文冠果壳苷抗阿尔茨海默病的炎症相关机制研究

发布时间：2018-05-17 14:13

本文选题：文冠果壳苷 + 小胶质细胞　；参考：《沈阳药科大学》2013年博士论文

【摘要】：目的：文冠果壳苷是一种从文冠果果壳中提取出的三萜皂苷类化合物,前期研究证实其对多种痴呆模型动物的学习记忆障碍具有改善作用。为进一步探讨文冠果壳苷对阿尔茨海默病(AD)防治作用及作用机制,本文依据AD的炎症假说,从整体水平及细胞分子水平考查了文冠果壳苷对β-淀粉样蛋白(Aβ)致AD模型的改善作用及炎症相关机制。方法：①以β淀粉样蛋白25-35肽段(Aβ25-35)/γ-干扰素(IFN-γ)诱导小胶质细胞活化后,考察文冠果壳苷对Aβ25-35/IFN-γ激活的小胶质细胞NO释放量的影响；通过ELISA实验检测IL-1β及TNF-α的含量；采用western blot方法检测iNOS、COX-2、NF-κB及MAPK通路相关蛋白表达的情况；采用免疫荧光方法检测NF-κB P65的核转移情况；采用RT-PCR方法检测IL-1β、TNF-α、 iNOS、COX-2及TLR2mRNA表达的情况；通过MTT法考察小胶质细胞条件培养液对SH-SY5Y神经元生存率的影响。②侧脑室注射Aβ1-42制备痴呆小鼠模型,通过Y迷宫、Morris水迷宫实验考察文冠果壳苷对模型小鼠学习记忆障碍的影响；通过免疫组织化学方法考察了小鼠海马及皮层区CD11b的表达情况；利用ELISA方法考察了小鼠海马组织中IL-6及IL-4表达情况；利用western blot方法考察了小鼠海马组织中iNOS、COX-2、NF-κB及MAPK通路相关蛋白表达的情况；利用RT-PCR方法考察了小鼠海马组织中iNOS、COX-2及TLR2mRNA表达的情况。结果：①文冠果壳苷可显著降低Aβ25-35/IFN-γ诱导的N9细胞及原代小胶质细胞培养液中NO、TNF-α、IL-1β的含量,抑制iNOS及COX-2蛋白的表达,抑制(?)TNF-α、 IL-1β、iNOS、COX-2及TLR2mRNA表达;显著抑制NF-κB、MAPK蛋白的激活及NF-κB P65的核转移,抑制活化的小胶质细胞条件培养液引起的SH-SY5Y神经元的死亡。②文冠果壳苷(0.08～0.32mg/kg)显著提高侧脑室注射Aβ1-42致痴呆模型小鼠Y迷宫实验中小鼠自发交替反应率；显著缩短水迷宫实验中小鼠到达安全台的逃避潜伏期及游泳路程,并显著增加小鼠在原安全台所在象限游泳时间和游泳路程百分比；文冠果壳苷可显著抑制模型小鼠海马促炎症因子IL-6含量的增加及炎症抑制因子IL-4含量的减少；抑制大脑皮层及海马CD11b蛋白的表达；Western blot结果显示,文冠果壳苷可显著降低iNOS、COX-2蛋白的表达；抑制NF-κB及MAPK蛋白的激活；RT-PCR结果显示,文冠果壳苷可显著抑制iNOS、 COX-2及TLR2mRNA表达。结论：文冠果壳苷下调小胶质细胞TLR2,抑制IKK表达、IκB蛋白的降解及NF-κB p50和p65亚单位的核转录,抑制MAPKs信号转导通路,进而抑制炎症因子释放的作用可能与其神经元保护作用及对侧脑室注射Ap1-42致痴呆模型小鼠的学习记忆障碍改善作用有关。本研究及前期试验结果提示,文冠果壳苷具有显著的改善学习记忆障碍及神经保护作用,作为AD防治候选化合物,具有进一步研究开发价值。
[Abstract]:Objective: to study the effects of triciterpenoid saponins extracted from the fruit shell of Coryocera chinensis on learning and memory disorders in various dementia model animals. In order to further investigate the preventive and therapeutic effects and mechanism of arachidonin on Alzheimer's disease (AD), the inflammatory hypothesis of AD was used in this paper. The amelioration of amyloid A 尾 (尾 -amyloid A 尾) -induced AD model and the mechanism of inflammation were investigated at the whole level and cell molecular level. Methods the microglial cells were activated by 尾 amyloid 25-35 peptide A 尾 25-35 / IFN- 纬), and the effects of Coryocephalin on the release of no from A 尾 25-35% IFN- 纬 activated microglia cells were investigated, and the contents of IL-1 尾 and TNF- 伪 were detected by ELISA assay. Western blot method was used to detect the expression of NF- 魏 B and MAPK pathway related proteins, immunofluorescence method was used to detect the nuclear metastasis of NF- 魏 B p65, and RT-PCR method was used to detect the expression of IL-1 尾 -TNF- 伪, iNOS-1 and TLR2mRNA. The effects of microglia conditioned medium on survival rate of SH-SY5Y neurons were investigated by MTT method. 