香烟烟雾提取物通过蛋白激酶 C-核因子相关因子2调节大鼠气道上皮细胞血红素加氧酶1表达

摘要

目的：探讨蛋白激酶C（PKC）-红系衍生的核因子相关因子2（Nrf2）对香烟烟雾提取物（CSE）诱导的大鼠气道上皮细胞血红素加氧酶1（HO-1）表达的影响。方法：通过CSE刺激雄性SD大鼠气道上皮细胞，使用PKC抑制剂RO318220和Nrf2 siRNA，将细胞分为对照组、CSE 3 h组、RO318220组、Nrf2 siRNA组和RO318220＋Nrf2 siRNA组，用Western blotting法分别检测HO-1、Nrf2和p-PKC蛋白表达，免疫细胞化学法观察HO-1蛋白表达，逆转录-聚合酶链反应（ RT-PCR）法检测HO-1 mRNA表达，免疫荧光法检测Nrf2蛋白定位，测定HO-1活性。结果：暴露CSE 3 h后，Nrf2蛋白主要表达在胞核，胞核蛋白表达增强，p-PKC蛋白、HO-1的mRNA和蛋白高表达， HO-1活性增强。预先给予RO318220，PKC蛋白、Nrf2胞浆和胞核蛋白、HO-1的mRNA和蛋白表达均明显减弱， HO-1活性显著降低。预先用siRNA沉默Nrf2，胞浆和胞核的Nrf2蛋白表达均减弱，HO-1活性、mRNA和蛋白水平明显降低。 RO318220联合Nrf2 siRNA处理后，PKC蛋白、Nrf2胞浆和胞核蛋白、HO-1 mRNA和蛋白表达均明显降低，HO-1活性明显降低。结论：CSE通过PKC激活Nrf2，诱导Nrf2核转位，从而上调HO-1的表达水平。%[ABSTRACT]AIM:ToobservetheeffectofthesignalingpathwayofproteinkinaseC(PKC)-nuclearfactor-ery-throid 2-related factor 2 (Nrf2) on the expression of heme oxygenase-1 (HO-1) induced by cigarette smoke extract (CSE) in the rat airway epithelial cells .METHODS:After exposed to CSE , the airway epithelial cells of male SD rats were di-vided into control group , CSE3 group, RO318220 group, Nrf2 siRNA group and RO318220+Nrf2 siRNA group.The pro-tein levels of HO-1, Nrf2 and PKC were semi-quantified by Western blotting .The protein expression of HO-1 was assessed by immunocytochemistry .The mRNA expression of HO-1 was detected by reverse transcription-polymerase chain reaction ( RT-PCR) .Nrf2 protein location was observed by immunofluorescence staining .HO-1 activity was also determined .RE-SULTS:After exposure to CSE for 3 h, the Nrf2 protein was mainly located in the nucleus , and the expression level of Nrf2 protein in the nucleus was stronger than that in the control cells .CSE also significantly enhanced the levels of p-PKC protein, and HO-1 mRNA, protein and activity .Pretreatment with RO318220 significantly decreased the levels of PKC pro-tein, Nrf2 protein, and HO-1 mRNA, protein and activity.Meanwhile, Nrf2 protein significantly decreased , and the activ-ity, mRNA and protein levels of HO-1 were also significantly attenuated by pretreatment with siRNA to knock down Nrf2. Pretreatment with RO318220 and Nrf2 siRNA significantly decreased the PKC protein , Nrf2 protein, and HO-1 mRNA, protein and activity .CONCLUSION:CSE up-regulates the HO-1 expression through PKC-induced nuclear translocation of Nrf2 in the rat airway epithelial cells .