The Effect And Mechanism Of Pirfenidone On Fiber Hyperlpasia Of Airway

Objectives: As one type of airway stenosis,BCAS(Benign cicatricial airway stenosis)is caused by a variety of reasons.The postoperative restenosis of BCAS is a clinically urgent problem to be solved.Fibrosis may play an important role in BCAS,while biomarkers for early diagnosing and monitoring of BCAS fibrosis and effective drugs for BCAS are lacking.The purpose of this study was to initially evaluate the significance of fibrosis in BCAS,and further explore the role of PFD(Pirfenidone)in airway fibrous hyperplasia and the underlying molecular mechanisms.Finally,the role of PFD in BCAS was evaluated through a prospective clinical study.Methods: Firstly,clinical data,airway scar tissue samples and blood samples of BCAS patients were collected.HE staining,Masson staining and IHC(Immunohistochemistry)were used to analyze the fibrosis of scar tissue.Serum TGF-β1(transforming growth factor-β1)and PINP(Procollagen type I N propeptide)levels were measured using ELISA(Enzyme linked immunosorbent assay)and compared among the groups.Then,to study the effect of PFD on BEAS-2B,we used LDH release assay,CCK-8 assay,flow cytometry and scratch assay to analyze the cytotoxicity,cell proliferation,cell cycle and cell migration,respectively.Epithelial mesenchymal transformation(EMT)of BEAS-2B cells was induced by TGF-β1.The expression of N-cadherin,E-cadherin,vimentin,a-SMA and p-SMAD2/SMAD2 were determined by q RT-PCR(Real-time fluorescence quantitative polymerase chain reaction),WB(Western blot)and IF(Immunofluorescence).Finally,through a prospective observational study of 24 weeks,the efficacy of PFD combined with interventional bronchoscopy on airway fibrosis was observed in patients with BCAS.Results: Of all BCAS patients hospitalized due to dyspnea,there were87.4% with cicatrices stricture type TBTB(Tracheobronchial tuberculosis).The principal histological finding was squamous epithelialisation of the bronchial epithelial cells,fibrotic lesions with thickened submucosal layers and a large amount of collagen deposition in airway scar tissue.Compared with the control group,airway scar tissue had higher levels of TGF-β1,collagen I,N-cadherin,vimentin and α-SMA,and lower levels of E-cadherin.Furthermore,bronchoscope interventional therapy resulted in marked cell coagulative necrosis and collagen degeneration.Serum TGF-β1(Transforming growth factor-β1)and PINP(Procollagen type I N-propeptide)levels in TBTB group and non-TBTB group were both significantly higher than those in the control group(P<0.05),and there was no significant difference between the TBTB group and the non-TBTB group(P>0.05).Clinical significance of serum TGF-β1 and PINP levels in PTTS(Post-tuberculosis tracheobronchial stenosis):(1)Serum TGF-β1 and PINP levels in the airway stenosis group were both significantly higher than those in non-stenosis group(P<0.05);(2)Serum TGF-β1 and PINP levels in patients with airway stenosis and different clinical characteristics: Patients with atelectasis exhibited significantly higher serum TGF-β1 and PINP levels than those without atelectasis(P<0.05).Patients with mucus plugging had significantly higher TGF-β1 and PINP levels than those without mucus plugging(P<0.05).Patients with left main bronchus stenosis and those with right main bronchus stenosis had comparable TGF-β1 levels(P>0.05),while patients with right main bronchus stenosis had higher serum PINP levels than those with left main bronchus stenosis(P<0.05).Serum TGF-β1 levels were comparable between severe airway tracheal stenosis and mild-to-moderate airway tracheal stenosis(P>0.05),while serum PINP levels were higher in severe airway tracheal stenosis than those in mild-to-moderate airway tracheal stenosis(P<0.05).(3)The levels of serum TGF-β1 and PINP both increased significantly after the interventional bronchoscopy therapy(P<0.05).In the post-interventional bronchoscopy period,the serum TGF-β1 and PINP levels in the severe stenosis group at the baseline were also higher than those in the mild-to-moderate stenosis group.Bronchoscope interventional therapy resulted in increased serum TGF-β1and PINP levels(P<0.05).In the post-interventional bronchoscopy period,the serum TGF-β1 and PINP levels in the severe stenosis group at the baseline were both higher than those in the mild-to-moderate stenosis group,while none of these was statistically significant(P>0.05).When comparison of post-and pre-interventional bronchoscopy data was performed in each group,the increase in TGF-β1 and PINP levels was both revealed to be correlated with the amount of biopsies and median duration of the bronchoscopy procedure per patient,while none of these was statistically significant(P>0.05).(4)The baseline levels of serum TGF-β1and PINP in the subgroup with recurrence were both significantly higher than those in the non-recurrent subgroup(P<0.05).(5)The serum PINP levels were positively correlated with TGF-β1 levels in patients with airway stenosis(P<0.05),whereas the correlations were not significant in the non-stenosis group(P>0.05).(6)The area under the ROC curve for serum TGF-β1 levels to distinguish patients with airway stenosis from those without stenosis was 0.824(95% CI: 0.748-0.900)and that for PINP was 0.863(95% CI: 0.796-0.930).PFD had no cytotoxicity to BEAS-2B,and could inhibit cell proliferation,cell cycle,and cell migration of BEAS-2B.For TGF-β1-induced EMT in BEAS-2B cells,q RT-PCR showed that PFD inhibited the m RNA expression of N-cadherin,vimentin,a-SMA and total SMAD2,while promoted the expression of E-cadherin;WB and IF analyses showed that PFD inhibited the protein expression of N-cadherin,vimentin,a-SMA and p-SMAD2/SMAD2,while promoted the protein expression of E-cadherin.In addition,compared with interventional bronchoscopy,PFD combined with interventional bronchoscopy could significantly prolonged the average duration of airway intervention for cicatrices stricture type TBTB(P<0.05).Conclusions: Fibrosis is the main manifestation of BCAS,higher levels of TGF-β1 and EMT may involve in the occurrence and development of BCAS.Serum TGF-β1 and PINP levels may be used as fibrosis markers for the diagnosis and monitoring of BCAS.PFD inhibits TGF-β1-induced EMT in BEAS-2B cells through TGF-β1/SMAD2 signaling pathway.PFD significantly prolonged the average airway intervention period in patients with cicatrices stricture type TBTB.PFD is expected to be a new drug for the treatment of BCAS.