Role Of Lipid Raft-ceramide In DUOX1 Activation Induced By TNF-α In Human Bronchial Epithelial

Respiratory diseases such as allergic rhinitis and asthma are common allergic disease. According to the recent investigations, we found airway hyperresponsiveness are the common characteristics of the pathophysiology in the pathogenesis. When the airway epithelial cells are stimulated by inflammatory factors, the airway epithelium will produce large amounts of reactive oxygen species via oxidase, then causing airway hyper-responsiveness. DUOX1 is one of the most important oxidases, which produces reactive oxygen species in airway epithelial cells, and which not noly makes the airway epithelial cells produce superoxide anion but also plays the role of SOD. The data suggest that DUOX1 is the major NADPH oxidase expressed in airway epithelium, therefore becomes a major contributor of H2O2 production.Lipid raft is defined as membrane microdomains of cells which enriched in cholesterol and sphingolipids. Stimulated by inflammatory factors, the sphingolipids are hydrolyzed into ceramide, which has the spontaneous aggregation nature, those membrane microdomains can be assembled into a large platform induced by the ceramide, which can play an important role in the activation of bio-oxidation enzymes and the interreaction of themselves.In this work,we will to examine if lipid raft plays an important role in TNF-αleading to DUOX1 and P47^phox activation, which should provide new targets for novel therapeutic interventions for allergic disease .OBJECTIVE To investigate the role of lipid raft-ceramide in TNF-αleading to DUOX1 activation in human bronchial epithelial.METHODS 1. Fractionation by sucrose gradient centrifugation and measure the protein DUOX1 and P47^phox by Western blotting. 2. Confocal analysis of lipid raft clusters and its colocalization with DUOX1 or P47^phox. 3. Measurement of intracellular ROS accumulation by measuring fluorescence of DCF.RESULTS The levels of P47^phox and DUOX1 expression were increased significantly (p<0.05) and which was inhibited by the pretreatment with MCD and DES. (p<0.05); In the confocal microscope, TNF-αcan induce DUOX1 and P47^phox significantly increase on the cell membrane, which will be prevented by Lipid raft interventors. Meanwhile, DUOX1 and P47^phox will show co-localization with Lipid raft marker cholera toxin and ceramide; TNF-αwill increase ROS levels (p<0.05) which was inhibited by pretreatment with MCD, DES, APO, and DPI (p<0.05).CONCLUSIONS TNF-αcan induce DUOX1 and P47^phox expression increasing in Lipid raft, then the DUOX1 and P47^phox 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 of the ceramide of Lipid raft.