Function Of Obesity-related Diseases In Airway Epithelial Cells Stimulated By Cigarette Smoke Extract And The Related Machanism

Objective:Recent clinical researches demonstrated "obesity paradox" in chronic obstructive pulmonary disease(COPD)patients.However,why obesity is beneficial to COPD development remains unclear.Obesity is distinguished by hyperinsulinemia,which was defined as insulin concentration over than 0.01 μg/ml in blood.Meanwhile,cellular senescence of lung cells is involved in COPD progression.In this study,we aim to investigate the roles of insulin in high concentration in cellular senescence.Nevertheless,besides obesity,hyperinsulinemia is also a typical feature of type 2 diabetes mellitus.Researches have shown that type 2 diabetes mellitus is one of the hazards of COPD,patients having COPD and have type 2 diabetes mellitus as a complication are suffering a more progressive disease than patients without type 2 diabetes mellitus.Apart from hyperinsulinemia,another characteristic of type 2 diabetes mellitus is hyperglycemia.It has been proven that epithelial-mesenchymal transition of airway epithelial cells were up-regulated in COPD patients,both cigarette smoke and hyperglycemia could promote this progression.Hence,we aim to discuss the function of cigarette smoke and hyperglycemia in the progression of airway epithelial cells epithelial-mesenchymal transition.Methods:Human bronchial epithelial cells(BEAS-2B)and small airway epithelial cells(HSAEpiC)were treated with 0.1,1 or 5μg/ml insulin for 48 hours,or with 5μg/ml insulin for 8,24 or 48 hours,autophagy activator(rapamycin)or inhibitor(chloroquine)with or without exposed to 1%cigarette smoke extract(CSE)for 48 hours.The expression of senescence protein(p21),cell cycle related protein(cyclinE),and autophagy protein(LC3B,Beclin-1 and p62)were determined by western blot.Cellular senescence was detected by Senescence-β-Galactosidase staining.Autophagy level was detected by GFP-LC3B fluorescent light.Human bronchial epithelial cells(BEAS-2B)and small airway epithelial cells(HSAEpiC)were treated with 25mM glucose for 24 hours,or TGF-β1 signaling pathway activator(TGF-β1)or inhibitor(SB431542),with or without 1%CSE exposed for 24 hours.The expression of epithelial associated protein(Z01),mesenchymal associated protein(vimentin),TGF-β1 associated protein(SMAD2/3/4,P-SMAD2/3)were determined by western blot.The transcription of epithelial associated protein(Z01),mesenchymal associated protein(vimentin,a-SMA),TGF-β1 associated protein(TGF-β1,SMAD2/3/4,P-SMAD2/3)were determined by qRT-PCR.Resu ts:Insulin receded cellular senescence in a time-and dose-dependent manner,as indicated by decreased expression of p21,increased expression of cyclinE and down-regulated ratio of SA-β-Gal stained cells in BEAS-2B and HSAEpiC under physiological or CSE exposure condition.Activating autophagy could decrease cellular senescence,while suppressing autophagy worked the opposite way.5μg/ml insulin treated for 48 hours up-regulated the ratio of LC3B Ⅱ/Ⅰ,Beclin-1 and number of spot dot of GFP-LC3B,and down-reg ulated the level of p62 in AEC cells with or without 1%CSE exposure.Meanwhile,the effect of 5μg/ml insulin on receding cellular senescence could be alleviated by autophagy inhibitor chloroquine or reinforced by autophagy activator rapamycin.Further investigation proved that 25mM glucose and 1%CSE treated for 24 hours seperately could promote airway epithelial cells epithelial-mesenchymal transition,as indicated by increased expression and transcription of vimentin,transcription of a-SMA5 decreased expression and transcription of e-cadherin.Meanwhile,the TGF-β1 signaling pathway was up-regulated,as the expression and transcription of SMAD2/3/4,transcription of TGF-β1,phosphorylation of SMAD2/3 were up-regulated.Moreover,treatment with TGF-β1 activator could increase airway epithelial cells epithelial-mesenchymal transition,while TGF-β1 inhibitor worked the opposite way.Conclusion:Insulin in high concentration could recede the CSE induced cellular senescence of human airway epithelial cells through autophagy pathway,and hyperglycemia could reinforce the CSE induced airway epithelial cells epithelial-mesenchymal transition through TGF-β1 signaling pathway.