PI3K/Akt-aPKC_(ι/ζ)-Nrf2调控大鼠气道上皮细胞γ-谷氨酰半胱氨酸合成酶表达

The pathway of PI3K/Akt-aPKC_(ι/ζ)-Nrf2 regulating the expression of γ-glutamylcysteine synthetase in the bronchial epithelial cells of rats

目的:观察香烟烟雾提取物(CSE)是否通过磷酯酰肌醇-3-激酶(PI3K)/Akt-不典型蛋白激酶Cι/ζ(aP-KCι/ζ)-核因子相关因子2(Nrf2)信号通路调控大鼠气道上皮细胞γ-谷氨酰半胱氨酸合成酶(-γGCS)表达。方法:用Western blot法检测-γGCS、Nrf2、p-Akt和p-aPKCι/ζ蛋白质,细胞免疫化学法观察-γGCS蛋白质表达,反转录-聚合酶链反应(RT-PCR)法检测-γGCSmRNA,免疫荧光法检测Nrf2蛋白质,流式分析法检测p-Akt阳性细胞率,双酶法测定-γGCS活性,酶循环分析法测定总还原型谷胱甘肽(GSH)含量。结果:暴露CSE 3 h,GSH含量显著升高,Nrf2胞核蛋白质、p-aPKCι/ζ蛋白质、p-Akt蛋白质及其阳性细胞率、-γGCS蛋白质及其mRNA和活性均显著增强。aPKCι/ζ抑制剂RO813220明显减弱p-aPKCι/ζ蛋白质、-γGCS蛋白质及其mRNA和活性表达,但增强Nrf2胞浆蛋白质表达,对p-Akt无影响。p-Akt抑制剂LY294002及RO813220+LY294002均降低p-aPKCι/ζ蛋白质、p-Akt蛋白质及阳性细胞率、-γGCS蛋白质及其mRNA和活性表达,增强Nrf2胞浆蛋白质表达。直线相关性分析显示Nrf2与-γGCS、p-Akt及p-aPKCι/ζ呈正相关,p-Akt与Nrf2、p-aPKCι/ζ及-γGCS呈正相关,p-aPKCι/ζ与Nrf2、p-Akt及-γGCS呈正相关(P<0.05)。结论:CSE可能通过PI3K/Akt-aPKCι/-ζNrf2调节-γGCS表达。

Objective： To observe the effect of the signal pathway of phosphoinositol-3-kinase（PI3K）/Akt-antypical protein kinase Cι/ζ（aPKCι/ζ）-Nuclear factor-E2 related factor（Nrf2） on γ-glutamylcysteine synthetase（γ-GCS） of the bronchial epithelial cells of rats after exposure to cigarette smoke extracts（CSE）.Methods： γ-GCS,Nrf2,p-Akt and p-aPKCι/ζ proteins were semi-quantified by Western blot.γ-GCS protein expression was assessed by immunocytochemistry. γ-GCS mRNA expression was detected by reverse transcription-polymerase chain reaction（RT-PCR）.Nrf2 protein was observed by immunofluorescence.The rate of the cells expressed p-Akt were analyzed by flow cytometry.GSH content and γ-GCS activity were measured.Results： GSH content,Nrf2 protein of nucleus,p-aPKCι/ζ protein,p-Akt protein and positive cells,γ-GCS protein and mRNA and activity were significantly increased after exposure to CSE for 3 hours.aPKCι/ζ inhibitor RO813220 significantly reduced the expression of p-aPKCι/ζ protein,γ-GCS protein and mRNA and activity,but enhanced Nrf2 protein of cytoplasm expression,had no effect on p-Akt.p-Akt inhibitor LY294002 and RO813220 ＋ LY294002 decreased p-aPKCι/ζ protein,p-Akt protein and positive cells,γ-GCS protein and mRNA and activity expression,increased Nrf2 protein of cytoplasm expression.The correlation analysises demonstrated that there were a positive correlation between Nrf2 and γ-GCS,p-Akt,p-aPKCι/ζ,between p-Akt and Nrf2,p-aPKCι/ζ,γ-GCS,between p-aPKCι/ζ and Nrf2,p-Akt,γ-GCS.Conclusion： CSE might upregulate γ-GCS expression through PI3K/Akt-aPKCι/ζ-Nrf2 signaling pathway in the bronchial epithelial cells of rats.