复方二精灵多糖对谷氨酸诱导的海马神经元凋亡的保护作用

Neuroprotective effect of polysaccharides from Erjingling prescription on hippocampal neuron apoptosis induced by glutamate

目的探讨中药复方二精灵多糖(EJL)预防阿尔茨海默病的机制。方法实验分为正常对照组、模型(100μmol.L-1谷氨酸诱导)组、阳性对照组(50μmol.L-1多奈哌齐)及EJL实验组(0.5,1.0,2.0及3.0g.L-1)。体外培养9d的海马神经元先用各药物预处理一定时间后,加入谷氨酸。采用MTT法、Hoechst 33258荧光染色、流式细胞术和Western蛋白质印迹法等方法检测细胞凋亡。结果MTT检测发现,随着EJL浓度的增加,细胞的存活率也增加;与模型组〔(59.6±4.6)%〕比较,EJL2.0及3.0g.L-1组细胞存活率明显升高〔(82.8±2.8)%和(89.4±4.1)%;n=3,P<0.05〕。荧光染色观察形态学变化发现,EJL预处理组凋亡细胞明显减少。FITC-Annexin Ⅴ和PI双染,在直方图中可以发现,模型组的凋亡率为32.33%,EJL预处理组则分别为24.33%,22.01%及12.12%。Western蛋白质印迹法结果显示,与模型组比,EJL2.0g.L-1预处理可以显著提高Bcl-2蛋白表达量,减少Bax蛋白表达量。结论EJL对海马神经元有一定的保护作用。

AIM To explore the mechanism of polysaccharides from Erjingling（EJL） , a traditional Chinese medicine prescription preventing Alzheimer disease. METHODS Cultured hippocampal cells were randomly divided into seven groups ： normal group, glutamate （ 100 μmol·L^-1） group （ model ）, donepezil （ 50 μmol·L^-1） group and EJL （0.5 , 1.0 , 2.0 and 3.0 g· L^-1 ） groups. After culturing 9 d in vitro, hippocampal cells were pretreated with different concentration of EJL or donepezil respectively for a period of time, then exposed to glutamate. The apoptosis effects were detected by using MTT, Hoechst 33258, flow cytometry and Western blot assays. RESULTS The survival rate of hippocampal cells was raised with the increasing in EJL＇s concentration. Compared with model group [（ 59. 6 ± 4.6 ） % ） , 2.0 and 3.0 g. L^-1 EJL had significant effect on the cell survival rate [ （ 82.8± 2.8）% and （89.4±4. 1）%, n=3, P〈0.05） 1. The Hoechest 33258 staining was re- vealed that EJL-pretreatment could obviously reduce the apoptosis of neurons. By FITC- Annexin V/PI staining, the data demonstrated that apoptosis rate of glutamate group was 32.33%, but EJL-pretreated was 24. 33%, 22.01% and 12.12% , respectively. Moreo- ver, 2.0 g.L^-1 EJL could remarkably increase the expression of Bcl-2 protein and decrease Bax protein. CONCLUSION EJL could pro- tect the apoptosis of hippocampal neurons induced by glutamate.