| Background:Chronic obstructive pulmonary disease(COPD),mainly caused by cigarette smoke(CS),is a common and progressive disease.Oxidative stress,inflammatory response and cell apoptosis caused by CSE are known as the critical mechanisms in COPD pathogenesis.Recently several studies suggested that IL-22 promote the proliferation and repair of various epithelial cells.Whether IL-22 protects human bronchial epithelial cells from CSE-induced oxidative stress,inflammatory response and cell apoptosis,has remained agonistic.In this study,we attempt to explore the protective effect of IL-22 on human bronchial epithelial cells by a COPD cellular model consist of BEAS-2B cells exposed with CSE.Methods:1.BEAS-2B cells were exposed to various concentrations of CSE(0,2,4,6,8,10%)for 12 h,24 h,48 h,respectively.Cell viability was measured using Cell Counting Kit‐8 assay.2.The contents of MDA and SOD in the supernatants were determined by using malondialdehyde(MDA)and superoxide dismutase(SOD)assay kits.3.The levels of IL-18,IL-1β and Lactate dehydrogenase(LDH)were examined by enzyme-linked immunosorbent assay(ELISA),respectively.4.Western blotting was used to detect the apoptosis-related proteins.Annexin V-APC and7-AAD double staining and flow cytometry were adopted to measure cell apoptosis rate.5.Ceramide fluorescence intensity was observed by immunofluorescence microscopy,meanwhile ceramide level in cellular supernatant was determined by a Human ceramide ELISA kit.Results:1.The survival rate of BEAS-2B cells decreased gradually with increasing CSE concentration and incubation time(P<0.05).When BEAS-2B cells were stimulated with5% CSE for 24 h,cell viability was decreased to 70% ～80%.Hence,CSE(5%,24h)was selected for subsequent experiments.2.With the increase of CSE concentration,the content of MDA in cell supernatant gradually increased(P<0.05),whereas the content of SOD gradually decreased(P<0.05).However,given different concentrations of IL-22(100,50 and 10ng/m L),the level of MDA decreased and the SOD content increased in a concentration-dependent way(P<0.05).3.CSE exposure dose-dependently increased inflammatory cytokines IL-18 、IL-1β and LDH secretion in BEAS-2B cells(P<0.05).Compared with the CSE-treated group,IL-22 concentration-dependently reversed these changes(P<0.05).4.With the increase of the concentration of CSE treatment,flow cytometry indicated that the apoptosis rate increased gradually(P<0.05).Meanwhile,western blots demonstrated that the expression levels of apoptosis-related proteins Bax,Bcl-2 and Cle-capase3 were up-regulated(P<0.05).When100ng/ml IL-22 was co-incubated with 5%CSE,compared with the CSE group,the protein expression level of Cle-capase3 was down-regulated(P<0.05),and the rate of apoptosis decreased(P<0.05).5.CSE stimulation a dose-and time-dependently increased the content of ceramide in BEAS-2B cells(P<0.05).Pharmacological inhibitors of ceramide synthesis,either Myriocin,Imipramine or GW4869,blocked this effect of CSE(P<0.05).6.When given Myriocin,Imipramine or GW4869,the level of MDA,IL-18,IL-1β and LDH in the supernatant decreased(P<0.05),while the content of SOD increased(P<0.05).Moreover,the protein expression level of Cle-capase3 and the apoptosis rate decreased(P<0.05).7.As the concentration of IL-22 increased,ceramide synthesis induced by CSE was significantly inhibited(P<0.05).Conclusions:1.CSE suppresses BEAS-2B cells viability,and induces oxidative stress,inflammatory response and cell apoptosis.2.Ceramide mediates CSE-induced oxidative stress,inflammatory response and cell apoptosis in BEAS-2B cells.Inhibition of ceramide production can ameliorate cell damage induced by CSE.3.Exogenous administration of IL-22 can protect bronchial epithelial cells from CSEinduced oxidative stress,inflammatory response and cell apoptosis.The protective effect of IL-22 may be associated with inhibition of ceramide generation by CSE. |
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