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Role of Lipid Raft-ceramide in TNF-α-induced NF-κB Activation and DUOX-1 Expression in Human Bronchial Epithelial Cells

Author: LvLiHua
Tutor: JinSi
School: Huazhong University of Science and Technology
Course: Pharmacology
Keywords: Lipid rafts Ceramide Tumor necrosis factor -α Airway epithelial cells Nuclear transcription factor -kappa B Methyl-β- cyclodextrin Philippines streptozotocin Desipramine Dual oxidase -1
CLC: R562.25
Type: Master's thesis
Year: 2009
Downloads: 27
Quote: 0
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Abstract

, Has many remarkable achievements in genomics and proteomics. As lipidomics concept proposed, people increasingly found the lipids function is not only limited to previously aware of the role they play in the biological field may be beyond the imagination of the people. Located in lipid rafts is a functional unit body constituted by the lipid membrane. Rich in sphingolipids, cholesterol and specific proteins dynamically gathered together to form relatively stable with certain functions of rafts floating in the flow cell membranes. When the cells by certain stimuli, lipid rafts acidic sphingomyelinase (ASM) activated and release large amounts of ceramide from sphingomyelin hydrolysis, which quickly coupled to each other to form a micro-area, these micro area can be quickly links formed one or several of the platform area. Ceramide rich membrane area to provide a platform for the interaction between the protein and other biologically active molecules. The lipid rafts widely involved in various biological behavior of the cells, play a particularly important role in signal transduction. Bronchial asthma is a chronic inflammatory disease of the airways associated with a variety of inflammatory cells and inflammatory mediators, airway epithelial cells are the main effector cells of airway inflammation. Tumor necrosis factor-α (TNF-α) is important in the inflammatory process of asthma initiation factor, it can be a series of inflammatory mediators induced by activation of NF-κB, resulting in airway epithelial cell damage and excessive airway mucus secretion. NF-κB is widely present in various tissues and cells, κB sequence binding with a variety of gene promoter to promote transcription, through the regulation of many important cytokines, adhesion molecules, chemokines gene expression involved in the body's various immune and inflammatory response. DUOX-1 is glycoprotein p91Phox the homologues, can be expressed in the airway epithelial cells, able to catalyze its substrate to produce hydrogen peroxide, and to cause cell damage and stimulate the cells to secrete large amounts of mucus. This study was to investigate the lipid rafts - ceramide pathway whether the expression of TNF-α induced NF-κB activation and its downstream target genes play a role in order to provide a new strategy for the prevention and treatment of asthma and other airway inflammatory diseases. After the destruction of the purpose of human airway epithelial cell membrane lipid rafts - ceramide, TNF-α to stimulate the cell induced NF-κB activation and DUOX1 expression by the impact. DUOX1 the expression is dependent on activation of NF-κB. Method 1 transcription factor activity assay method based on ELISA detection of NF-κB activity in the cells. 5ng/ml, 10ng/ml, 15ng/ml TNF-α treatment of airway epithelial cells 15min, 30min, 60min. Select the greatest degree of activation of NF-κB TNF-α concentration and stimulation time to conduct the cell grouping experiments and detection of NF-κB activity in cells. ① control group ② TNF-α stimulation group: 10ng/mlTNF-α treated cells 15min. ③ the cholesterol removed agent methyl-β-cyclodextrin (M-β-CD) M-β-CD group: 10mM M-β-CD processing Cell 30min. The ④ MT group: M-β-CD treatment of cells and then the cells were stimulated with TNF-α. ⑤ cholesterol binding agent Philippines streptozotocin Ⅲ (Filipin Ⅲ), Filipin groups: cells were treated with 6ug/ml Filipin one hour. The ⑥ FT group and then with TNF-α: Filipin processing cells stimulate the cells. ⑦ acidic sphingomyelinase inhibitor desipramine (Desipramine), Desipramine group: 10 μM Desipramine processing cells one hour. The ⑧ DT Group: Desipramine treated cells after cytokine stimulation of cells. 2. Western blot to detect the expression of cell DUOX-1. The cells are grouped into: ① control group (2) TNF-α stimulation group: 10ng/ml TNF-α-treated cells. ③ M-β-CD group: 10mM M-β-CD processing cell 30min. ④ MT group: MβCD processing cells then the cells were stimulated with TNF-α. The ⑤ Desipramine group: 10μM Desipramine the cells were treated for 1 hour. The ⑥ DT group: Desipramine treatment of cells and then the cells were stimulated with TNF-α. 3 immunohistochemical observation of DUOX-1 expression in the cytoplasm. The cells are grouped into: ① control group (2) TNF-α stimulation group: 10ng/ml TNF-α-treated cells. ③ MT group: 10mM M-β-CD processing cells 30min after cells were stimulated with TNF-α. ④ The DT: 10 μM Desipramine processing cells 1 hour cells and then the cells were stimulated with TNF-α. 4. DNA decoy. The cells are grouped into: ① control group. ② TNF-α stimulation groups: 10ng/ml TNF-α treatment cells 3 hours. The ③ transfection reagents groups: transfection reagent treated cells overnight. ④ DNA transfection groups: transfection reagent to DNA duplexes transfected into the cell, and then 10ng/ml TNF-α treated cells. 5 Statistical analysis: one-way ANOVA. Results 1.10ng/ml TNF-α treatment of airway epithelial cells 15min activation of NF-κB in cells is the most significant (P lt; 0.05). Lipid rafts interference agent may be inhibited TNF-α stimulation of NF-κB activity was increased (P lt; 0.05). 2. TNF-α stimulation of cells caused DUOX-1 expression increased (P lt; 0.05). The lipid rafts interference agents can inhibit TNF-α stimulation DUOX-1 expression (P lt; 0.05). 3. TNF-α stimulate cell DUOX-1 particles in the electron microscope, the cytoplasm increased significantly. The suppression of the lipid rafts interference agent may increase in TNF-α stimulation DUOX-1 particles. Increased expression of TNF-α stimulation of 4.NF-κB DNA binding sequence is transfected into cells can be suppressed the cells DUOX-1 (P lt; 0.05). Obvious conclusion 1.TNF-α can activate NF-κB; airway epithelial cells lipid rafts - ceramide signaling pathways involved in TNF-α carry signals to the cell transduction. The 2.TNF-α can cause increased airway epithelial cells DUOX-1 expression; DUOX-1 expression is dependent activation of NF-κB, this process is also dependent on the lipid rafts - ceramide signaling pathway. 3 the lipid rafts disruptors M-β-CD Filipin Ⅲ well Desipramine promise for the treatment of bronchial asthma and other inflammatory diseases of the airway.

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CLC: > Medicine, health > Internal Medicine > Respiratory system and chest diseases > Trachea and bronchial disease > Bronchial disease > Bronchial asthma
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