Study On The Mechanism Of CTHRC1 Promoting Airway Remodeling In Asthma By Modulating Epithelial-Mesenchymal Transition

Objective: Asthma is a chronic respiratory disease.In recent years,the incidence of asthma is increasing in China,leading to a public health problem.Although only 5% to 10% may suffer from severe asthma,it leads to 60% of the medical costs and a huge burden on society and families.Asthma is a heterogeneous disease characterized by chronic airway inflammation,airway hyperresponsiveness,and airway remodeling.Airway remodeling can lead to irreversible lung function impairments,airway obstruction,loss of elastic recoil,glucocorticoid resistance,and the occurrence of severe asthma.Epithelial-mesenchymal transition(EMT)is an important mechanism of tissue repair after damage,and also considered to be a hallmark pathological change and a classic mechanism of fibrosis and airway remodeling in asthma.Understanding the molecular mechanism of EMT in airway remodeling is an important scientific issue in the field of respiratory health.Collagen triple helix repeat containing-1(CTHRC1),a secreted glycoprotein,plays extensive roles in various pathological processes,including malignant tumors,fibrosis,and inflammatory diseases.CTHRC1 is significantly upregulated in several fibrosis and inflammatory diseases,which may provide a new target for therapy.However,the results of related studies are controversial and lack of studies on chronic inflammation and fibrosis in airway remodeling of asthma.In addition,bioinformatics analysis of publicly available databases showed that CTHRC1 expression was increased in bronchial epithelial cells of patients with allergic asthma,especially in patients with severe asthma.Therefore,it is speculated that CTHRC1 may be involved in the pathogenesis of chronic airway inflammation,fibrosis and airway remodeling in asthma.CTHRC1 is implicated in cancer related EMT by activating TGF-β/Smad,Wnt/β-catenin,and Akt/Erk/Snail signaling pathways.However,the functional role and molecular mechanism of CTHRC1 in asthma EMT and airway remodeling has not been explored.Snail,a core EMT transcription factor,can be stabilized by GSK-3β.The interaction between GSK-3β and Snail is regulated by a variety of factors.Activated Akt has been reported to inhibit GKS-3β activation by the induction of GKS-3β phosphorylation.Activation of the Akt/GSK-3β/Snail pathway is involved in the occurrence of EMT in tumors and pulmonary fibrosis.CTHRC1 knockdown has been reported to inhibit phosphorylation of Akt and Erk in tumor,leading to EMT reduction.Therefore,CTHRC1 may be involved in asthma EMT via activation of Akt/GSK-3β/Snail signaling pathway.In the present study,OVA-induced asthmatic mouse model and TGF-β1-stimulated human bronchial epithelial cells(BEAS-2B)were conducted to investigate:(1)The expression and localization of CTHRC1 in vitro and in vivo;(2)Whether CTHRC1 regulates airway inflammation,EMT,fibrosis,and airway remodeling in asthma;(3)The potential molecular mechanism by which CTHRC1 regulates bronchial epithelial EMT and whether by activating the Akt/GSK-3β/Snail signaling pathway.Methods: 1.To conduct asthmatic airway remodeling mouse model,16 mice werrandomly assigned to two groups including the control group and OVA group.In the OVAgroup,mice were intraperitoneal injection of 10 μg of ovalbumin and 1 mg of aluminumhydroxide gel on days 1,7,and 14.Subsequently,the mice were received with aerosolized5% OVA in saline 3 days/week from day 21 to day 77 for 30 min per day,while the contromice were injected with normal saline and challenged with saline in the same manner.Thealveolar lavage fluid(BALF)was collected.Total and differential counts of inflammatorycells in BALF were performed.Cytokine levels including IL-4,IL-5,IL-13,and IFN-γ inBALF supernatants were measured by ELISA.Total protein level in BALF was alsomeasured.Mouse blood was collected and serum OVA specific Ig E content was assayedby ELISA.The lung tissues were collected and pathological changes were evaluated byHE,PAS,and Masson staining analysis to assess