Primary mechanism of the role of dual oxidase-1 causing airway allergic diseases in human bronchial epithelium
10.3760/cma.j.issn.1673-0860.2013.10.007
- VernacularTitle:双重氧化酶1引起变应性疾病发生机制的初步研究
- Author:
Li-Fen WANG
1
;
Zhi-Chun HUANG
;
Xiu-Fa WU
;
Hai-Fei WANG
Author Information
1. 东南大学附属中大医院耳鼻咽喉头颈外科
- Keywords:
NADPH oxidase;
Respiratory hypersensitivity;
Membrane microdomains;
Epithelial cells;
Tumor necrosis factor-alpha;
Desipramine;
Ceramides
- From:
Chinese Journal of Otorhinolaryngology Head and Neck Surgery
2013;48(10):823-829
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the role of dual oxidase-1 (DUOX-1) inducing airway hyperresponsiveness in human bronchial epithelium.Methods The human bronchial epithelial cells were divided into several groups:control group,tumor necrosis factor-α (TNF-α) group,methyl-β-cyclodextrin (M-β-CD) + TNF-α group,desipramine (DES) + TNF-α group,diphenylene iodonium (DPI) + TNF-α group and apocynin (APO) + TNF-α group.Fractionation was performed by sucrose gradient centrifugation and the protein DUOX-1 was measured by western blotting.The lipid raft clusters and its colocalization with DUOX-1 were confocal analysed.The intracellular reactive oxygen species (ROS) accumulation was measured by fluorescence of reactive oxygen probe of intracellular measurement.Sigmastat 3.02 software was used to analyze the data.Results (1) Detection of ROS,control group:1.00± 0.00 ; TNF-α group:1.95± 0.16 ; M-β-CD + TNF-α group:0.91 ± 0.16 ; DES + TNF-α group:1.49± 0.20 ; DPI + TNF-α group:1.03 ± 0.16 ; APO + TNF-α group:1.47 ± 0.26.The difference was statistically significant (F =3.83,P ＜0.05).(2) Extracts in rafts to lipid rafts region represents the ratio of total protein,protein content DUOX-1each group,control group:0.21 ± 0.02; TNF-α group:0.49 ± 0.04; M-β-CD + TNF-α group:0.08 ±0.02 ; DES + TNF-α group:0.09 ± 0.03 ; the difference was statistically significant (F =3.96,P ＜ 0.05).(3) DUOX-1 protein fluorescence values,control group:1.72 ± 0.21; TNF-α group:8.11 ± 1.23; M-β-CD + TNF-α group:1.51 ± 0.32; DES + TNF-α group:1.43 ± 0.11 ; the difference was statistically significant (F =4.87,P ＜ 0.05).(4) DUOX-1 gene detection,control group:1.00 ± 0.00 ScrRNA +TNF-α group:1.75 ± 0.04; DUOX-1siRNA + TNF-αgroup:1.15 ± 0.02; the difference was statistically significant (F =4.19,P ＜ 0.05).Conclusion TNF-α can induce DUOX-1 expression increasing in lipid raft,then the DUOX-1 can be activated to increase reactive oxygen species level; acidic sphingomyelinase inhibitor desipramine can inhibit this process,the results disclose that the process will depend on the ceramide of lipid raft.
