参知健脑片组分配伍对拟阿尔茨海默病细胞模型的保护作用及机制探讨

【作者】 杨傲然；

【导师】 范吉平；

【作者基本信息】 北京中医药大学 ， 中医内科学， 2007， 博士

【摘要】 目的：依据阿尔茨海默病（AD）发病机理中的“Aβ级联假说”，从Aβ产生的氧化损伤毒性的环节的各个靶点入手，并与神经营养肽APP17进行对比，探讨参知健脑方组分对Aβ的神经元毒性的保护作用。方法：1．选择参知健脑方组成中药的有效组分（即人参皂苷Rg1、知母皂苷元Sar、芍药苷Pae），首先利用细胞毒性试验确定各组分有效且无毒的作用浓度，再利用正交设计的方法，将各个浓度按不同配比组合，通过对正常SH-Y5Y细胞和拟AD细胞模型SH-Y5Y细胞增殖率的观察，比较各组配比组合后对细胞增殖率的影响，筛选出最佳配比组合用于下一步实验。2．用25μmol／L的Aβ25-35孵育SH-Y5Y细胞复制AD细胞模型，分别在6h、24h、48h、72h观察β淀粉样蛋白对于细胞的活性和存活率、乳酸脱氢酶的释放量、细胞内活性氧的产生量、细胞周期的变化、细胞凋亡率的变化及6h和24h细胞内产生的凋亡相关信号转导蛋白Bcl-2、Bax、Caspase-3的变化来观察Aβ产生的神经毒性的作用，在各个时间点分别给予中药有效组分配伍和APP17肽10μmol·L^-1，通过与模型组对比观察对Aβ产生的细胞毒性的抑制作用。结果：1．人参皂苷、知母皂苷元、芍药苷三种组分对细胞有增殖作用且无毒的作用浓度分别是人参皂苷(1.5625，3.1256，6.25μg·mL^-1)知母皂苷元(6.25，12.5，25μg·mL^-1)，芍药苷(6.25，12.5，25μg·mL^-1)，利用正交实验设计所得的最佳配比是(人参皂苷1.5625μg·mL^-1，知母皂苷元6.25μg·mL^-1，芍药苷6.25μg·mL^-1)。3．Aβ作用细胞后，细胞的存活率在随着作用时间的延长表现出了下降的趋势，与此相应，细胞的LDH释放量表现为随着作用时间的延长逐渐增加的趋势。参知健脑方组分组对细胞的存活率有一定的提高作用，对LDH释放量起到了一定的抑制作用，与APP17肽组比较无显著性差异。4．细胞的凋亡率在6h开始升高，至24h达峰值，此后凋亡率又逐渐降低，但与正常对照组始终差异显著。细胞周期表现为细胞被阻抑于G0—G1期的比例增加，S期的比例下降。参知健脑方组分组对细胞的凋亡率有一定的抑制作用，能促进细胞周期S期的比例增加，与APP17肽组比较无显著性差异。5．细胞内的活性氧随作用时间的延长逐渐增加，参知健脑方组分组表现出了一定的抑制作用，与APP17肽组比较无显著性差异。6．细胞内的凋亡信号转导蛋白Bax和Caspase逐渐增加，而Bcl-2表现为逐渐增加。参知健脑方组分组可以促进Bcl-2表达的增加，促进Bax和Caspase表达减少，与APP17肽组比较无显著性差异。结论：参知健脑方组分对Aβ引起的神经元毒性损害有保护作用，其机制可能与抑制了Aβ产生的氧化应激损伤和细胞周期异常有关，同时对凋亡信号转导蛋白的产生也有一定的抑制作用，与APP17肽的神经营养作用相似。更多还原

【Abstract】 Neuroprotective effects and possible mechanisms of SZJN on neunonsdamaged by fragment of beta-amyloid proteinObjective: According to the beta-amyloid （Aβ） cascade hypothesis inAlzheimer’s disease（AD）,the purpose of this study was to investigate thepossible effective mechanism of SZJN on neuronal toxicity of Aβ.Methods: 1. Chose the constituents of herbs in SZJN, （that means ginsengsaponin; anemarrhenae saponin; paeonia saponin）. First, determin theeffective but toxicantless constituents. Then combinate varisconcentration according thogonal design. And through observing growth rateof cell to choose the most effective combination which will used in thenext experiment. 2. SH-Y5Y cell lines were incubated with 25-35 fragmentsof Aβat a concentration of 25μmol/L to duplicate AD model used to assessthe effect and possible mechanism of SZJN. To analyze the effect, theobservation time was at 6 hour, 24 hour, 48 hour and 72 hour. The activityand survival rate was counted, morph of the cells was observed and the amountof LDH and ROS released by cell was also observed. At last the apoptosisrate and the protein of apoptosis signal transduction were also observed.Through these methods we can evaluate the AD cell model and investigatepossible mechanisms of neuron protective.Results: 1. By turns the suitable concentration of three constituentswere 1.5625μg·mL^-1,3.1256μg·mL-^-1, 6.25μg·mL^-1（ginsengsaponin）; 3.125μg·mL^-1, 6.25μg·mL-^-1, 12.5μg·mL（-1）（anemarrhenae saponin and paeoniasaponin）. The most suitable matching by the experiment of orthogonal designis ginseng saponin (1.5625μg·mL^-1), anemarrhenae saponin (6.25μg·mL-^-1),paeonia saponin (6.25μg·mL^-1). 3. As SH-Y5Y cell lines were incubated with25-35 fragments of Aβ, the survival rate of cell showed a descendingtendency. And corresponding with this LDH is gradually increasing. Not onlycan SZJN increased the survival rate, but also inhibited the realeasingof LDH. The result is similar to APP17. 4. The apoptosis rate beginincreasing at first and reached peak value at 24h. Though decreasing after24h, it still higher than the normal group. Most of cells are repressed at period of G0-G1. And the number in the S period is decreased too. Theconstituents of SZJN have a certain depression to the rate of cell apoptosis.It can pwomote the percentage of cell cycle S. And there were nosignificantly different with the APP17 group. 5. Intracellular ROSgradually increased with the role of the extended time. SZJN demonstrateda certain depression and were not significantly different with the APP17group. 6. Intracellular signal transduction pathway of apoptosis proteinBax and caspasegradually increased, while Bcl-2 first increased and thenreduced. SZJN can promote Bcl-2 increased and reduced Bax and Caspase.There were no significantly different compared with the APP17 group.Conclusion: The compants of SZJN have a protection on the Aβinducedneuronal toxicity damage. The mechanism may be related to inhibition ofthe Aβoxidative stress injury and cell cycle abnormalities. Meanwhil italso have a certain depressed effect on the apoptosis signal transductionprotein. It have a simlar role with hPP17.更多还原