脂筏-双氧化酶信号平台在气道上皮细胞防御反应及介导上皮损伤中的作用和机制

项目名称： 脂筏-双氧化酶信号平台在气道上皮细胞防御反应及介导上皮损伤中的作用和机制

项目编号： No.30870997

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 金属学与金属工艺

项目作者： 金肆

作者单位： 华中科技大学

项目金额： 32万元

中文摘要： 在培养的气道上皮细胞中，以炎症介质肿瘤坏死因子α65288;TNF-α65289;刺激后，分离提取细胞膜脂筏，免疫印迹检测发现，TNF-α20351;双氧化酶1（Duox1）及其亚基P47向脂筏层转移。免疫荧光染色，激光共聚焦显微成像发现，TNF-α21487;使脂筏及神经酰胺（ceramide）发生聚集，并与Duox1及P47共定位。同时，TNF-α21487;引起细胞内活性氧水平和转录因子NF-κ#27963;性升高，但可被脂筏干扰剂甲基-β#29615;糊精（MCD）及酸性鞘磷脂酶（ASM）抑制剂地昔帕明（也是一种抗抑郁症药）所抑制，还可被ASM及Duox1小干扰RNA（siRNA）所抑制。这些结果表明，TNF-α21487;刺激Duox1及其亚基P47phox向脂筏聚集，形成全酶复合体，激活该酶，使细胞内活性氧生成增多，进而激活转录因子NF-κ#65292;介导炎症反应。在这一过程中，ASM发挥十分关键的作用，该酶可催化脂筏内的鞘磷脂水解生成ceramide，进而促进脂筏聚集，并募集Duox1及P47，最终导致Duox1激活。这些研究基本阐明了双氧化酶激活的机制，达到预期目标。根据这些发现，进一步开展了地昔帕明抗过敏性鼻炎的动物水平转化医学研究，也取得良好效果。

中文关键词： 双氧化酶1;脂筏;酸性鞘磷脂酶;活性氧;炎症

英文摘要： Airway epithelial cells were stimulated by tumor necrosis factor αTNF-αand membrane lipid rafts were isolated and examined by Western blot. It was found that TNF-αtimulated a translocation of dual oxidase 1 (Duox1) and its subunit P47 to lipid raft layers. Immunofluorescence staining and confocal imaging showed that TNF-αtimulated the aggregation of lipid rafts and ceramide, which co-localized with Duox1 and P47. Meanwhile, TNF-αnduced the increase of intracellular reactive oxygen species (ROS) and the transcription factor NF-κactivity, which were not only blocked by raft disruptor, methyl-βyclodextrin (MCD) and acidic sphingomyelinase (ASM) inhibitor desipramine (also an antidepressant drug), but also by ASM and Duox1 small interfering RNA (siRNA). These results suggest that, TNF-αan stimulate the aggregation of Duox1 and its subunits P47phox to lipid rafts, forming a holoenzyme complex, thus the enzyme is activated, followed by an increase in ROS and NF-κactivity, contributing to the occurrence of inflammation. In this process, ASM plays a crucial role in catalyzing the hydrolysis of sphingomyelin to generate ceramide, thus facilitating lipid raft aggregation and recruiting Duox1 and P47, leading to Duox1 activation. Basically, these research define the mechanism of dual oxidase activation and achieve the desired objectives. Based on these findings, further translational medical research of using desipramine to treat allergic rhinitis in animal level was conducted and also achieved good results.

英文关键词： dual oxidase;lipid raft;acid sphingomyelinase;reactive oxygen species;inflammation