Hypoxia-mediated Autophagy Inhibits Collagen Deposition In Human Tracheal Fibroblasts

BackgroundAcquired tracheoesophageal fistulas（TEFs）are abnormal fistulas formed by communication between the esophagus and the respiratory tract due to various causes such as malignant tumors of the digestive tract or respiratory tract and iatrogenic trauma,which are difficult to diagnose and treat and have a high mortality rate.Among them,tracheoesophageal fistula after endotracheal intubation is a common cause of benign acquired tracheoesophageal fistula.Tracheal ischemia-hypoxia caused by compression of tracheal tissue by endotracheal tube cuff plays an important role in the formation of tracheoesophageal fistula.Airway injury repair is involved by two important airway cells:airway fibroblasts and airway epithelial cells.Previous studies by our group have found that severe hypoxia can reduce the proliferation of human tracheal fibroblasts and affect the production of collagen and fibrous tissue.Recent studies have shown that autophagy,as a downstream target gene of hypoxia,can mediate the involvement of fibroblasts in the development of fibrotic diseases in a variety of organs.Therefore,we suspect that hypoxia is involved in the formation of benign acquired tracheoesophageal fistulas by inducing autophagy.This study intends to further explore the formation mechanism of benign acquired tracheoesophageal fistula,which is expected to provide new therapeutic ideas for the diagnosis and treatment of future diseases.ObjectiveTo explore the mechanism of hypoxia-mediated autophagy in tracheoesophageal fistula formation after endotracheal intubation.Methods1.Detect the expression of HIF-1αandα-SMA by immunohistochemistry in normal airway tissue samples and tracheoesophageal fistula samples;2.Divide the tracheal fibroblasts into 2 groups:Normoxia group（21%O₂）and hypoxia group（1%O₂）.The migration ability of the two groups of cells at 0h,12h,and 24h was determined by scratch assay;after the two groups of cells were cultured for 12h,HIF-1αandα-SMA and autophagy-related protein and Col-1 expression were determined by western blot;α-SMA expression was analyzed by cell immunofluorescence.3.The experiment was divided into four groups:Normoxia group（21%O₂）;hypoxia group（1%O₂）;chloroquine（10μM,24 h）+hypoxia group（1%O₂,12 h）;and rapamycin（1μM,24 h）+hypoxia group（1%O₂,12 h）.α-SMA and Col-1 expression were determined by immunofluorescence and western blot,respectively.Results1.Immunohistochemistry revealed increased expression of HIF-1αandα-SMA in tracheoesophageal fistula specimens.2.Western blot and immunofluorescence revealed that HIF-1αandα-SMA expression was increased in human tracheal fibroblasts under hypoxia,and the increase of HIF-1αwas correlated with the duration of hypoxia.3.Detection of autophagy activity and collagen expression in human tracheal fibroblasts under normoxic and hypoxic conditions revealed that the expression of autophagy marker protein p62 was decreased,the conversion of LC3-I to LC3-II was increased,that is,autophagy activity was increased,while the expression of Col-1 protein was decreased under hypoxic conditions.4.To verify the effect of autophagy on Col-1 expression in human tracheal fibroblasts,autophagy activity modulator was used.The results suggested that the expression ofα-SMA in human tracheal fibroblasts increased in each hypoxia group after hypoxia treatment.Col-1 expression was increased in human tracheal fibroblasts in normoxia and chloroquine+hypoxia groups,and decreased after hypoxia conditions and the use of rapamycin（RP）to promote autophagy.5.Scratch assay was used to explore the effect of hypoxia on fibroblast migration,and it was found that the average migration rate of human tracheal fibroblasts was 15.85%at 12 hours and 34.64%at 24 hours under normoxic conditions.Under hypoxia,the mobility was 39.01%at 12 h and 41.83%at24 h.Conclusion1.Hypoxia promotes the differentiation of human tracheal fibroblasts into myofibroblasts;2.Hypoxia induces autophagy in human tracheal fibroblasts,which in turn inhibits the deposition of Col-1.3.Hypoxia-mediated autophagy inhibiting collagen deposition in tracheal fibroblasts may be one of the important molecular mechanisms of tracheoesophageal fistula formation.