The Preliminary Study On The Role Of SIRT1 In Tracheal Stenosis Induced By Tracheal Injury

Benign tracheal stenosis may be due to a variety of diseases.The iatrogenic etiologies such as tracheal intubation,tracheostomy and surgical airway injury,as well as tracheobronchial tuberculosis,benign tumors and trauma are all common causes of benign tracheal stenosis.Benign tracheal stenosis frenquently leads to airway inflammtion,granulation tissue proliferation and hypertropic scars,which is often accompanied by severe airway stenosis and even lead to life-threatening symptoms.Although the treatment techniques of benign tracheal stenosis continue to be improved and there are many procedures including endotracheal ballon dilatation,thermal ablation and stent implantation,there is still a high recurrent rate up to 40-70%.How to prevent repeated recurrences of acquired tracheal stenosis is a challenging problem for clinical doctors.Therefore it makes a lot of sense to explore pathogenesis and prophylaxis as well as treatment of tracheal stenosis.Researches have shown that there are inflammation and fibrosis injuries in tracheal stenosis induced by tracheal injury in the rabbit model.The14-member-ring macrolide antibiotic EM has anti-inflammtory properties and can effectively alleviate tracheal fibrosis that may be beneficial in tracheal stenosis,but the efficacy of this medicine for the treatment of tracheal stenosishas not yet been fully characterized.Recent researches have shown that class III histone deacetylase SIRT1 plays a pivotal role in inhibiting inflammation and fibrosis.SIRT1 may be a therapeutic target for some inflammatory and fibrotic diseases.However,it is rarely reported about the role of SIRT1 in tracheal stenosis induced by tracheal injury.It remains unclear whether SIRT1 involves in the pathogenesis of tracheal stenosis and whether EM plays a role in mitigating tracheal stenosis induced by tracheal injury via regulating the expression of SIRT1.Therefore,the above issues are still to be studied.The aim of this preliminary study was to investigate the pathogenesis of tracheal stenosis induced by tracheal injury and observe the expression of SIRT1 in tracheal stenosis tissues and tracheal fibroblasts,in addition,this study had a further discussion about the effectiveness and possible underlying mechanism of EM and BUD.The main contents of this study are as follows:PARTⅠ: ROLES OF INFLAMMATION AND FIBROSIS FACTORS AND DRUGS INTERVENTION IN TRACHEAL STENOSIS INDUCED BY TRACHEAL INJURY IN THE RABBIT MODELObjectives: The pathological mechanisms for tracheal stenosis induced by tracheal injury remain not fully understood.This study aimed to establish an animal model to investigate the roles of inflammation and fibrosis factors in tracheal stenosis induced by tracheal injury,and to explore the effectiveness of drugs intervention on tracheal stenosis.Methods: Twenty four specific pathogen-free rabbits were enrolled in thisstudy.Trancheal stenosis was induced in rabbits by a trauma injury to the mucosa with a nylon brush scraping of the tracheal mucosa and suture.The rabbits were divided into four groups,model group(A group),EM group(B group),BUD group(C group),EM combined BUD group(D group).Rabbits were given daily medication from 1 week before operation to the 9th day after operation,rabbits of each group were euthanized on the 10 th day after the operation.Serum TGF-β1,VEGF,IL-6,IL-8 levels were evaluated by ELISA.HE staining was used to assess pathological changes in the airway mucosa and to measure the tracheal lumen in order to evaluate the degree of stenosis in each group.IHC was used to detect the expression of COL-I and COL-III.Results: Serum TGF-β1,VEGF,IL-6,IL-8 levels of B group and D group decreased significantly compared with A group(P<0.05).H&E staining revealed significant cross-sectional tracheal stenosis with proliferation of granulation tissue caused by variable inflammatory cell infiltration,angiogenesis,epithelial erosion and hyperplasia in A group,the tracheal stenosis degree of B group(24.3±4.4)% and D group(15.6± 2.0)% decreased substantially compared with A