P13K／Akt—Nrf2信号通路调节大鼠气道上皮细胞γ-谷氨酰半胱氨酸合成酶表达的研究  

Cigarette smoke extract regulating the expression of T-glutamylcysteine synthetase through the pathway of PI3K/Akt-Nrf2 in the bronchial epithelial cells of rats

作　　者：江刚[1,2] 戴爱国[1] 胡瑞成[1] 

机构地区：[1]湖南省人民医院呼吸内科,长沙410005 [2]湖南省老年医院湖南省老年医学研究所呼吸疾病研究室

出　　处：《中国医师杂志》2010年第1期9-13,共5页Journal of Chinese Physician

基　　金：基金项目：湖南省科技计划资助项目（01JZY2017,05JT1023）;湖南省医药卫生科研课题资助项目（B2005125,B2005292）

摘　　要：目的观察香烟烟雾提取物（CSE）通过磷酯酰肌醇-3-激酶（PBK）／Akt—Nrf2（Nuclearfactor—E2relatedfactor）信号通路对大鼠气道上皮细胞γ-谷氨酰半胱氨酸合成酶（γ-GCS）的影响。方法用浓度为10％的CSE对体外原代培养的大鼠气道上皮细胞进行不同时间处理及使用P13K特异抑制剂LY294002进行干预。观察CSE在不同的时间及使用LY294002干预后对Nrt2的核转位及γ-GCS的影响，用细胞免疫化学、流式术、免疫荧光及Westernblot方法检测Nrf2、p-Akt及γ-GCS的表达水平；用逆转录-聚合酶链反应（RT—PCR）方法检测γ-GCSmRNA的表达水平；检测还原型谷胱甘肽（GSH）的含量及γ-GCS的活性。结果暴露CSE后1h，GSH含量明显低于对照组，在暴露CSE后3h和6h，GSH含量明显增高。Nrf2蛋白质在对照组主要表达在胞浆，其蛋白质表达增强，而在CSE1h、CSE3h和CSE6h组主要在胞核中表达，其蛋白质表达增强。p-Akt蛋白质在暴露CSE后1h增强，3h最强，6h减弱。p-Akt阳性细胞百分比变化趋势与其蛋白质变化趋势相同。γ—GCSmRNA和蛋白质在CSE1h、CSE3h、CSE6h组表达逐渐增强。γ—GCS活性在CSE1h、CSE3h、CSE6h组逐渐增强。预先使用LY292002，p-Akt阳性细胞百分比及蛋白质表达明显弱于CSE3h组，Nrf2胞浆蛋白质表达增强，胞核表达减弱，吖-GCSmRNA、蛋白质和活性及GSH含量均明显低于CSE3h组。相关性分析显示Nri2与γ-GCS、γ-GCS活性呈正相关（r=0．81，0．77，P〈0．05），p-AM与γ-GCS、γ-GCS活性、GSH及Nrf2呈正相关（r=0．373，0．44，0．63，0．56，P〈0．05）。结论P13K／Akt信号通路可能参与Nrf2核转位及其对抗氧化基因γ-GCS的调控。Objective To observe the effect of PI3K （phosphoinositol -3-kinase, PI3K）/Akt-Nrf2 （Nuclear factor -E2 related factor） signal pathway on γ-glutamylcysteine synthetase（γ-GCS）in the bronchial epithelial cells of rats treated with cigarette smoke extract （CSE）. Methods The bronchial epithelial cells were deah with 10% concentration of CSE for different time and pretreated with PI3k inhibitor （ LY.294002 ）. The expressions of Nrf2, p-Akt and T-GCS proteins were examined by immunocytochemistry, flow cytometry, immunofluorescence and western blot. The expressions of γ-GCS mRNA were detected by reverse transcription-polymerase chain reaction （RT-PCR）. Reduced glutathione（GSH） content and the lev-el ofγ-GCS activi-ty were examined. Results GSH content in CSElh group was significantly decreased, but still higher in CSE3 and 6 groups compared to the control. Nrf2 protein mainly located in the cytoplasm. Nrf2 plasmosin mainly increased in the nucleus in control group, and Nrf2 nucleic protein significantly enhanced in CSE1, 3 and 6 groups. P-Akt protein was up-regulated at lh, reached its peak at 3h, declined slightly at 6h after exposure to CSE. The tendency of the percentage of p-Akt positive cells was as same as p-Akt protein. γ-GCS mRNA, protein and activity, gradually increased in CSE I h, CSE 3h, CSE 6h groups. Pretreated with LY294002, the expression of p-Akt protein was markedly decreases, while Nrt2 plasmosin expressed strongly, and γ-GCS mRNA, protein, activity and GSH content were significantly decreased compared to CSE3h group. Linear correlation analysis demonstrated that there were a positive correlation among Nrf2 and γ-GCS, 3,-GCS activity, and among p-Akt and Nrf2, GSH,γ-GCS,γ-GCS activity. Conclusion PI3K/Akt signal path might participate in Nrf2 nuclear translocation via regulating the expression of γ-GCS.

关 键 词：1-磷脂酰肌醇3-激酶/代谢 DNA结合蛋白质类/代谢 谷氨酸-半胱氨酸连接酶/代谢 烟雾/副作用 信号传导 

分 类 号：R563.9[医药卫生—呼吸系统]