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目的研究低剂量脂多糖(lipopolysaccharide,LPS)诱导的PC12细胞中核因子E2相关因子2(nuclear factor-erythroid 2-related factor-2,Nrf2)的表达及其抗活性氧(reactive oxygen species,ROS)的作用。方法分别以浓度为0.01、0.025、0.05、0.1、0.25、0.5、1μg/μl的LPS刺激PC12细胞24 h,MTT法检测细胞活性确立对细胞没有毒性作用的最大安全剂量;采用此剂量刺激PC12细胞,RT-PCR、Western blot检测细胞内Nrf2 mRNA、总蛋白及细胞质、细胞核内Nrf2蛋白的表达;流式细胞仪检测LPS处理8 h细胞内ROS的平均荧光强度。结果 LPS浓度小于0.1μg/μl对细胞没有明显毒性作用(P>0.05);采用0.1μg/μl LPS刺激PC12细胞,24 h内细胞内Nrf2 mRNA及总蛋白表达逐渐升高,其中18 hmRNA及24 h mRNA和总蛋白与空白对照组比较差异显著(P<0.01),12 h内胞质Nrf2蛋白表达先降低后似有恢复趋势,6 h最低(P<0.01),胞核蛋白表达升高,各时相组与空白对照组比较差异显著(P<0.01),3 h达到峰值;0.1μg/μlLPS刺激的8 h,PC12细胞内ROS的平均荧光强度明显减弱(P<0.01)。结论低剂量LPS具有时间依赖性方式促进PC12细胞内Nrf2转录、翻译和核转位的作用,下调细胞内ROS表达水平,并可能藉此参与应激耐受。
Objective To investigate the expression of nuclear factor-erythroid 2-related factor-2 (Nrf2) induced by low dose lipopolysaccharide (LPS) in PC12 cells and its role in reactive oxygen species effect. Methods PC12 cells were stimulated with LPS at the concentrations of 0.01, 0.025, 0.05, 0.1, 0.25, 0.5 and 1 μg / μl for 24 h, respectively. MTT assay was used to determine the maximum safe dose with no cytotoxic effect on cells. The expression of Nrf2 mRNA, total protein, cytoplasm and Nrf2 protein in cells were detected by RT-PCR and Western blot. The average fluorescence intensity of ROS in LPS-treated cells was detected by flow cytometry. Results LPS concentration of less than 0.1μg / μl had no significant cytotoxic effect on cells (P> 0.05). After stimulated with 0.1μg / μl LPS, the expression of Nrf2 mRNA and total protein in 24 hours was significantly increased, among which 18 h mRNA and 24 (P <0.01). The expression of Nrf2 protein in cytoplasm appeared to be recovering at first and then decreased at 6 h (P <0.01), while the expression of nuclear protein increased at 6 h The average fluorescence intensity of ROS in PC12 cells was significantly reduced at 0.1μg / μl LPS stimulation for 8 h (P <0.01). Conclusion Low-dose LPS can promote the transcription, translation and nuclear translocation of Nrf2 in PC12 cells in a time-dependent manner and downregulate the expression of ROS in cells, and may take part in the stress tolerance.