本文選題：文冠果殼苷 + 小膠質(zhì)細胞�。� 參考：《沈陽藥科大學》2013年博士論文

【摘要】：目的：文冠果殼苷是一種從文冠果果殼中提取出的三萜皂苷類化合物,前期研究證實其對多種癡呆模型動物的學習記憶障礙具有改善作用。為進一步探討文冠果殼苷對阿爾茨海默病(AD)防治作用及作用機制,本文依據(jù)AD的炎癥假說,從整體水平及細胞分子水平考查了文冠果殼苷對β-淀粉樣蛋白(Aβ)致AD模型的改善作用及炎癥相關機制。方法：①以β淀粉樣蛋白25-35肽段(Aβ25-35)/γ-干擾素(IFN-γ)誘導小膠質(zhì)細胞活化后,考察文冠果殼苷對Aβ25-35/IFN-γ激活的小膠質(zhì)細胞NO釋放量的影響；通過ELISA實驗檢測IL-1β及TNF-α的含量；采用western blot方法檢測iNOS、COX-2、NF-κB及MAPK通路相關蛋白表達的情況；采用免疫熒光方法檢測NF-κB P65的核轉移情況；采用RT-PCR方法檢測IL-1β、TNF-α、 iNOS、COX-2及TLR2mRNA表達的情況；通過MTT法考察小膠質(zhì)細胞條件培養(yǎng)液對SH-SY5Y神經(jīng)元生存率的影響。②側腦室注射Aβ1-42制備癡呆小鼠模型,通過Y迷宮、Morris水迷宮實驗考察文冠果殼苷對模型小鼠學習記憶障礙的影響；通過免疫組織化學方法考察了小鼠海馬及皮層區(qū)CD11b的表達情況；利用ELISA方法考察了小鼠海馬組織中IL-6及IL-4表達情況；利用western blot方法考察了小鼠海馬組織中iNOS、COX-2、NF-κB及MAPK通路相關蛋白表達的情況；利用RT-PCR方法考察了小鼠海馬組織中iNOS、COX-2及TLR2mRNA表達的情況。結果：①文冠果殼苷可顯著降低Aβ25-35/IFN-γ誘導的N9細胞及原代小膠質(zhì)細胞培養(yǎng)液中NO、TNF-α、IL-1β的含量,抑制iNOS及COX-2蛋白的表達,抑制(?)TNF-α、 IL-1β、iNOS、COX-2及TLR2mRNA表達;顯著抑制NF-κB、MAPK蛋白的激活及NF-κB P65的核轉移,抑制活化的小膠質(zhì)細胞條件培養(yǎng)液引起的SH-SY5Y神經(jīng)元的死亡。②文冠果殼苷(0.08～0.32mg/kg)顯著提高側腦室注射Aβ1-42致癡呆模型小鼠Y迷宮實驗中小鼠自發(fā)交替反應率；顯著縮短水迷宮實驗中小鼠到達安全臺的逃避潛伏期及游泳路程,并顯著增加小鼠在原安全臺所在象限游泳時間和游泳路程百分比；文冠果殼苷可顯著抑制模型小鼠海馬促炎癥因子IL-6含量的增加及炎癥抑制因子IL-4含量的減少；抑制大腦皮層及海馬CD11b蛋白的表達；Western blot結果顯示,文冠果殼苷可顯著降低iNOS、COX-2蛋白的表達；抑制NF-κB及MAPK蛋白的激活；RT-PCR結果顯示,文冠果殼苷可顯著抑制iNOS、 COX-2及TLR2mRNA表達。結論：文冠果殼苷下調(diào)小膠質(zhì)細胞TLR2,抑制IKK表達、IκB蛋白的降解及NF-κB p50和p65亞單位的核轉錄,抑制MAPKs信號轉導通路,進而抑制炎癥因子釋放的作用可能與其神經(jīng)元保護作用及對側腦室注射Ap1-42致癡呆模型小鼠的學習記憶障礙改善作用有關。本研究及前期試驗結果提示,文冠果殼苷具有顯著的改善學習記憶障礙及神經(jīng)保護作用,作為AD防治候選化合物,具有進一步研究開發(fā)價值。
[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- 緯), and the effects of Coryocephalin on the release of no from A 尾 25-35% IFN- 緯 activated microglia cells were investigated, and the contents of IL-1 尾 and TNF- 偽 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- 偽, 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- 偽 iNOS and IL-1 尾 in A 尾 25-35% IFN- 緯 -induced N9 cells and primary microglia cells were significantly decreased, the expression of iNOS and COX-2 protein, the expression of TNF- 偽, 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.
【學位授予單位】：沈陽藥科大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R749.16

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相關期刊論文 前1條

1 王莉莉;馬挺;巴俊杰;;蒙藥文冠木研究進展[J];內(nèi)蒙古醫(yī)學院學報;2008年S2期

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本文編號：1901686