蛇床子素对Aβ_(25-35)诱导的星形胶质细胞NF-κB活化机制的影响

Effects of Osthole on Activation of Nuclear Factor-kappa B in Astrocytes Induced by Amyloid Protein Beta 25-35

【目的】探讨蛇床子素（Osthole，Ost）拮抗β淀粉样蛋白25—35（Ap25-35）毒性损伤、保护神经细胞的作用及其与核因子-κB（NF—κB）活化机制的关系。【方法】以原代培养的大鼠星形胶质细胞（astrocytes，AS）为靶标建立阿尔茨海默病（Alzheimer＇s disease，AD）细胞模型。采用CCK-8（cell counting kit-8）法检测细胞活力，进行AB25-35造模浓度、时间以及Ost预处理最适宜浓度的选择。经Aβ25-35和Ost干预后，采用激光共聚焦显微镜检测分析异硫氰酸荧光素／碘化丙啶（FITC／PI）双重标记的NF—κB与其抑制蛋白（IKBot）在细胞内的激活程度，结合形态学观察分析Ost对星形胶质细胞的保护作用。【结果】Ost可拮抗Aβ25-35的神经毒性作用，尤其以低浓度（0．01-1μmol／L）为佳，随着浓度的升高，对细胞的保护作用呈减弱趋势。40μmol／L的Aβ25-35作用于As24h可过度活化NF—κB的表达（P〈0．01），激活IκBα发生磷酸化及降解（P〈0．01）。低浓度的Ost可显著抑制NF—κB的过度活化（P〈0．01），上调细胞核内IκBα的表达（P〈0．01）。【结论】Ost抑制Aβ25-35的神经毒性作用，延缓AD发生发展的作用机理可能与NF—κB活化机制有关。

Objective To study the correlation between neuroprotective effect of osthole （Ost） and the activation of nuclear factor kappa B （NF-κB） in neurons induced by amyloid-beta peptide 25-35 （Aβ25-35）. Methods Rat cell model of Alzheimer＇s disease （AD） was induced by Aβ25-35. According to the results of cellular morphology and cell counting （CCK-8） colorimetric analysis, we optimized the concentration and duration of Aβ25-35 for establishment of AD cell model , and also determined the optimal pretreatment concentration and duration of Ost. The activities of NF- kappa B （NF-κB） and inhibin of NF-kappa B alpha （IκBα） in astrocytes were analyzed with laser scanning eonfocal microscope （LSCM） after fluorescencein-5-isothioeyanate/propidium iodide （FITC/PI） double labeling. Results Aβ25-35 of 40μmol/L was selected as the stable effective concentration and 24h was the optimal effective time for the establishment of AD cell model. Aβ25-35 of 40μmol/L up-regulated the activity of NF-κB （P 〈 0. 01 ） and activated the phosphorylation and degradation of IκBα （P 〈 0. 01 ） in astrocytes. Ost at lower concentrations （0. 01 - 1 μmol/ L） protected astroeytes by up-regulating the expression of IκBα （P 〈0. 01 ） and by inhibiting over-activation of NF-κB （P 〈 0. 01 ）, and the protective effect of Ost showed a decreasing trend when the Ost concentration increased. Conclusion The mechanism of Ost in inhibiting Aβ-induced neurotoxieity and in delaying the progress of AD is probably related with the regulation of the activity of NF-κB.