【摘要】：目的:探討蛋白激酶C(PKC)-紅系衍生的核因子相關(guān)因子2(Nrf2)對(duì)香煙煙霧提取物(CSE)誘導(dǎo)的大鼠氣道上皮細(xì)胞血紅素加氧酶-1(HO-1)表達(dá)的影響。方法:將25只SD大鼠的氣道上皮細(xì)胞隨機(jī)分為對(duì)照組、CSE3h組、RO318220(PKC抑制劑)組、Nrf2 siRNA組和Nrf2 siRNA+RO318220組,每組5只。對(duì)照組用DMEM/F12正常培養(yǎng),CSE3h組用10%CSE共培養(yǎng)3 h;RO318220組則用3μmol/L RO318220預(yù)處理0.5h,余處理同CSE3h組;Nrf2 siRNA組先用Nrf2 siRNA預(yù)處理,余處理同CSE3h組;Nrf2 siRNA+RO318220組則先用3μmol/L RO318220和Nrf2 siRNA預(yù)處理,余處理同CSE3h組。用Western印跡法分別檢測(cè)HO-1,Nrf2和PKC蛋白表達(dá),細(xì)胞免疫化學(xué)法觀察HO-1蛋白表達(dá),反轉(zhuǎn)錄-聚合酶鏈反應(yīng)(RT-PCR)法檢測(cè)HO-1mRNA表達(dá),免疫熒光法觀察Nrf2核轉(zhuǎn)位,測(cè)定HO-1活性。結(jié)果:CSE明顯誘導(dǎo)Nrf2核轉(zhuǎn)位,其誘導(dǎo)作用可被RO318220阻斷。HO-1蛋白在CSE3h組強(qiáng)陽(yáng)性表達(dá),在對(duì)照組、RO318220組、Nrf2 siRNA組和Nrf2siRNA+RO318220組均弱陽(yáng)性表達(dá)。CSE3h組PKC蛋白、HO-1 mRNA和蛋白表達(dá)較對(duì)照組顯著升高,HO-1活性較對(duì)照組明顯增強(qiáng)(P0.05)。PKC蛋白表達(dá)水平在Nrf2 siRNA組和CSE3h組無(wú)明顯差異(P0.05)。結(jié)論:CSE通過(guò)PKC信號(hào)通路誘導(dǎo)Nrf2核轉(zhuǎn)位,上調(diào)HO-1的表達(dá)水平。
[Abstract]:Aim: to investigate the effect of protein kinase C (PKC)-erythroid derived nuclear factor-associated factor 2 (Nrf2) on the expression of heme oxygenase-1 (HO-1) in rat airway epithelial cells induced by cigarette smoke extract (CSE). Methods: twenty-five SD rats were randomly divided into three groups: control group, CSE3h group, RO318220 (PKC inhibitor) group, Nrf2 siRNA group and Nrf2 siRNA RO318220 group. The control group was cultured with DMEM/F12, the CSE3h group was co-cultured with 10%CSE for 3 h, the RO318220 group was pretreated with 3 渭 mol / L RO318220 for 0.5 h, the rest was pretreated with CSE3h, the Nrf2 siRNA group was pretreated with Nrf2 siRNA, and the other was treated with CSE3h. Nrf2 siRNA RO318220 group was pretreated with 3 渭 mol / L RO318220 and Nrf2 siRNA, and the others were the same as CSE3h group. The expression of HO-1,Nrf2 and PKC protein was detected by Western blot, the expression of HO-1 protein was observed by immunocytochemistry, the expression of HO-1mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR), and the nuclear translocation of Nrf2 was observed by immunofluorescence. HO-1 activity was determined. Results: CSE significantly induced nuclear translocation of Nrf2, which could be blocked by RO318220. Ho-1 protein was strongly expressed in CSE3h group, and weakly expressed in control group, RO318220 group, Nrf2siRNA group and Nrf2siRNA RO318220 group. The expression of HO-1 mRNA and protein was significantly higher than that of the control group, and the activity of HO-1 was significantly higher than that of the control group (P0.05), but there was no significant difference in the expression level of PKC protein between the Nrf2 siRNA group and the CSE3h group (P0.05). Conclusion: CSE induces nuclear translocation of Nrf2 through PKC signaling pathway and up-regulates the expression of HO-1.
【作者單位】： 湖南省人民醫(yī)院呼吸內(nèi)科;
【分類號(hào)】：R563.9

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