the degree of inflammatory celinfiltration,goblet cell hyperplasia,and collagen deposition,respectively.The protein andm RNA expression levels of CTHRC1 were analyzed by Western blot and q RT-PCRrespectively.The expression and localization of CTHRC1 were detected byimmunofluorescence staining.In TGF-β1-stimulated BEAS-2B cells,the protein andm RNA expression levels of CTHRC1 were analyzed by Western blot and q RT-PCRrespectively,and the expression and location of CTHRC1 were detected byimmunofluorescence staining.2.To knockdown CTHRC1 expression in asthmatic airway remodeling mouse model,lentivirus containing sh RNA specific for CTHRC1 was intratracheally delivered into the OVA mice.32 mice were randomly assigned to four groups including the control group and OVA group,OVA+sh-NC group,and OVA+sh-CTHRC1 group.Counts of inflammatory cells,cytokine levels and total protein level in BALF,and OVA specific Ig E level in serum were measured.The pathological changes of lung tissues were evaluated by HE,PAS,and Masson staining analysis to assess the degree of inflammatory cell infiltration,goblet cell hyperplasia,and collagen deposition,respectively.The protein and m RNA expression levels of CTHRC1 in lung tissue were measured by Western blot and q RT-PCR,respectively,and the expression and location of CTHRC1 were detected by immunofluorescence staining.E-cadherin,N-cadherin,α-SMA,fibronectin,and Snail protein expressions in lung tissue were measured by Western blot.The expressions of Ecadherin,N-cadherin,and α-SMA were also detected by immunofluorescence staining.The BEAS-2B cells were transfected with si RNA specific for CTHRC1 to knockdown CTHRC1 expression,and were stimulated by TGF-β1.The cells were divided into four groups: control group,TGF-β1 group,TGF-β1+si-NC group,and TGF-β1+si-CTHRC1 group.The BEAS-2B cells were transfected with CTHRC1 overexpression plasmid to form the CTHRC1-overexpression group,and were stimulated by TGF-β1.The cells were divided into six groups: control group,Vector group,CTHRC1-OE group,TGF-β1 group,TGF-β1+Vector group,TGF-β1+ CTHRC1-OE group.The protein and m RNA expression levels of CTHRC1 in cells were measured by Western blot and q RT-PCR,respectively,and the expression and location of CTHRC1 were detected by immunofluorescence staining.E-cadherin,N-cadherin,α-SMA,and fibronectin protein expressions in cells were measured by Western blot.The expressions of E-cadherin and N-cadherin were also detected by immunofluorescence staining.3.The lung tissues of control group,OVA group,OVA+sh-NC group and OVA+shCTHRC1 group were used to measure p-Akt,Akt,p-GSK-3β,GSK-3β and nuclear Snail protein expressions by Western blot.The BEAS-2B cells were transfected with si RNA specific for CTHRC1 and stimulated by TGF-β1.The cells were divided into four groups: control group,TGF-β1 group,TGF-β1+si-NC group,and TGF-β1+si-CTHRC1 group.The BEAS-2B cells were transfected with CTHRC1 overexpression plasmid and stimulated by TGF-β1.The cells were divided into four groups: control group,TGF-β1 group,TGF-β1+Vector group,TGF-β1+ CTHRC1-OE group.P-Akt,Akt,p-GSK-3β,GSK-3β and nuclear Snail protein expression were measured by Western blot.The BEAS-2B cells were transfected with CTHRC1 overexpression plasmid,stimulated by TGF-β1,and treated by GSK-3β inhibitor(SB216763)and Akt inhibitor respectively.The cells were divided into four groups: TGF-β1+Vector group,TGF-β1+CTHRC1-OE group,TGF-β1+CTHRC1-OE+SB216763 group,TGF-β1+CTHRC1-OE+Akt inhibitor group.E-cadherin,Ncadherin,α-SMA,fibronectin,p-Akt,Akt,p-GSK-3β,GSK-3β and nuclear Snail protein expression were measured by Western blot.The expression of E-cadherin and N-cadherin were detected by immunofluorescence staining.Results: 1.(1)Compared to the control group,the inflammatory cell infiltration around bronchus and vascular,Th2 cytokine levels(IL-4,IL-5 and IL-13)in BALF,total protein level in BALF,serum OVA specific Ig E,goblet cell hyperplasia,mucus secretion,collagen deposition,and thickness of the bronchial wall and smooth muscle cell layer were obviously increased in the OVA-induced asthmatic mice,indicating that the asthmatic airway remodeling mouse model was successfully conducted.