绿原酸对Aβ_(25-35)诱导小脑颗粒细胞损伤的保护作用机制

Influence of Chlorogenic Acid on Aβ_(25-35)-induced Damage to Granular Cell in Cerebellum

目的:探讨绿原酸(chlorogenic acid,CHA)对β淀粉样蛋白25-35(amyloidβ-protein 25-35,Aβ25-35)激活小鼠巨噬细胞引起的小脑颗粒细胞(cerebellar granular cell,CGC)损伤的保护作用机制。方法:实验分为空白对照组、Aβ25-35处理组和Aβ25-35+CHA共同处理不同时间组;空白对照组:加入等量培养基;Aβ25-35损伤组:加入10μmol·L-1 Aβ25-35;Aβ25-35和CHA处理不同时间组:将10μmol·L-1 Aβ25-35和1 000μmol·L-1 CHA同时加入共同培养2 d的小脑颗粒细胞和巨噬细胞,分别作用6,12,24,48,96 h。应用倒置相差显微镜观察小脑颗粒细胞的形态变化并计数;应用Western blot检测p38分裂原激活蛋白激酶(p38 mitogen-activated protein kinase,p38MAPK)、下游底物有丝分裂原激活蛋白激酶活化的蛋白激酶2(mitogen-activated protein kinase activating protein kinase-2,MAPKAPK-2)、热休克转录因子(heat-shock protein27,HSP27)三种蛋白的磷酸化表达的改变。结果:CHA保护组的小脑颗粒细胞数量较Aβ25-35损伤组明显增加;CHA作用96 h可明显抑制Aβ25-35引起的巨噬细胞磷酸化的p38MAPK表达增加,逆转Aβ25-35引起的联合培养的小脑颗粒细胞和巨噬细胞磷酸化的MAPKAPK-2和磷酸化的HSP27表达降低的变化。结论:绿原酸是通过抑制Aβ25-35激活巨噬细胞p38MAPK信号通路的蛋白表达起到保护小脑颗粒细胞的作用。

Objective：To investigate the effects of chlorogenic acid on granular cell in cerebellum induced by amyloidβ-protein-induced macrophage activation in vitro. Methods：The experiment was divided into blank control group,Aβ25-35 treatment group and Aβ25-35＋CHA co-processing of different time group. Blank control group：Equal amount of culture medium;Aβ25-35 treatment group：with Aβ25-3510μmol＆#183;L-1;Aβ25-35 and different time of CHA treatment group：The co-cultured system of cerebellar granular cells and macrophages were cultivated for 2 days and then the 10μmol＆#183;L-1 Aβ25-35 and 1 000μmol＆#183;L-1 CHA were added into this system for 6,12,24,48,96 h respectively. The growth phynotype of neurons and macrophages were detected by inverted phase contrast microscope;The expressions of p38MAPK,MAPKAPK-2,HSP27 protein were tested by Western blot. Results：The numbers cerebellar granule cell of chlorogenic acid protection group was increased significantly compared with Aβ25-35 injury group. CHA could inhibite increased expression of phosphorylated p38MAPK of macrophage induced by Aβ25-35 at difference time;Decreased expression of phosphorylated MAPKAPK-2 and phosphorylated HSP-27of macrophage induced by Aβ25-35 could increased after the 1 000μmol＆#183;L-1 CHA added into the medium. Conclusion：The protection of Chlorogenic acid for cerebellar granular cells induced by Aβ25-35 via inhibition the activation of p38MAPK signaling pathway,which decreased the expression of p38MAPK and increased the expression of MAPKAPK-2,HSP-27.