Qiu Qiao-meng, Zheng Jin-tao, Nan Chao, Zhao Yuan-yuan, Zhao Guang-ju, Hong Guang-liang, Liang Huan, Li Meng-Fang, Lu Zhong-Qiu
Department of Emergency Medicine, Wenzhou Medical College, Wenzhou, China.
Zhonghua Yi Xue Za Zhi. 2013 Apr 9;93(14):1114-7.
To explore the effects of NF-E2-related factor-2 (NRF2)-617C/A promoter polymorphism on NRF2 expression as well as lipopolysaccharide-induced inflammatory responses in macrophages.
NRF2-617C/A promoter fragments were synthesized by chemical method and cloned into a pUC57 vector. The dul-luciferase reporter assay was employed to determine the activity of promoters. Then recombinant adenoviral vectors were constructed and transfected into macrophages. The expression of Nrf2 was examined by Western blotting and reverse transcription (RT)-PCR. The expressions of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-10 (IL-10) in macrophages after the stimulation of LPS were determined by enzyme-linked immunosorbent assay (ELISA).
The activity of NRF2-617C promoter-luciferase reporter (FLuc/RLuc activity ratio) was significantly higher than that of NRF2-617A group (0.584 ± 0.016 vs 0.258 ± 0.018, P < 0.05).The NRF2 protein and mRNA levels in -617C group were much higher than those of 617A group (1.123 ± 0.080 vs 0.951 ± 0.057,1.889 ± 0.031 vs 1.647 ± 0.323, both P < 0.05). After the stimulation of LPS, the NRF2 protein expression in macrophages significantly increased (0.584 ± 0.016 vs 0.258 ± 0.018, P < 0.05). Compared with -617A group, there was a significantly higher expression of NRF2 in -617C group (0.671 ± 0.033 vs 0.751 ± 0.014, P < 0.05). Additionally, the productions of IL-6 and IL-10 in -617C group were markedly lower than those in -617A group as well as IL-6/IL-10 (both P < 0.05). However, no significant difference existed in the levels of TNF-α between -617C and -617A groups (P > 0.05).
The -617C/A promoter polymorphism of NRF2 may influence the NRF2 expression. And it appears to be associated with the LPS-induced inflammatory responses in macrophages.
探讨核因子E2相关因子2（NRF2）-617C/A启动子多态性对NRF2表达以及脂多糖诱导的巨噬细胞炎症反应的影响。
采用化学方法合成NRF2-617C/A启动子片段，并克隆至pUC57载体。采用双荧光素酶报告基因检测法测定启动子活性。然后构建重组腺病毒载体并转染至巨噬细胞。通过蛋白质免疫印迹法和逆转录（RT）-PCR检测Nrf2的表达。采用酶联免疫吸附测定（ELISA）法测定脂多糖刺激后巨噬细胞中肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）和白细胞介素-10（IL-10）的表达。
NRF2-617C启动子-荧光素酶报告基因的活性（荧光素酶/海肾荧光素酶活性比值）显著高于NRF2-617A组（0.584±0.016比0.258±0.018，P<0.05）。-617C组的NRF2蛋白和mRNA水平均显著高于617A组（1.123±0.080比0.951±0.057，1.889±0.031比1.647±0.323，均P<0.05）。脂多糖刺激后，巨噬细胞中NRF2蛋白表达显著增加（0.584±0.016比0.258±0.018，P<0.05）。与-617A组相比，-617C组的NRF2表达显著更高（0.671±0.033比0.751±0.014，P<0.05）。此外，-617C组的IL-6和IL-10产生量以及IL-6/IL-10均显著低于-617A组（均P<0.05）。然而，-617C组和-617A组之间的TNF-α水平差异无统计学意义（P>0.05）。
NRF2的-617C/A启动子多态性可能影响NRF2表达。并且它似乎与脂多糖诱导的巨噬细胞炎症反应有关。
