雷公藤甲素对Aβ25-35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-β25-35 via apoptosis pathway

摘要:

探讨雷公藤甲素对β- 淀粉样蛋白25-35（Aβ25-35）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Aβ25-35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Aβ25-35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、Aβ25-35处理组、Aβ25-35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-2 和的表达水平。结果Aβ25-35在10 μmol/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。Aβ25-35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Aβ25-35同时处理细胞时，前者能够降低Aβ25-35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-3、及-2 基因的表达以抑制细胞凋亡，从而对Aβ25-35诱导PC12细胞损伤发挥保护作用。

Abstract:

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 PC12 cell injury model of Alzheimer's disease could be established by Aβ25-35 at a concentration of 10 μmol/L with a survival rate of(58 ±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 dosedependent 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.