低剂量脂多糖诱导PC12细胞中Nrf2的表达及其抗ROS作用的实验研究

Effects of low-dose lipopolysaccharide on up-regulating Nrf2 expression and reducing ROS in PC12 cells

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 study the effects of low-dose lipopolysaccharide（LPS） on nuclear factor-erythroid 2-related factor-2（Nrf2） expression and reactive oxygen species（ROS） level in PC12 cells.Methods LPS solutions with concentrations of 0.01,0.025,0.05,0.1,0.25,0.5 and 1 μg/μl were used to treat PC12 cells for 24 h,and then cell viability was tested by MTT assay to determine the maximum safe dose.PC12 cells treated by LPS of the determined dose were subjected to detection of Nrf2 mRNA and protein expression in whole cell and Nrf2 protein expression in cytoplasm and nuclei through RT-PCR and Western blotting.The average fluorescence intensity of ROS in the cells was tested by flow cytometry as well.Results LPS with a concentration lower than 0.1 μg/μl had no obvious toxicity to PC12 cells（P0.05）.In PC12 cells treated by 0.1 μg/μl LPS for 24 h,the expression of Nrf2 mRNA and protein in whole cell increased gradually.During the process,there were significant differences for mRNA expression at 18 h as well as mRNA and total protein expression at 24 h,as compared with those of the control group（P0.01）.In PC12 cells treated by 0.1 μg/μl LPS for 1,3,6 and 12 h,the Nrf2 protein expression in cytoplasm decreased gradually and then recovered slightly,with the lowest expression at 6 h,as compared with that of the control group（P0.01）.The Nrf2 protein expression in the nuclei increased,with a significant difference from that of the control group（P0.01）,and showed an expression peak at 3 h.In PC12 cells treated by 0.1 μg/μl LPS for 8 h,the average fluorescence intensity of ROS decreased significantly（P0.01）.Conclusion Low-dose LPS can promote the transcription,translation and nuclear translocation of Nrf2 in PC12 cells with time dependence and reduce ROS level,and probably participates in stress tolerance.