Autophagy Regulates Glucose Metabolism To Govenrn Airway Stem Cell Function

Background: Numerous studies have shown that there are many mechanisms for the repair of airway epithelium,including extrapulmonary stem cells(bone marrow,peripheral blood,embryos,et al.)and intrapulmonary stem cells,but extracellular stem cells contribute less to airway epithelial repair than intrapulmonary stem cells.There are self-renewing stem cells in the airway epithelial mucosa,which can effectively repair the damaged airway epithelium.In 1995,Stripp et al first discovered that after Naphthalene damages the airway epithelium,a small number cells called v Club(formerly known as v Clara cells)survive at the end of the airway(near the alveolar region),and the surviving cells can be repaired.These airway epithelial stem cells were identified to express the secreted globin family member 1A1(Secretoglobin family 1A member1,Scgb1a1).The stem cells expressing Scgb1a1 were specifically labeled with EGFP using the Cre ER-flox transgenic strategy,and it was confirmed in vivo and in vitro that the airway stem cells positively expressed by Scgb1a1 were able to differentiate into Club cells(formerly known as Clara cells).Whether the timely repair of airway epithelial damage in asthma patients is related to the low differentiation function of airway stem cells requires further experimental research.The role of autophagy-related functions in asthma physiology is gradually being revealed.Related studies have shown that childhood asthma is directly related to the variation of autophagy related 5(Atg5).The failure to repair the airway epithelium in acute asthma can lead to a shift to chronic asthma.At the same time,a large number of studies have proved that there is a relationship between autophagy and glucose.Low glucose promotes autophagy,and autophagy also feedbacks the function of airway stem cell sugar uptake,which in turn affects airway stem cell proliferation and differentiation,and ultimately affects airway epithelial damage.Whether the subsequent repair process,in turn,affects the conversion of acute asthma to chronic asthma,we will study these issues.Methods: 1.Acute asthma model was established using Atg5f/f mice and Scgb1a1-cre ER;Atg5f/f mice;autophagy in v Club cells after detection of acute asthma model(OVA modeling)in Atg5f/f mice using CYTO-ID autophagy detection kit changes,analysis of abundance changes in v Club cells of airway epithelium in Atg5f/f and Scgb1a1-cre ER;Atg5f/f after acute asthma modeling by flow cytometry.2.The wild-type mouse v Club cells were sorted by flow cytometry and cultured in vitro.The culture conditions included autophagy activator(Spermidine)and autophagy inhibitor(3-MA,Bafilomycin).Conditional gene knockout mice Scgb1a1-cre ER;Atg5f/f mice were sorted into v Club cells,wild-type mice were used as control group and organ-like organs were cultured in vitro to study the effects of autophagy on the proliferation and differentiation of v Club cells.influences.At the same time,Atg5f/f mice and Scgb1a1-cre ER;Atg5f/f mice were used to make naphthalene model.During the modeling period,the body weight changes of the mice were monitored,and the body weight changes of the control group and the experimental group were analyzed.At D0,D2 D20 was used to collect mouse lung tissue,and the differentiation of v Club cells was analyzed by immunofluorescence staining(Cyp2f2).3.Using 2-NBDG fluorescence to detect glucose uptake in v Club cells,2-NBDG is a fluorescently labeled glucose analog that detects glucose uptake in v Club cells using Atg5f/f mice and Scgb1a1-cre ER;Atg5f/f mice.The v Club cells were selected and incubated for 1 hour in vitro,and the proportion of GFP-positive cells was shown by v Club cells in the flow analysis experimental group and the control group.4.The wild-type mouse v Club cells were sorted by flow cytometry and cultured in vitro.The culture conditions were different concentrations of glucose(4.5g/L,1.5g/L,0.5g/L and 0g/L).And glycolytic pathway inhibitor 2-DG.Conditional gene knockout mice Scgb1a1-cre ER;Glut1 f/f mice were sorted into v Club cells,wild type mice were used as control group,and organoids were cultured in vitro to study the effect of glucose on proliferation and differentiation of v Club cells.Results: 1.After modeling acute asthma,the airway epithelial v Club cells were sorted.The proportion of CAT+ cells in PBS group and OVA group was analyzed by CYTO-ID autophagy detection kit.It was found that CAT+ v Club cells decreased and autophagy level after acute asthma modeling.In addition,the abundance of v Club cells in the lungs of mice was significantly decreased in Scgb1a1-cre ER;Atg5f/f mice after acute asthma modeling,indicating that autophagy is beneficial to maintain the pool of v Club cells in mouse lungs.Deletion of airway stem cells with Atg5 has no effect on the recruitment of OVA-induced inflammatory cells.2.The v Club cells were obtained by flow sorting.The organ-like culture found that autophagy activator(Spermidine)promoted the proliferation and differentiation of v Club cells,and autophagy inhibitors(3-MA,Bafilomycin)inhibited the proliferation and differentiation of v Club cells.The knockout mouse Scgb1a1-cre ER;Atg5f/f mice sorted v Club cells and cultured in vitro,and it was found that Atg5 deletion inhibited the proliferation of v Club cells.The Naphthalene lung injury model was used to analyze the changes of body weight of Atg5f/f mice and the experimental group Scgb1a1-cre ER;Atg5f/f mice.The weight loss rate of D2 in the experimental group was significantly higher than that of the control group,and the control group and the experimental group D20 were analyzed.The weight loss rate of the experimental group was significantly lower than that of the control group.The number of Cyp2f2 positive cells in the lung epithelial airways of D0,D2 and D20 was analyzed by immunofluorescence staining(Cyp2f2).The differentiation of v Club in the experimental group was significantly lower than the control group.3.The v Club cells were extracted from Atg5f/f mice and Scgb1a1-cre ER;Atg5f/f mice,and the effect of autophagy on the uptake of glucose by v Club cells was analyzed by 2-NBDG fluorescence method.The results showed that knockdown of the Atg5 v Club cells decreased the ability to take up glucose.4.Through v Club cell organ culture,low glucose(0.5g/L)promoted the proliferation of v Club cells,but did not have much effect on the differentiation of v Club cells.Glucose-free(0g/L)inhibited the proliferation and differentiation of v Club cells.The glycolysis inhibitor 2-DG inhibits the proliferation of v Club cells and promotes differentiation.Conclusion: Autophagy promotes the proliferation and differentiation of airway stem cells.After OVA-induced acute asthma modeling,the level of autophagy in mouse airway stem cells decreased and the abundance decreased.Autophagy promotes glucose uptake by airway stem cells,while glucose deprivation,inhibition of glycolysis,and loss of Glut1 inhibit proliferation of airway stem cells.Although blocking autophagy of airway stem cells does not affect the OVA-induced acute asthmatic inflammatory response,autophagy maintains the regenerative function of airway stem cells through reprogramming of glucose metabolism,which can alleviate the acute inflammatory response of asthma to a chronic inflammatory process.