(2)The expression of CTRHC1 protein and m RNA was significantly increased in lung tissues of OVA-induced asthmatic mice compared with the control group.Immunofluorescence showed that CTHRC1 was mainly expressed in the bronchial epithelium.(3)The expression of CTRHC1 protein and m RNA was significantly increased in the TGF‐β1‐stimulated BEAS-2B cells compared with the control group.Immunofluorescence showed that CTHRC1 was mainly expressed in the cell membrane and cytoplasm of bronchial epithelial cells.2.(1)In the lung tissues of OVA-induced airway remodeling mice,CTHRC1 knockdown attenuated airway inflammation,goblet cell hyperplasia,mucus secretion,and thickness of the bronchial wall and smooth muscle cell layer.(2)The OVA induction resulted in a significantly decreased expression of epithelium marker E-cadherin,and increased expressions of transcription factor Snail,mesenchymal marker N-cadherin,and fibrosis marker α-SMA and fibronectin,indicating occurrence of EMT and fibrosis.Expression of E-cadherin was significantly upregulated,while expressions of Snail,Ncadherin,α-SMA,and fibronectin were downregulated in OVA-induced asthmatic mouse tissues following CTHRC1 knockdown,indicating suppression of EMT and fibrosis.(3)In BEAS-2B cells,TGF-β1 treatment caused a significant decreased expression of Ecadherin,and increased expressions of N-cadherin,α-SMA,and fibronectin,indicating occurrence of EMT and fibrosis.CTHRC1 knockdown markedly attenuated the decreased expression of E-cadherin,and increased expression of N-cadherin,α-SMA,and fibronectin in TGF‐β1‐stimulated BEAS-2B cells,indicating suppression of EMT and fibrosis.In BEAS-2B cells,the overexpression of CTHRC1 caused a significant decreased expression of E-cadherin,and increased expressions of N-cadherin,α-SMA,and fibronectin,indicating occurrence of EMT and fibrosis,regardless of TGF-β1 treatment.3.(1)The expressions of p-Akt,p-GSK-3β and nuclear Snail were increased in the lung tissue of OVA mice.CTHRC1 knockdown significantly reduced p-Akt,p-GSK-3β,and nuclear Snail levels,indicating suppression of Akt/GSK-3β/Snail pathway.(2)In TGF-β1-stimulated BEAS-2B cells,the expressions of p-Akt,p-GSK-3β,and nuclear Snail were increased.CTHRC1 knockdown markedly attenuated the TGF-β1-stimulated expressions of p-Akt,p-GSK-3β,and nuclear Snail,indicating suppression of Akt/GSK-3β/Snail pathway.CTHRC1 overexpression further aggravated the TGF-β1-stimulated expressions of p-Akt,p-GSK-3β and nuclear Snail,indicating activation of Akt/GSK-3β/Snail pathway.(3)In TGF-β1-stimulated BEAS-2B cells,SB216763 treatment further aggravated the increased expressions of p-Akt,p-GSK-3β,and nuclear Snail,decreased expression Ecadherin,and increased expressions of N-cadherin,α-SMA,and fibronectin,indicating SB216763 further promoted Akt/GSK-3β/Snail pathway activation and EMT caused by CTHRC1 overexpression.Akt inhibitor treatment,attenuated the increased expressions of p-Akt,p-GSK-3β,and nuclear Snail,decreased expression E-cadherin,and increased expressions of N-cadherin,α-SMA,and fibronectin,indicating Akt inhibitor suppressed Akt/GSK-3β/Snail pathway activation and EMT caused by CTHRC1 overexpression.Conclusions: 1.CTHRC1 expression was significantly increased in lung tissues of OVAinduced asthmatic airway remodeling mice and TGF-β1-stimulated BEAS-2B cells,and was predominantly detected in the cell membrane and cytoplasm of bronchial epithelial cells.2.In asthmatic airway remodeling mice,CTHRC1 knockdown indicated a significant reduction of airway inflammation,goblet cell hyperplasia,thickness of the bronchial wall and smooth muscle cell layer,fibrosis,and EMT,thereby reducing airway remodeling.In BEAS-2B cells,CTHRC1 knockdown attenuated TGF-β1-induced EMT and fibrosis,while CTHRC1 overexpression induced EMT and fibrosis,and promoted TGF-β1-induced EMT and fibrosis.3.CTHRC1 promoted EMT of bronchial epithelial cells in asthma,partially through activating the Akt/GSK-3β/Snail signaling pathway.