2 the dementia mice were induced by intraventricular injection of A 尾 1-42, and the effects of arachidonin on learning and memory impairment in model mice were investigated by Y-maze Morris water maze test. The expression of CD11b in hippocampus and cortex of mice was investigated by immunohistochemical method, and the expression of IL-6 and IL-4 in hippocampus was investigated by ELISA method. The expression of iNOS COX-2 and TLR2mRNA in mouse hippocampal tissues was investigated by western blot method and the expression of iNOS COX-2 and TLR2mRNA in hippocampus by RT-PCR method. Results the content of NO-TNF- 伪 iNOS and IL-1 尾 in A 尾 25-35% IFN- 纬 -induced N9 cells and primary microglia cells were significantly decreased, the expression of iNOS and COX-2 protein, the expression of TNF- 伪, IL-1 尾 iNOSCOX-2 and TLR2mRNA were inhibited, and the activation of NF- 魏 BmMAPK protein and the nuclear transfer of NF- 魏 B p65 were also inhibited. Inhibiting the death of SH-SY5Y neurons induced by activated microglia conditioned medium. 2. The rate of spontaneous alternating reaction in Y maze test of dementia model mice induced by intracerebroventricular injection of A 尾 1-42 was significantly increased. In the water maze test, the escape latency and swimming distance of the mice to the safety station were significantly shortened, and the swimming time and the swimming distance percentage of the mice in the quadrant of the original safety platform were significantly increased. The expression of CD11b protein in cerebral cortex and hippocampus was inhibited by the increase of pro-inflammatory factor IL-6 and the decrease of IL-4 in hippocampus, and the expression of CD11b protein in cerebral cortex and hippocampus was also inhibited by the inhibition of CD11b protein expression in cerebral cortex and hippocampus of the model mice by Western blot. The results of RT-PCR showed that the expression of iNOS, COX-2 and TLR2mRNA were significantly inhibited by the inhibition of the activation of NF- 魏 B and MAPK protein. Conclusion: cecarboside down-regulates TLR2, inhibits the degradation of IKK expression of I 魏 B and the nuclear transcription of NF- 魏 B p50 and p65 subunits, and inhibits the MAPKs signal transduction pathway. The effect of inhibiting the release of inflammatory factors may be related to the protective effect of neurons and the improvement of learning and memory impairment in dementia model mice induced by contralateral ventricular injection of Ap1-42. The results of this study and previous experiments suggest that the ciguloside can significantly improve the learning and memory impairment and neuroprotective effect, as a candidate compound for AD prevention and treatment, which has the value of further research and development.
【学位授予单位】：沈阳药科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R749.16

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