Study On The Mechanism Of CeRNA Mediated β-Elemene’s Preventive And Therapeutic Effects On Benign Airway Stenosis

Background and purpose: With the increasing incidence of endotracheal tuberculosis,post-traumatic tracheal intubation and open tracheotomy,the incidence of benign airway stenosis also increased.With the rapid development of interventional pulmonology,bronchoscopic interventional technique provides a new and effective treatment for patients with benign airway stenosis and has become one of the most important treatment.When some patients undergo bronchoscopic interventional treatment,the vicious cycle of "stenosis-secondary injury after interventional therapy-restenosis after repair" often appears,which pose a difficult problem to be solved urgently in the field of respiratory interventional therapy.The development and evolution of granulation tissue is the key link in the whole process of injury repair and tissue remodeling,and fibroblast,as one of the major constituent cells of granulation tissue,plays a leading role.Therefore,positive regulation of the function of airway granulation fibroblasts to improve the repair process and optimize the repair outcome is a key to prevent restenosis after endobronchial interventional therapy.It has been found that the active ingredient β-elemene extracted from Chinese herbal medicine has anti-fibrosis effect,and our previous study also found that β-elemene can inhibit the proliferation of airway granulation fibroblasts and promote their apoptosis.However,the exact mechanism and the efficacy of β-elemene in the prevention and treatment of airway stenosis still need to be further studied.Long non-codingRNAs(lncRNAs)can affect gene expression at multiple levels.As competitive endogenousRNAs(ceRNAs)of microRNAs(miRNAs),it regulates the expression of downstream genes,which plays an important role in the occurrence and development of many diseases.It has been reported that β-elemene can inhibit the proliferation of malignant cells by targeting lncRNAs.This study intend to take lncRNAs as a starting point to elucidate the possible mechanism of ceRNA mediated β-elemene regulating the function of airway granulation fibroblasts and thus inhibiting airway granulation hyperplasia,so as to provide theoretical basis and experimental evidence for the clinical application of β-elemene in the prevention and treatment of benign airway stenosis.Methods: 1.Primary human airway granulation fibroblasts(PHAGF)and primary human airway normal fibroblasts(PHAF)were cultured,purified and identified to establish in vitro cell models.High-throughput lncRNA Microarray(containing mRNAs)were used to screen lncRNAs and mRNAs differentially expressed before and after IC50-β-elemene treatment in PHAGF.Bioinformatics analysis was performed to enrich the pathways related to cell proliferation and apoptosis and construct the ceRNA network regulating the pathway.2.The target ceRNA axis to be studied was selected,and the expression differences of ceRNA axis and target pathway in PHAF and PHAGF were compared.Then dual luciferase reporter assay,RT-q PCR,Western blotting and flow cytometry were used to verify that β-elemene regulates the activity of the target pathway by aiming the target lncRNA and thereby influences the cell proliferation and apoptosis of PHAGF through the ceRNA mechanism.3.A New Zealand rabbit model of benign airway stenosis was established by our improved method,then a simple endoscope was introduced for observation and evaluation,and the pathological evolution curve of the model was drawn.The feasibility of endotracheal topical injection assisted by simple endoscope was also explored.4.After endotrecheal topical application of β-elemene in the airway granulation hyperplasia stage in animal models,the effect ofβ-elemene on inhibiting airway granulation hyperplasia and reducing airway stenosis rate was determined at the overall level in vivo through endoscopic evaluation and pathological HE staining,and the mechanism of β-elemene was further verified by immunofluorescence detection of cell proliferation marker Ki67,Tunel test of cell apoptosis phenotype and examining of target ceRNA axis and target pathway molecule expression by immunohistochemistry,RT-q PCR and Western blotting.Results: 1.PHAF and PHAGF were successfully cultured in vitro,and theβ-elemene regulated ceRNA network which containing lncRNA-MIR143HG/microRNA-miR-1275/ILK target axis of interest in PHAGF was constructed.2.Compared with PHAF,there were abnormal changes in the MIR143HG/miR-1275/ILK axis and the ILK/Akt pathway in PHAGF.MIR143 HG can competitively bind to miR-1275 to release its inhibition of ILK,and MIR143 HG and ILK are mutual ceRNA of miR-1275.By down-regulating the expression of MIR143 HG in PHAGF,β-elemene can affect the expression of Cyclin D1 and Bcl-2,which are effector genes of the ILK/Akt pathway,and finally inhibit the proliferation of PHAGF and promote its apoptosis.3.The modified method can effectively construct the New Zealand rabbit model of benign tracheal stenosis,with high stability and high stenosis rate.The pathologic evolution curve of the model can accurately reproduce the pathologic process of human benign airway stenosis.The simple endoscope can be used for model’s follow-up,and endotrecheal topical injection assisted by it is feasible.4.Topical injection ofβ-elemene can effectively inhibit the hyperplasia of airway granulation tissue and finally alleviate airway stenosis in model rabbits.Histological analysis showed that the proliferation and apoptosis of fibroblasts in the airway granulation tissues were decreased after β-elemene treatment,and the expression of MIR143 HG,miR-1275 and ILK/Akt pathway molecules in the granulation tissues were consistent with the in vitro experiment.Histological analysis showed that the proliferation and apoptosis of fibroblasts in the airway granulation tissues were decreased afterβ-elemene treatment,and the expression of MIR143 HG,miR-1275 and ILK/Akt pathway molecules in the granulation tissues were consistent with the in vitro experiment.Conclusion: The ILK/Akt pathway of PHAGF was abnormally activated,and lncRNA-MIR143 HG and ILK are mutual ceRNA of microRNA-1275.β-elemene can inhibit the cell cycle progression of PHAGF and promote its apoptosis by regulating the MIR143HG/miR-1275/ILK axis,thus effectively inhibiting the proliferation of airway granulation tissue and ultimately alleviating airway stenosis.The MIR143HG/miR-1275/ILK axis is a potential therapeutic target for benign airway stenosis,and β-elemene is a potential drug for clinical treatment of benign airway stenosis.