A New Method For Compresssion-established Animal Model And The Effect Of HIF-1α On Pathogenesis Of Benign Tracheoesophageal

Objective1.To explore the method of constructing rabbit model of tracheoesophageal fistula by magnetic compression.2.To screen the differentially expressed genes in rabbit tracheoesophageal fistula by bioinformatics method and provide meaningful biological targets for clinical prevention and treatment of tracheoesophageal fistula.3.By intervening HIF-1α,the formation of fistulas was observed in living animals and the formation of myofibroblasts was observed at the molecular level,so as to verify the effect of HIF-1α and myofibroblasts on the formation of tracheoesophageal fistulas.Methods1.The animal model of tracheoesophageal fistula was constructed by magnetic compression using flexible fiberoptic bronchoscopy to verify the feasibility of this modeling approach.2.Before fistula formation,the compression tissue between the two magnets was taken at the time points of 3,5,7,and 8 days after magnets placement,and the fistula tissue was detected using transcriptome sequencing technology,and the normal airway tissue 4 cm away from the fistula was used as a control to select the differential genes and signaling pathways that may be associated with benign acquired tracheoesophageal fistula formation by bioinformatics analysis.3.According to the results of transcriptome sequencing analysis,we selected the HIF-1α signaling pathway most related to ischemia,hypoxia,cell proliferation and other processes as the study subjects,and added HIF-1α inhibitors for intervention.The experiment was divided into four groups:blank control group,DMSO intravenous injection group,5 mg/kg YC-1 intravenous injection group,and 1 mg/kg YC-1 intravenous injection group.The fistula formation in each group was compared,and immunoblotting was used to detect the expression of α-SMA.Results1.Bronchoscope-assisted magnetic compression is not only feasible but also noninvasive in constructing a rabbit model of tracheoesophageal fistula;after fistula formation,Masson staining shows significant collagen deposition in the tissue around the fistula,HE staining shows increased neutrophils at the fistula,and significant inflammatory response.2.By RNA-seq,we found 2056,2235,166 and 3383 differentially expressed genes in compressed tissues on days 3,5,7 and 8 before fistula formation,respectively.KEGG enrichment analysis of these differentially expressed genes was performed using clusterProfiler software to obtain signaling pathway annotation information of differential genes,and the results revealed that HIF-1α signaling pathway,TNF-1 signaling pathway and JAK-STAT signaling pathway,MAPK signaling pathway-related proteins were significantly differentially expressed.3.Compared with the blank control group,the expression of α-SMA was significantly increased in the DMSO intravenous injection group(P<0.01),decreased after the addition of HIF-1α inhibitor,and significantly decreased in the fistula in the 5 mg/kg YC-1 intravenous injection group(P<0.05).Conclusion1.The study proves that it is feasible to construct tracheoesophageal fistula model by magnetic pressure assisted by bronchoscopy.2.The process of benign acquired tracheoesophageal fistula formation may be regulated by signaling pathways such as HIF-1α,TNF-1,JAK-STAT,and MAPK.3.HIF-1α promotes the activation and formation of myofibroblasts at the tracheoesophageal fistula,and the increased formation of myofibroblasts may be an important mechanism of fistula formation.