摘要
目的:观察人参皂苷Rg1对淀粉样β蛋白25～35诱导的海马神经元细胞损伤是否具有保护作用,并分析其作用机制。方法:实验于2004-03/2005-03在广州中医药大学临床药理研究所DME中心完成。①取新生24h清洁级SD大鼠,无菌环境下取出海马组织进行贴壁培养并以B27无血清培养基添加剂抑制非神经元细胞的生长。②海马神经元形态及细胞活力观察:细胞培养7d至分化成熟状态,然后被随机分为3组:人参皂苷Rg1预处理组(分为1,2,4,8,16μmol/L5个浓度组),首先每加入各浓度的Rg1(中国药品生物制品检定所提供,批号:0703-200221)预作用24h后,再加入40μmol/L淀粉样β蛋白25～35共孵育24h;模型组:每孔加入40μmol/L淀粉样β蛋白25～35作用24h;空白组:正常细胞培养用液;每组均为8孔。运用倒置显微镜进行海马神经元形态学观察,四甲基偶氮唑盐比色法法检测细胞活力,即吸光度(A值),该值越高说明细胞活力越强。③检测核因子κB活性:调整细胞悬液密度为2×108L-1,接种于6孔板(内置已用多聚赖氨酸包被的盖玻片),1mL/孔。随机分为5组(3孔/组):正常组,模型组,2,4,8μmol/L人参皂甙Rg1预处理组。细胞培养7d后,2,4,8μmol/L人参皂甙Rg1预处理组加终浓度为2,4,8μmol/L的人参皂甙Rg1预作用24h后,与模型组一起加40μmol/L淀粉样β蛋白,作用24h。激光共聚焦显微镜检测核因子κB在细胞内的激活程度,以图像分析仪计算核区荧光与胞浆区荧光比值,比值越高说明核因子κB活性越高。④计量结果差异比较采用单因素方差分析。结果:①神经元细胞形态:相差显微镜下可见人参皂甙Rg1预处理组细胞损伤相对较模型组轻,但较正常组严重。②海马神经元细胞活力:正常组和2,4μmol/L人参皂甙Rg1预处理组明显高于模型组(P<0.05),以4μmol/L人参皂甙Rg1预处理组最高;1,8,16μmol/L人参皂甙Rg1预处理组虽然也高于模型组,但差异不明显(P>0.05)。③神经元细胞核因子κB活性:正常组和2,4,8μmol/L人参皂甙Rg1预处理组明显高于模型组(P<0.01),以4μmol/L人参皂甙Rg1预处理组最高;4,8μmol/L人参皂甙Rg1预处理组明显高于正常组(P<0.01)。结论:人参皂甙Rg1对淀粉样β蛋白25～35诱导的大鼠海马神经元损伤具有保护作用,尤以4μmol/L人参皂甙Rg1作用效果最明显。核因子κB信号通路可能是人参皂甙Rg1对抗淀粉样β蛋白25～35细胞毒性作用的重要途径之一。
AIM： To explore primarily if ginzenoside Rg1 is protective for hippocampal neuronal damage induced by amyloid β protein 25-35 （Aβ25-35）, and analyze the mechanism of this effect.
METHODS： The experiment was finished in DME Center, Institute of Clinical Pharmacology, Guangzhou University of Traditional Chinese Medicine from March 2004 to March 2005. ①Hippocampal neurons were separated at sterile condition and cultured adhesively from newborn （less than 24 hours） SD rats of cleaning grade. Meanwhile, B27 zerum-free medium was used to inhibit the growth of non-neurons.②Hippocampus neuronal morphology and cell activity： After seven-day culture, the differentiated cells were divided randomly into 3 groups： Rg1 pretreatment group： After pretreated with Rg1 （1, 2, 4, 8 and 16 μmol/L, provided by China Materia Medica Biological Product Inspection Institute, batch code： 0703-200221） for 24 hours, the cells were incubated in 40 μmol/L Aβ25-35, totally for 24 hours. Model group： The cells in each hole were put in 40 μmoL/L Aβ25-35 to culture for 24 hours. Control group： Normal cell culture solution, with 8 holes in each group. Hippocampal neurons were observed morphologically by inverted phase contrast microscope. MTT colorimetrie analysis was used to measure the cell activity, that was absorbance （A value）, and the higher A value indicated the stronger cell activity. ③Assay the activity of nuclear factor-kappa B （NF-κB）： With the adjusted suspension concentration of 2×10^8 L^-1, the cells were incubated on the 6-hole plate （cover glass coated with polylysine）, 1 mL per hole. Then the cells were randomized into 5 groups with 3 holes in each： control group, model group, and Rg1 pretreatment groups of 2, 4 and 8μmol/L. After seven-day culture, the cells of Rg1 pretreatment groups were treated with Rg1 （2, 4 and 8μmoL/L） for 24 hours, and then put in 40μmol/L Aβ 25-35 to co-culture for 24 hours at the same time with model group, laser scanning confecal microscope （LSCM） was performed to assay the activation of NF-κB in hippecampal neurons, while the image analyzer was applied to calculate the ratio of fluorescence in nuclear area and fluorescence in plasm area, and the higher ratio implied the higher activity of NF-κB.④The difference of measurement was compared with one-way analysis of variance （ANOVA）.
RESULTS： ①Ncuronal morphology： Observed by phase contrast microscope, the cellular damage of Rg1 groups were better than that of model group, but severer than that of control group.②Hippocampus neuronal cell activity： The cell activity was obviously higher in control group and Rg1 pretreatment groups of 2 and 4μmol/L than in model group （P 〈 0.05）, and the highest in Rgl pretreatment group of 4μmol/L; The cell activity was also elevated in Rgl pretreatment groups of 1, 8 and 16μmol/L compared with model group, with the insignificant difference （P 〉 0.05）.③Activity of NF-κB： The activity of model group was significantly lower than that of control group and Rg1 pretreatment groups of 2, 4 and 8μmol/L （P〈 0.01）, and the highest in Rg1 pretreatment group of 4μmol/L; In addition, the activity of control group was decreased obviously compared with Rg1 pretreatment group of 4 and 8μmol/L （P 〈 0.01）.
CONCLUSION： Ginseneside Rg1 can protect the hippecampus neuronal damage induced by Aβ 25-35, especially 4μmol/L Rgl obviously. Rg1 also inhibits the neurotoxicity of Aβ 25-35 through NF-κB signal pathway.
出处
《中国临床康复》
CSCD
北大核心
2006年第23期39-42,i0001,共5页
Chinese Journal of Clinical Rehabilitation
基金
广东省自然科学基金资助项目(31479)~~