group(53.3 ± 4.4)%(P < 0.05).COL-I and COL-III expressed in each group,Rabbits in B group and D group had significantly less COL-I and COL-III expression compared with A group(P<0.05).Conclusions: This study demostrates that increased inflammation and fibrosis factors involved in the pathological mechanisms of tracheal stenosis induced by tracheal injury.EM combined with glococorticoid might play a synergistic role in the treatment of tracheal stenosis by reducing inflammation and fibrosis.PART Ⅱ: EXPRESSION OF SIRT1 IN TRACHEAL STENOSIS TISSUES AND DRUGS INTERVENTION IN TRACHEAL STENOSIS INDUCED BY TRACHEAL INJURY IN THE RABBIT MODELObjectives: This study aimed to explore the expression of SIRT1 in the context of tracheal stenosis in the rabbit animal experimental model,and to explore the effectiveness of drugs intervention on tracheal stenosis.Methods: Twenty four rabbits were enrolled in this study.Trancheal stenosis was induced in rabbits by a trauma injury to the mucosa with a nylon brush scraping of the tracheal mucosa and suture.The rabbits were divided into four groups,model group(A group),EM group(B group),budesonide group(C group),EM combined with budesonide(D group).Rabbits were given daily medication from 1 week before operation to the 9th day after operation,rabbits of each group were euthanized on the 10 th day after the operation.The expression of SIRT1 were evaluated by IHC.Results: Expression of SIRT1 was weakly detected in A group,expression of SIRT1 in B group and D group increased significantly compared with A group(P<0.05).Conclusions: EM and EM combined with budesonide increased SIRT1 expression in tracheal stenosis tissues of rabbits with tracheal stenosis induced by tracheal injury.EM reduced the inflammation and fibrosis of tracheal stenosis induced by tracheal injury,which maybe partly through enhancing the expression of SIRT1.PART Ⅲ : A PRELIMINARY MECHANISTIC STUDY OF THE EFFECTS OF EM ON MITIGATING INFLAMMATION AND FIBROSIS IN TRACHEAL FIBROBLASTSObjectives: This study aimed to investigate the effects of EM or EM combined with BUD on TGF-β1 induced fibrosis and inflammation of tracheal fibroblasts and explore the possible underlying molecular mechanism.Methods: Fibroblasts isolated from biopsies of patients with acquired tracheal stenosis were cultured.The cytokines in cell culture supernatant and cell gene expression of SIRT1,COL-1,IL-6 were assessed in 6 conditions:(1)fibroblast growth medium(NC group),(2)fibrobalst growth medium with10ng/m L TGF-β1(TGFβ1 group),(3)fibroblast growth medium with 10ng/m L TGF-β1 and 1mg/L erythromycin(EM1 group),(4)fibroblast growth medium with 10ng/m L TGF-β1 and 10mg/L erythromycin(EM10 group),(5)fibroblast growth medim with 10ng/m L TGF-β1 and 10 n M budesonide(BUD group),(6)fibroblast growth medium with 10ng/m L TGF-β1 and 10mg/L erythromycin and 10 n M budesonide(EM10+BUD group).The production of cytokines in cell culture supernatant were evaluated by ELISA,The expressions of SIRT1 and IL-6,COL-1 were determined by immunofluorescence or real time-PCR.Results:(1)TGF-β1 increased IL-6 and IL-8 level in fibroblasts culture supernatant,EM1 group,EM 10 group,BUD group and EM10+BUD group reduced TGF-β1 induced inflammation in tracheal fibroblasts(P < 0.05).(2)TGF-β1 also increased IL-6 and COL-1 m RNA expression.EM1 group,EM 10 group and EM10+BUD group increased SIRT1 m RAN expression compared with TGF-β1 group(P < 0.05).EM1 group,EM 10 group,BUD group and EM10+BUD group reduced TGF-β1 induced Changes of IL-6 and COL-1m RNA in tracheal fibroblasts(P<0.05).(3)There was no expression difference of SIRT1 between TGF-β1 group and NC group.EM10 group and EM10+BUD group increased SIRT1 expression.Conclusions:(1)EM demonstrated an anti-inflammation and anti-fibrosis effect by significantly reducing the expression of IL-6 and IL-8,and reducing collagen deposition of human acquired tracheal stenosis fibroblasts in vitro.This effect was most pronounced when glucocortitoid was present.SIRT1 may be involved in this protective effect.(2)EM combined with BUD indicate a promising adjuvant therapy for the treatment of acquired tracheal stenosis.