Role And Mechanism Of ORMDL3 In Pyroptosis Of Bronchial Epithelial Cells In Obese Asthma

BackgroundObese asthma is a new concept put forward in 2014.It is a chronic disease that seriously threatens children’s health and belongs to a new phenotype of asthma.Unlike eosinophilic inflammation in allergic asthma,obese asthma is characterized by neutrophil infiltration,which is more severe in wheezing,earlier in airway remodeling,more obvious in airway obstruction,and less sensitive to hormone therapy.At present,the mechanism of airway remodeling in obese asthma is still unclear,and it is very important to clarify the initiation factors and mechanism.pyroptosis is a new programmed cell death mode discovered in recent years,which is involved in airway remodeling of asthma.It belongs to a new way of inflammatory cell death,and is closely related to the inflammatory response of Nod-like receptor pyrin domain 3(NLRP3).The role and mechanism of pyroptosis in airway remodeling of obese asthma is not clear.NLRP3 protein,apoptosis-associated speck-like protein containing CARD(ASC)and Caspase-1 precursor are closely linked to form NLRP3 inflammasome to ensure its proper function.NLRP3 inflammasome is associated with the development of many inflammatory and immune diseases,such as metabolic syndrome,inflammatory bowel disease,asthma and so on.Gasdermin D(GSDMD)is also known as pyroptosis final executor protein.During pyroptosis process,NLRP3 inflammasome can indirectly activate GSDMD.After GSDMD is cleaved,it forms perforated protein embedded in cell membrane,promotes the release of inflammatory factors,and eventually leads to cell death.Serum ORMDL3 is an important regulatory factor involved in airway remodeling in asthma.Its high expression in vivo and in vitro causes endoplasmic reticulum related inflammatory response,which can directly promote the occurrence of asthma by regulating sphingomyelin.ORMDL3 belongs to Orm protein,which is involved in regulating sphingomyelin metabolism.Sphingomyelin is the key lipid family of membrane structure and intracellular and intercellular signal transduction.There are abnormal metabolism of sphingomyelin in obese people.It is found that the high expression of ORMDL3 in lung epithelial cells and macrophages promotes the production of ceramide,which is an important intermediate and inflammatory mediator of sphingomyelin metabolism.Cathepsin D(CTSD)is a direct downstream factor of ceramide.The entry of CTSD into the cytoplasm increases the permeability of mitochondria and triggers Caspase to induce cell damage.In the classic pathway,NLRP3 activates Caspase-1,which in turn stimulates GSDMD related pyroptosis.Caspase-8 is the molecular regulatory switch of programmed necrosis,apoptosis and pyroptosis.These results suggest that the overexpression of ORMDL3 may lead to cell death.In conclusion,ORMDL3 can induce cell damage by promoting the expression of downstream CTSD,and NLRP3 and GSDMD may play an important role in cell damage.In obese asthma,we investigated the expression of ORMDL3 and its mechanism in promoting cell damage.ObjectiveThe purpose of this study was to investigate whether airway remodeling and pyroptosis exist in bronchial epithelial cells of obese asthma,and whether cell death promotes airway remodeling and its possible pathogenesis.To observe the expression of ORMDL3,NLRP3 and GSDMD in bronchial epithelial cells of obese asthma,and to explore whether ORMDL3 participates in airway remodeling of obese asthma through CTSD/NLRP3/GSDMD.So as to provide new ideas for the treatment and prevention of obesity asthma in the future.MethodsForty female BALB/c mice of SPF grade were purchased and randomly divided into four groups:normal group,asthma group,obese group and obesity asthma group,with 10 mice in each group.Mice in normal group were given normal diet and normal saline inhalation,mice in obese group were given high-fat diet and normal saline inhalation,mice in asthma group were given normal diet and ovalbumin inhalation,mice in obese asthma group were given high-fat diet and ovalbumin inhalation.After the 14 weeks,we removed mice that did not meet the obesity criteria in the obese group and the obese asthma group.For the successful model mice,we use protein chip to observe their difference of protein expression in lung tissue.HE staining and Masson staining were used to observe the airway inflammation and collagen deposition.Furthermore,the expressions of ORMDL3,NLRP3 and GSDMD in the lung tissues were detected by immunofluorescence and immunohistochemistry.RT-qPCR was used to detect the expression of NLRP3,GSDMD and CTSD in human bronchial epithelial cells infected by ORMDL3 overexpressing lentivirus.ResultsCompared with the normal group and asthma group,the body weight of obese group and obese asthma group increased significantly(P<0.05).There were significant airway inflammation,airway remodeling and collagen deposition in asthma,obese asthma and obese mice,and in which the obese asthma group was the most serious.With protein chip,we found that CTSD,Caspase-1,Caspase-8 and GSDMD expression were 3.1,1.5,1.5 and 1.3 times increased compared with those in normal mice.In mice of asthma,obese and obese asthma group,the expression of ORMDL3,NLRP3 and GSDMD were increased,especially in obese asthma group.In the vitro cell experiment,we found that the mRNA levels of CTSD,NLRP3 and GSDMD were significantly increased in human bronchial epithelial cells infected with ORMDL3 overexpressing lentivirus.Conclusions1.In animal experiments,airway remodeling and cell death were found in obese asthma mice,and pyroptosis aggravated airway remodeling.2.In vitro experiments showed that the expression of NLRP3 and GSDMD increased after the expression of ORMDL3 increased,which suggested that ORMDL3 could promote lung epithelial cell pyroptosis and aggravate airway remodeling in obese asthma by regulating CTSD/NLRP3/GSDMD pathway.