文冠果壳苷抗阿尔茨海默病的炎症相关机制研究

【作者】 齐越；

【导师】 邹莉波；

【作者基本信息】 沈阳药科大学 ， 药理学， 2013， 博士

【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。更多还原

【Abstract】 Aim:Xanthoceraside is a novel triterpenoid saponin extracted from the shell of the fruit of Xanthoceras sorbifolia Bunge. Our previous study showed that xanthoceraside could significantly improve learning and memory impairment in several Alzheimer’s disease (AD) animal models. In this study, we further investigate the effect of xanthoceraside on AD model in vitro and in vivo according to inflammation hypothesis, which aims to explore its anti-AD effects and possible related mechanisms of action.Methods:①We investigated the effect of xanthoceraside on Aβ25-35/IFN-γ-induced microglia activation. The NO production was measured by Griess regent. The levels of IL-1β and TNF-a protein expression were measured by ELISA assay according to the manufacturer’s instructions; NF-κB P65nuclear translocation were measured with the immunofluorescence assay, the expression of iNOS、COX-2、 NF-κB and MAPKs were measured with western blot assay; iNOS、COX-2and TLR2mRNA expression were measured with RT-PCR.②We studied the effect of xanthoceraside on learning and memory impairments in mice induced by intracerebroventricular (i.c.v.) injection of the aggregated Aβ by using Y-maze and Morris water-maze test. The expression of CD11b in the hippocampus and cortex of mice were measured with immunohistochemistry assay; IL-6and IL-4protein expression were measured with ELISA assay according to the manufacturer’s instructions; The expression of iNOS、COX-2、NF-κB and MAPKs were measured with western blot assay; iNOS、COX-2and TLR2mRNA expression were measured with RT-PCR.Results:①Compared with model group, Pretreatment with xanthoceraside (0.01,0.1μM) significantly suppressed the increase of NO, TNF-α and IL-1β production inprimary microglia and N9microglia, respectively, down-regulated TNF-α and IL-1β mRNA expression, decreased Aβ25.35/IFN-γ-induced iNOS and COX-2protein and mRNA expression in a concentration-dependent manner; Xanthoceraside inhibited the AP25-35/IFN-γ-induced translocation of NF-κB p50and p65into the nucleus, blocked IκB degradation and increased IKK level. Pretreatment with xanthoceraside significantly attenuated the Aβ25-35/IFN-y-induced phosphorylation of ERK, JNK and p38, whereas their non-phosphorylated forms were not changed, and decreased TLR2gene expression. Xanthoceraside protected neuron damage by the inhibition of microglia activation.②Compared with model group, xanthoceraside (0.08～0.32mg/kg) significantly decreased the escape latency and swimming distance in Morris-water maze test, and increased the swimming time and percentage of the swimming distance in the fourth quadrant where the platform had been locatedin the probe test; Xanthoceraside increased the percentage of alternation behaviors in Y-maze test; Xanthoceraside suppressed the release of IL-6and increased the IL-4production, decrease in CD11b positive particles in both cerebral cortex and hippocampus, attenuated iNOS and COX-2protein and mRNA expression, inhibited the translocation of NF-κB p50and p65into the nucleus, attenuated the phosphorylation of ERK, JNK and p38; and decreased TLR2gene expression.Conclusion:Xanthoceraside has the effect of improving learning and memory impairment in mice induced by i.c.v. of Aβ and inhibited pro-inflammation expression in Aβ25-35/IFN-γ-stimulated microglia via TLR2/MAPKs and NF-κB pathways. The neuroprotective effects of xanthoceraside may include the inhibition of pro-inflammation and TLR2expression, and attenuated the NF-κB and MAPKs activity. Therefore, xanthoceraside may provide a useful therapeutic strategy for the treatment of AD.更多还原