摘要
目的:观察人参皂苷Rg1对H2O2诱导HEK293T细胞损伤过程中NF-κB转录活性的影响,探讨人参皂苷Rg1的抗氧化机制。方法:H2O2模拟氧化应激条件,MTT法和台盼蓝染色法检测人参皂苷Rg1对细胞生长的影响;以DCFH-DA为探针流式细胞仪检测细胞内ROS水平;利用双荧光素酶顺式报告系统,检测人参皂苷Rg1对荧光素酶报告基因NF-κB-Luc相对荧光素酶值的影响,以检测人参皂苷Rg1是否具有下调H2O2诱导的NF-κB转录激活的作用。结果:H2O2诱导损伤的293T细胞存活率随H2O2浓度的增高而下降,相同H2O2浓度下,人参皂苷Rg1预保护组细胞存活率较损伤组显著提高(P<0.05);H2O2处理后细胞内自由基明显增加,其荧光强度增加了40%～50%,而给予有效浓度的人参皂苷Rg1后,荧光强度降低35%～40%;与正常对照组比较,H2O2模拟氧化应激条件下,HEK293T细胞中NF-κB荧光素酶报告活性明显升高(P<0.05),而在人参皂苷Rg1预保护组则明显受到抑制(P<0.05)。结论:人参皂苷Rg1对H2O2所致的细胞氧化应激损伤具有明显的保护作用,其可能机制是有效地清除了细胞内过多的自由基,下调了转录因子NF-κB的转录活性,继而抑制了NF-κB通路的激活。
Objective：To observe the influence of ginsenoside Rgl on transcriptional activation of NF-kB induced by hydrogen peroxide （H2O2） in 293T cell, and probe into the antioxidant mechanism of ginsenoside Rgl. Methods： In the experiment, cells was exposed to H2O2 after pretreatment with Rgl, cell proliferation and cytotoxicity studies were detected by MTT and Trypan blue. The quantities of generation of intracellular reactive oxygen species （iROS） was analyzed by flow cytometric analysis measured with fluorescent probe 2,7-dichlorofluorescin diacetate （DCFH-DA）. NF-kB-responsive element-luciferase reporter gene was transfected and dual-luciferase cis-reporting systems were used to assay the transcriptional activity of NF-kB under the stimulated circumstance of oxidative stress induced by H2O2.Results：The results of MTT showed that ginseneside Rgl apparently protected the proliferation of 293T cell, which were repressed by H2O2 （ P 〈 0.05）. The results by try- pan blue showed that H2O2 stimulated substantial cytotoxicity. This effect was markedly attenuated by treatment with ginsenoside Rgl. Oxidant production,measured as the fluorescence of diehlorofluorescein, was significant increased by 40% -50% through H2O2 stimulation. The decrease in iROS generation was significant blocked by 35% -40% through Rgl and antioxidant.The relative luciferase reporter assay of NF-kB was apparently improved by H2O2-induced（ P 〈 0.05）, but Giusenoside Rgl significantly repressed the relative value of luciferase （P 〈 0.05）. Conclusion： Ginsenoside Rgl has the obvious protective function from the damage of oxidative stress damage, whose possible mechanism is to eliminate excessive free radicals of the cells effectively, to reduce transcriptional activation of nuclear factor kappa B（ NF-kB）, and subsequently to suppress the NF-kB circuit activation.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2009年第11期991-995,共5页
Chinese Journal of Immunology
基金
国家自然科学基金(No.30772872)
青年科学基金资助项目(No.30701105)
关键词
人参皂苷RG1
氧化应激
NF-ΚB
双荧光素酶报告系统
Ginsenoside Rg1
Oxidative stress
Nuclear factor-kappa B（NF-κB）
Dual-luciferase reporter system
