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 的表达

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：将25 只SD 大鼠的气道上皮细胞随机分为对 照组、CSE3h 组、RO318220(PKC 抑制剂) 组、Nrf2 siRNA 组和Nrf2 siRNA+RO318220 组，每组5 只。对照组 用DMEM/F12 正常培养，CSE3h 组用10% CSE 共培养3 h；RO318220 组则用3 mol/L RO318220 预处理0.5 h，余处理同CSE3h 组；Nrf2 siRNA 组先用Nrf2 siRNA 预处理，余处理同CSE3h 组；Nrf2 siRNA+RO318220 组则先用3 mol/L RO318220 和Nrf2 siRNA 预处理，余处理同CSE3h 组。用Western 印迹法分别检测HO-1， Nrf2 和PKC 蛋白表达，细胞免疫化学法观察HO-1 蛋白表达，反转录- 聚合酶链反应(RT-PCR) 法检测HO-1 mRNA 表达，免疫荧光法观察Nrf2 核转位，测定HO-1 活性。结果：CSE 明显诱导Nrf2 核转位，其诱导作 用可被RO318220 阻断。HO-1 蛋白在CSE3h 组强阳性表达，在对照组、RO318220 组、Nrf2 siRNA 组和Nrf2 siRNA+RO318220 组均弱阳性表达。CSE3h 组PKC 蛋白、HO-1 mRNA 和蛋白表达较对照组显著升高，HO-1 活性较对照组明显增强(P<0.05)。PKC 蛋白表达水平在Nrf2 siRNA 组和CSE3h 组无明显差异(P>0.05)。结论： CSE 通过PKC 信号通路诱导Nrf2 核转位，上调HO-1 的表达水平。

Protein kinase C-nuclear factor-erythroid 2-related factor 2 regulating the expression of heme oxygenase-1 in rat airway epithelial cells

Objective: To observe the effect of the signaling pathway of protein kinase C (PKC)-nuclear factorerythroid 2-related factor 2 (Nrf2) on the expression of heme oxygenase -1 (HO-1) induced by cigarette smoke extract in rat airway epithelial cells. Methods: The airway epithelial cells of 25 male SD rats were randomly divided into a control group, a CSE3h group, a RO318220 group (PKC inhibitor), a Nrf2 siRNA group and a Nrf2 siRNA+RO318220 group, 5 rats in each group. The control group was incubated with DMEM/F12 alone. The CSE3h group was treated with 10% CSE for 3 h. The RO318220 group was pretreated with 3 μmol/L RO318220 for 0.5 h and subsequently treated with 10% CSE for 3 h. The Nrf2 siRNA group was pretreated with Nrf2 siRNA, and then treated with 10% CSE for 3 h. The Nrf2 siRNA+RO318220 group was pretreated with Nrf2 siRNA and 3 μmol/L RO318220 for 0.5 h, and then treated with 10% CSE for 3 h. The protein levels of Nrf2 in the nucleus and cytoplasm, and HO-1 and PKC in the whole cells were semi-quantified by Western blot. The protein expression of HO-1 was measured by immunocytochemistry. HO-1 mRNA expression was detected by RTPCR. Immunofluorescence staining was used to observe the nuclear translocatin of Nrf2. Results: CSE markedly induced Nrf2 nuclear translocation in the rat airway epithelial cells, and RO318220 pretreatment blocked the CSE induced Nrf2 nuclear translocation. Immunocytochemistry showed that HO-1 protein expression was strongly positive in the CSE3h group, weakly positive in the other 4 groups. The expression of PKC protein, HO-1 mRNA and protein significantly increased in the CSE3h group, and HO-1 activity markedly improved in the CSE group (P<0.05). The level of PKC protein expression was not significantly different in the Nrf2 siRNA group compared with that in the CSE3h group (P>0.05). Conclusion: CSE induces the nuclear translocation of Nrf2 by PKC signaling pathway, thus upregulating the HO-1 expression in the rat airway epithelial cells.