越南人参皂苷R7抑制LPS及TNF-α联合诱导大鼠星形胶质细胞C6的激活作用研究  

Inhibitory effects of vina-ginsenoside R7 on activation of rat C6 astrocytes induced by LPS and TNF-α combination

作　　者：王晓双 黄韬 秦力悦 李鸿丽[1] 兰云意 吴辉[1] 张蓓蓓[1] 石海莲[1] 吴晓俊[1] 王峥涛[1] 

机构地区：[1]上海市复方中药重点实验室暨上海中医药大学中药研究所,上海201203

出　　处：《中国中药杂志》2016年第8期1498-1503,共6页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金重点项目(81530096);上海高校特聘教授(东方学者)岗位计划项目(2013-59);上海高校中药药效物质E研究院项目

摘　　要：该研究旨在探讨越南人参皂苷R7(R7)对LPS及TNF-α联合诱导下大鼠星形胶质细胞C6的激活抑制作用及机制。取对数生长的C6细胞,在无血清的DMEM培养基中饥饿培养24 h后,使用0.25%胰酶将其消化收集,按1.5×106个/mL密度接入培养板中培养过夜。实验分组如下:对照组(无药物处理),模型组(LPS 1 mg·L^(-1),TNF-α10μg·L^(-1)处理24 h),给药组(分别用6.25,12.5,25,50,75μmol·L-1R7,4μmol·L^(-1)L-NMMA预处理2 h后,再经过LPS 1 mg·L^(-1),TNF-α10μg·L^(-1)刺激24 h)。处理之后,CCK-8法检测细胞活力,Griess法检测培养基中NO含量,ELISA法检测培养基中IL-6,TNF-α浓度,实时定量PCR法检测细胞中相关炎症基因的表达,双荧光素酶报告基因法检测细胞中NF-κB信号分子的激活。研究发现,与模型组相对比,R7量效相关性降低C6细胞的NO释放水平,其IC50约为34μmol·L^(-1);同时,R7减少LPS及TNF-α刺激造成的细胞增殖。50μmol·L-1R7可以显著降低iNOS(P<0.001),TNF-α(P<0.001),IL-1β(P<0.05)以及COX-2(P<0.001)的基因表达,不能下调IL-6的基因表达,但可以减少TNF-α(P<0.001)及IL-6(P<0.001)的释放。进一步的研究表明,不同剂量R7(25,50,100μmol·L-1)均可显著抑制NF-κB转录活性(P<0.05,P<0.01及P<0.001)。研究表明,R7可以抑制LPS及TNF-α联合诱导炎症因子基因表达及分泌,其作用可能与其抑制NF-κB转录活性相关,提示其在神经炎症相关中枢神经系统疾病中可能的治疗作用。To investigate the inhibitory effect and mechanism of vina-ginsenoside R7（ R7） on the activation of rat C6 astrocytes cells induced by LPS / TNF-α,cells in logarithmic growth phase were cultured in DMEM medium without FBS for 24 h. After dissociated using 0. 25% EDTA-trypsin,the cells were seeded into respective plates at the density of 1. 5 × 10^6 cells per m L and cultured overnight. The cells were divided into the following groups： control group（ no treatment）,model group（ treated with LPS 1 μg·m L^-1 and TNF-α 10 μg·L^（-1）treated for 24 h）,R7 groups（ pre-treated with 6. 25,12. 5,25,50,and 75 μmol·L^-1 R7,4 μmol·L^-1 LNMMA for 2 h and then stimulated with LPS 1 mg·L^-1 and TNF-α 10 μg·L^-1 for 24 h）. Cell viability was analyzed by CCK-8 kit.Secretion of nitric oxide（ NO） in the medium was measured by Greiss method. Concentrations of interleukin-6（ IL-6） and tumor necrosis factor（ TNF-α） were assayed by ELISA kits. Gene expressions of inflammatory factors were examined by quantitative-PCR analysis. Activation of NF-κB was detected by dual luciferase reporter gene assay kit. The results showed that R7 could significantly inhibit the secretion of NO from C6 cells in a dose-effect manner,with an IC50 of 34 μmol·L^-1. And it could reduce cell proliferation induced by LPS / TNF-α stimulation. Furthermore,R7 at 50 μmol·L^-1 significantly down-regulated gene expressions of i NOS（ P〈0. 001）,TNF-α（ P〈0. 001）,IL-1β（ P〈0. 05）,and COX-2（ P〈0. 001）,but could not change gene expression of IL-6. However,R7 reduced the secretion of TNF-α（ P〈0. 001） and IL-6（ P〈0. 001）. Further studies disclosed that,different concentrations of R7（ 25,50,100 μmol·L^-1） could significantly inhibit the transcription activity of NF-κB（ P〈0. 05,P〈0. 01,and P〈0. 001）. In conclusion,R7 could inhibit inflammatory responses in C6 cells induced by LPS / TNF-α probably by inhibiting the transcription activity of NF-κB,which indicates its poss

关 键 词：神经炎症 越南人参皂苷R7 脂多糖 炎症因子 NF-κB 星形胶质细胞 

分 类 号：R285.5[医药卫生—中药学]