吴茱萸碱对人胶质瘤U251细胞增殖及凋亡影响的研究

目的研究吴茱萸碱(evodiamine)对人胶质瘤U251细胞增殖和凋亡的影响及其可能的作用机制。方法体外培养人胶质瘤U251细胞,并将其分为空白对照组及25、50、100μg/mL吴茱萸碱4组。应用MTT法检测吴茱萸碱对U251细胞的增殖抑制作用;Hoechst33258荧光染色法检测吴茱萸碱诱导胶质瘤U251细胞凋亡;采用Annexin V-FITC/PI双染法检测各组早期凋亡率;Western blot法分析凋亡相关蛋白的变化。结果与空白对照组同期比较,25、50、100μg/mL吴茱萸碱组生长抑制率在24、48、72 h均增加,差异有统计学意义。Hoechst 33258荧光染色显示吴茱萸碱作用24 h后U251细胞出现典型的细胞凋亡特征,各处理组均可见凋亡小体。与空白对照组自发早期凋亡率3.12%比较,25、50、100μg/mL吴茱萸碱组早期凋亡率分别为8.65%、19.47%及28.97%,差异均有统计学意义。Western blot实验显示,与空白对照组同期比较,25、50、100μg/mL吴茱萸碱上调了FAS、FADD、Caspase-8及Caspase-3蛋白表达,Bcl-2蛋白表达明显下降,Bax蛋白表达明显上升,差异均有统计学意义。上述指标均呈时间和剂量依赖性。结论吴茱萸碱对U251细胞具有明显的抑制细胞增殖和促进细胞凋亡的作用,其机制可能与上调Fas途径和下调Bcl-2/Bax有关。

Effects of evodiamine on proliferation and apoptosis of human glioma cell

Objective To study the effect of evodiamine on human glioma cell U251 and explore the mechanism. Methods Human glioma cells U251 were in vitro cultured. They were divided into the control group and 25, 50 and 100 μg/mL evodiamine groups. The proliferation rate was detected at 24, 48, and 72 h using MTT assay. Hoechst 33258 staining was used to visualize nuclear changes and apoptotie body formation. The early apoptosis rate at 24 h was detected by flow cytometry. The expressions of apoptosis-related proteins were detected at 48 h respectively using western blot analysis. Results Compared with the control group, the growth inhibition rate were significantly increased in all evodiamine groups at 24, 48, and 72 h. Compared to the control cells, cells exposed to evodiamine presented typical apoptotic morphology and formation of apoptotic bodies. The early apoptosis rate was 8.65% , 19.47% , and 28.97% in 25, 50 and 100 μg/mL evodiamine groups, respectively, showing statistical difference when compared with the control group. Western blotting analysis showed that the expression of apoptosis related proteins FAS, FADD, Caspase-8 and Caspase-3 were significantly upregulated, Bcl-2 was downreguated and Bax was upregulated. All the above experiments were in time- and dose-dependent manners. Conclusions Evodiarnine significantly inhibited the proliferation and promoted the apoptosis of glioma cancer cell U251 in a dose- and time-dependent manner. The mechanism may be related to the upregulation of Fas signaling pathways and downregulation of Bcl-2/ Bax.