Autophagy And Glucose Metabolism Regulate The Function Of Club Cells

Background:Bronchial asthma（asthma）is an intractable pulmonary disease affects hundreds of millions of patients worldwide.The continuous progress of airway inflammation is a major feature of asthma pathology and physiology such as airway remodeling.Studies have shown the failure to repair the airway epithelial mucosa in time is one of the reasons for the persistence of the inflammatory response.It is essential to restore intact airway epithelial mucosa and terminate the promoting progress of injured epithelial cells on the airway inflammatory response in asthma.Many studies have confirmed epithelial progenitor cells Club cells,can proliferate or differentiate into ciliated cells to rebuild the damaged bronchial epithelial barrier.Cell autophagy is a stress-induced cell self-protection mechanism.Studies have suggested the occurrence of asthma is related to the loss of autophagy.Glucose deprivation can induce cell autophagy,and following cell autophagy is activated,it can regulate cell glucose uptake in return.Therefore,during the inflammatory response of asthma,what kind of regulation effect of cell autophagy and glucose metabolism on Club cell function,whether they will affect its proliferation and differentiation function,and how they participate in affecting the repair process after airway epithelial injury,are the main content of the research.Method:In this study,fluorescence-associated cell sorting and in vitro 3D organoid culture were used to study the proliferation of Club cells by counting its colony forming efficiency and colony size.We analyze the differentiation of Club cells from m RNA level by RT-PCR;The wild type mice were used to isolate Club cells,then we add autophagy activator（Spermidine）or autophagy inhibitor（3-MA or Bafilomycin）into the culture medium or use Scgb1a1-cre ER;Atg5^f/ftransgenic mouse model to knock down Atg5 to investigate the effects of autophagy on the Club cells in;We use2-NBDG fluorescence tracer method to study the regulation of autophagy on Club cell glucose uptake;We use a Scgb1a1-cre ER;Glut1 ^f/f transgenic mouse model to knock down Glut1 or adjust the concentration of glucose in the medium to show how glucose uptake influence of Club cells;The effects of glucose metabolism on Club cells was studied by adding glycolytic pathway inhibitor 2-DG to the culture medium.Result:1.Fluorescence-associated cell sorting Club cells of wild type mice.In vitro3D organoid culture,it was found small molecule autophagy activator（Spermidine）promotes Club cell proliferation and inhibits its differentiation into ciliated and goblet cells;Autophagy inhibitors（3-MA or Bafilomycin）inhibit the proliferation of Club cells and promote their differentiation.Fluorescence-associated cell sorting Club cells of the Scgb1a1-cre ER;Atg5^f/fmice（tamoxifen treatment）for in vitro 3D organoid culture.Compared with the control Atg5^f/f mice,the knockdown of Atg5 inhibited the proliferation of Club cells but promote their differentiation.2.Club cells of the Scgb1a1-cre ER;Atg5^f/f and Atg5^f/f mouse were compared for glucose uptake with 2-NBDG fluorescence tracer method.The results showed the knocked down of Atg5 the glucose uptake capability of Club cells have a downtrend.3.We use a Scgb1a1-cre ER;Glut1 ^f/f transgenic mouse model to knock down the gene of Glut1 of Club cell,it was found that Glut1 knockdown resulted in a decline in Club cell proliferation and enhanced its differentiation into ciliated cells and goblet cells.4.In vitro 3D organoid culture,it was found that gradually decreasing the glucose concentration promoted the proliferation of Club cells,but decreased the differentiation into ciliated and goblet cells.But further reducing or even depriving glucose,the ability of Club cell proliferation is slowed,and differentiation is accelerated.The low-concentration glycolysis inhibitor 2-DG increased Club cells;under the high-concentration 2-DG,Club cells decreased,and the differentiation of ciliated cells and goblet cells increased.Conclusion:Autophagy promotes the proliferation of Club cells and inhibits their differentiation into goblet cells and ciliated cells.After Atg5 knockdown,the glucose uptake of Club cells tended to decrease.The knockdown of gene Glut1,blocking glycolysis or glucose deprivation suppressed the proliferative capacity of Club cells and increased their differentiation.These results demonstrate autophagy and glucose metabolism play important roles in the regeneration and repair of epithelial mucosa by airway Club cells.