Role Of Lipid Raft-ceramide In Tnf-α-induced Nf-κb Activation And Duox-1 Expression In Human Bronchial Epithelial Cells

In the fields of genomics and proteomics, we have a lot of predominant achievement. With lipiomics getting into the eyes of people, Scientists find that they play more important roles in biologic area than imagining. Lipid raft is a kind of function unit which lies on the cell membrane and is composed of lipid. Abundant sphingolipids ,cholesterols and acylated proteins assemble together dynamicly and form into some stable structure called as lipid raft which is floating on the two-dimensional fluid cell plasma membrane and do its function. Lipid raft plays an important role in many ways, especially in signal transduction.Bronchial asthma is a disease with chronic airway inflammation, it is associated with many inflammatory cells and mediators. The human bronchial epithelial cell which mediates the living and activation of many inflammatory cells in the airway is the main effector cell in airway inflammation. Tumor necrosis factor(TNF-а)is an important promoter in asthma inflammation, it can induce a series of cytokines to product by activating NF-κB , and lead to the damage of the human bronchial epithelial cells and excretion of over mucus. NF-κB exists in many kinds of tissues and cells and participates into the transcriptional control of many genes. It is a generic name of a group of proteins that can bind specially with theκB sequence in the promotor sites of many genes and then enhance transcription. NF-κB participates into all kinds of immune or inflammatory reactions by control the expression of many important genes like cytokines, adhesion molecules and chemotatic factors. Dual oxidase (DUOX-1), a homolog of glycoprotein p91Phox, is expressed in airway epithelium and generates reactive oxygen species(ROS)-hydrogen peroxidase, which may induce cell damage and also plays role in mucin expression.However, the role of lipid-ceramide passway in TNF-αinduced NF-κB activation and DUOX-1 expression is an important question and must be solved. ObjectiveHow the lipid raft of the human bronchial epithelial cell effect the activation of NF-κB and expression of DUOX1, and if the latter is dependent on the former.Methods1. ELISA based transcription factor activity assay: we use different TNF-αconcentrations as 5ng/ml, 10ng/ml and 15ng/ml to treat the human bronchial epithelial cells by different time as 15min, 30min and 60min. We chose the combination of time and concentration with which the cytokines can activate NF-κB mostly to take the cell group tests.①control.②treated with 10ng/ml TNF-αby 15min.③treated with 10mM M-β-CD by 30min.④treated with 10mM M-β-CD by 30min before treated with TNF-α.⑤treated with 6ug/ml Filipin by 1 hour.⑥treated with 6ug/ml Filipin by 1 hour before treated with TNF-α.⑦treated with 10μM Desipramine by 1 hour.⑧treated with 10μM Desipramine by 1 hour before treated with TNF-α.2. Measure protein DUOX-1 by Western blot: We take the cell group test like follows:①control.②treated with 10ng/ml TNF-αby 3 hours.③treated with 10mM M-β-CD by 30min.④treated with 10mM M-β-CD by 30min before treated with TNF-α.⑤treated with 10μM Desipramine by 1 hour.⑥treated with 10μM Desipramine by 1 hour before treated with TNF-α.3. Observe the granules of protein DUOX-1 in cytoplasm by Immunohistochemistry: We take the cell group test like follows:①control.②treated with 10ng/mlTNF-αby 3 hours.③t reated with 10mM M-β-CD by 30min before treated with TNF-α.④treated with 10μM Desipramine by 1 hour before treated with TNF-α. 4. DNA double strands transfection. We take the cell group test like follows:①control.②treated with 10ng/ml TNF-αby 3 hours.③treated with single LipofectamineTM2000 reagent by over night.④t reated with the mixture of diluted LipofectamineTM2000 reagent and diluted DNA double strands by overnight before treated with TNF-α.5. Statistical method: One way ANOVA.Results1. The maximum activity of NF-κB is produced by the cells treated with 10ng/ml TNF-α(p<0.05). Lipid raft interventors may prevented the increase of NF-κB activation induced by TNF-αstimulating (p<0.05).2. TNF-αcan induce DUOX-1 expression increasely (p<0.05), which may be prevented by lipid raft interventors (p<0.05).3. The granules of protein DUOX-1 in cytoplasm is signifitantly increase after cells were treated with TNF-α, which may be prevented by lipid raft interventors.4. The transfection of the DNA binding series of NF-κB into the cells can prevente the DUOX-1 expression induced by TNF-αstimulating.Conclusion1. TNF-αcan activate obviously NF-κB in the human bronchial epithelial cells to promote transcription, in which lipid raft paticipates.2. TNF-αcan induce DUOX-1 expression increasly, and the expression of DUOX-1 is dependent on NF-κB activation.3. Lipid raft interventors like M-β-CD,FilipineⅢa nd Desipramine may be used as drugs in airway inflammation, e.g. Bronchial asthma.