雷公藤甲素对Aβ_(25-35)诱导PC12细胞损伤的保护作用及其机制研究

Triptolide inhibits cytotoxicity of PC12 cells induced by amyloid-β_(25-35) via apoptosis pathway

目的探讨雷公藤甲素对β-淀粉样蛋白25-35(Aβ_(25-35))诱导PC12细胞损伤的保护作用及其机制。方法通过噻唑蓝(MTT)法确立Aβ_(25-35)诱导PC12细胞损伤模型的工作浓度;同时利用MTT法检测1×10^(-11)、1×10-^(10)和1×10-^(9)mol/L雷公藤甲素对细胞活性的影响,以及雷公藤甲素和Aβ_(25-35)同时处理细胞后对细胞活性的影响。PC12细胞随机分为3组:对照组、Aβ_(25-35)处理组、Aβ_(25-35)和雷公藤甲素处理组。细胞处理24 h后,利用免疫荧光法检测细胞中活化Caspase-3的表达,同时采用逆转录聚合酶链反应检测凋亡相关基因Bcl-2和Bax的表达水平。结果 Aβ_(25-35)在10μmol/L工作浓度下可诱导PC12细胞复制阿尔茨海默病体外细胞损伤模型,其细胞存活率为(58±6)%;单纯雷公藤甲素在1×10^(-11)、1×10-^(10)和1×10-^(9)mol/L浓度下处理细胞对细胞活性无显著影响。Aβ_(25-35)处理细胞后,细胞活性降低,活化Caspase-3在细胞中表达升高,同时Bax mRNA表达升高,而Bcl-2 mRNA的表达下降。然而,雷公藤甲素和Aβ_(25-35)同时处理细胞时,前者能够降低Aβ_(25-35)对PC12细胞的毒性损伤,且细胞活性随雷公藤甲素浓度的增加而升高;同时活化Caspase-3在细胞中的表达,Bcl-2和Bax mRNA表达水平也受抑制。结论雷公藤甲素可能通过调节Caspase-3、Bax及Bcl-2基因的表达以抑制细胞凋亡,从而对Aβ_(25-35)诱导PC12细胞损伤发挥保护作用。

Objective To investigate the protective effect of triptolide against amyloid β-peptide 25-35 （Aβ25-35） induced injury of PC12 cells. Methods MTT assay was conducted to determine the optimal working concentration of Aβ25-35 for inducing PC12 cell injury model, and the potential protective effect of different concentrations of triptolide （1×10-11, 1×10-10 and 1×10-9 mol/L） on the PC12 cells. PC12 cells were randomly divided into 3 groups： control group, Aβ25-35 treatment group, and Aβ25-35 and triptolide co-treatment group. After 24-h treatment, active caspase-3 expression was observed by cyto-immunofluorescence. Meanwhile, Bcl-2 and Bax mRNA expressions were measured by RT-PCR. Results The in vitro PC12 cell injury model of Alzheimer＆#39;s disease could be established by Aβ25-35 at a concentration of 10 μmol/L with a survival rate of （58 ＆#177; 6）%. Triptolide （1 ×10-11, 1 ×10-10 and 1 ×10-9 mol/L） alone had no significant effect on cell viability. Following the Aβ25-35 exposure, cell viability was significantly decreased. Furthermore, active caspase-3 protein and Bax mRNA expressions obviously increased after Aβ25 -35 treatment, while Bcl-2 mRNA expression decreased remarkably （P 〈 0.05）. Triptolide, however, could significantly increase cell viability in a dose-dependent manner when it was added together with Aβ25 -35. Moreover, triptolide could also significantly suppress the expressions of active caspase-3 protein, Bcl-2 and Bax mRNA induced by Aβ25-35 （P〈0.05）.Conclusions Triptolide may inhibit cell apoptosis through modulation of expressions of active caspase-3, Bax and Bcl-2 to protect PC12 cells from Aβ25-35-induced cytotoxicity.