RNA and protein expression of FN,Collagen and?-SMA in the cells which pretreated with inhibitor of ALK5,P38 and Akt then incubated with TGF-?₁were less than the TGF-?₁ treatment groups(P<0.05).4.TNC-si RNA attenuate TGF-?₁-induced proliferation,the expression of FN and the secretion of IL6,IL8 in HBSMC:compared with stimulated with TGF-?₁after transfected with NC-si RNA,the expression of FN,and the expression of IL6,IL8 in the cell incubated with TGF-?₁after transfected with TNC-si RNA were reduced,respectively(P<0.05).5.TNC-si RNA attenuate TGF-?₁-induced the levels of phosphorylation of SMAD2 and SMAD3 in HBSMC:compared with transfected with NC-si RNA,the cells transfected with TNC-si RNA downregulated the levels of phosphorylation of SMAD2 and SMAD3in response to TGF-?₁ in HBSMC(P<0.05).Summary TGF-?₁ can promote the proliferation and the expression ECM protein in HBSMC via the classical SMAD-dependent pathway and nonclassical SMAD-independent pathway.The matrix protein TNC was participated in the proliferation,ECM protein FN expression and the secretion of IL6 and IL8 induced by TGF-?₁ in HBSMC via affected the SMAD2/3 phosphorylation.Conclusions1.Chronic biomass fuel exposure can cause the emphysema and airway remodeling in rats,accompanied by inflammation of the lungs and systemic,and the abnormal deposition of ECM proteins in the lungs;2.TNC and TGF-?₁ highly expression in lungs of rats exposed to biomass fuel for 6months;3.TGF-?₁significantly induced the proliferation and differentiation,the increased expression of ECM proteins,and the secretion of IL6 and IL8 through the classical and non-classical pathways in HBSMC;4.TNC mediated TGF-?₁ induced the proliferation,the expression of FN and the secretion of IL6 and IL8 by acting on the phosphorylation of transcription factor SMAD2 and SMAD3;5.BRPM2.5 induced the secretion of IL6 and IL8 directly through the pathway of MAPK and AKt or indirectly through TGF-?that paracrine form airway epithelium in HBSMC.