【摘要】 目的研究绿原酸(CHA)对淀粉样β蛋白25-35(Aβ25-35)激活巨噬细胞导致大脑皮层细胞损伤的保护作用机制。方法采用生后0~3 d左右的Sprague-Dawley(SD)大鼠乳鼠,取出大脑皮层细胞然后做体外培养。培养8 d后与小鼠腹腔巨噬细胞共同培养2 d,再加入10μmol/LAβ25-35制作阿尔茨海默病细胞模型。实验分为单独培养组、共同培养组、单独培养Aβ组、共同培养Aβ组和共同培养CHA组分别作用0.5、1、2、4、6 h后检测各组的结果。细胞损伤程度通过观察细胞形态和记数判定。p38分裂原激活蛋白激酶(p38MAPK)、有丝裂原激活蛋白激酶活化的蛋白激酶2(MAPKAPK-2)和热休克转录因子(HSP27)三种蛋白磷酸化的表达水平由Western blot检测。通过免疫荧光观察微管相关蛋白-2(MAP-2)的表达。结果 CHA显著抑制Aβ25-35处理所致的颗粒细胞密度的减少并且改善细胞的形态。Aβ25-35可以使p38MAPK的磷酸水平明显增加,在2h的时候磷酸化水平达到最高峰,4h后就逐渐下降,而使MAPKAPK-2和HSP27的磷酸化水平降低,2h降低最明显。结论 CHA通过抑制Aβ25-35诱导巨噬细胞释放炎症因子激活的p38MAPK信号转导通路,从而可以起到保护神经细胞的作用。更多还原

【Abstract】 Objective To investigate the effect of chlorogenic acid on cerebral cortical neurons injury induced by amyloid β protein via macrophage activation in vitro. Methods Cerebral cortical neurons were obtained from the cerebellar cortex of Sprague-Dawley(SD) rats and cultured. At the day 8 of culture, peritoneal macrophage of mice were added to the cerebral cortical neurons cultures and continued to be cultured for additional 2 days. Thereafter, Aβ25- 35(10 μmol/L) 10 μl were added into the co- culture of cerebral cortical neurons and macrophages. The experiment was divided into blank isolated culture group, coculture group, isolated culture Aβ25-35group, co-culture Aβ25-35group and co-culture CHA-treated group for 6,12, 24, 48, 96h, respectively. The survival of neurons and macrophages was detected by an inverted phase contrast microscope. Expressions of p38MAPK, MAPKAPK-2, HSP27 protein were tested by Western blot.Results Application of chlorogenic acid significantly improved the morphology and survival of cerebral cortical neurons damaged by Aβ25- 35- induced macrophage activation. In macrophages, the Aβ25- 35- induced p38MAPK pathway activation was inhibited by CHA at different time points. Accordingly, both the decreased expressions of phosphorylated MAPKAPK-2 and phosphorylated HSP-27 in macrophages caused by Aβ25- 35exposure were suppressed by CHA. Conclusion Chlorogenic acid is effective in reducing Aβ25- 35-induced damage to cerebral cortical neurons co- cultured with macrophages. Its protective effect may be in part achieved by action against the upregulation of p38MAPK expression in macrophages.更多还原