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目的:研究3β,5α,6β-三羟基胆甾烷(Triol)诱导恶性胶质瘤细胞凋亡的作用及其机制。方法:以不同浓度的Triol作用于C6细胞和A172细胞不同时间。采用MTT法检测细胞存活率,Hoechst 33342染色和TUNEL法检测细胞凋亡,试剂盒检测caspase活性变化,蛋白免疫印记方法检测凋亡相关蛋白Bcl-2家族蛋白的变化。结果:Triol可呈剂量和时间依赖性降低C6细胞和A172细胞的存活率;Triol处理细胞48 h,C6细胞和A172细胞的IC50值分别为(17.8±0.6)μmol/L和(20.6±0.2)μmol/L。Hoechst 33342染色、TUNEL检测和凋亡执行酶caspase-3活性检测结果显示,给药组中2种细胞都出现明显凋亡核象、TUNEL阳性细胞数增多和caspase-3的激活。Triol作用于C6细胞12 h、24 h和48 h后,在凋亡外通路中激活的caspase-8和在凋亡内通路中激活的caspase-9活性均随时间升高,抗凋亡蛋白Bcl-2和Bcl-xL的表达量随时间降低,而促凋亡蛋白Bak的表达量随时间升高。结论:Triol通过激活内、外凋亡通路引起恶性胶质瘤细胞的凋亡,且Bcl-2家族蛋白在此过程中起重要的调控作用。
Objective: To investigate the effects of 3β, 5α, 6β-trihydroxytryptamine on the apoptosis of human glioblastoma cells and its mechanism. Methods: Different concentrations of Triol were applied to C6 cells and A172 cells at different times. Cell viability was detected by MTT assay. Apoptosis was detected by Hoechst 33342 staining and TUNEL assay. The caspase activity was detected by kit assay. The protein level of apoptosis related protein Bcl-2 was detected by Western blotting. Results: Triol could decrease the survival rate of C6 cells and A172 cells in a dose-and time-dependent manner. The IC50 values of Triol-treated cells for 48 h were (17.8 ± 0.6) μmol / L and (20.6 ± 0.2) μmol / L. Hoechst 33342 staining, TUNEL detection and apoptosis caspase-3 activity test showed that the apoptotic nuclei, the number of TUNEL-positive cells and the activation of caspase-3 of the two kinds of cells in the treated group were significantly increased. Triol treatment of C6 cells at 12 h, 24 h and 48 h after activation of caspase-8 in the apoptotic pathway and caspase-9 activity in the apoptotic pathway increased over time, the anti-apoptotic protein Bcl -2 and Bcl-xL expression decreased with time, while the expression of pro-apoptotic protein Bak increased with time. Conclusion: Triol can induce apoptosis of glioblastoma cells through activation of intra-and extra-apoptotic pathways, and Bcl-2 family protein plays an important regulatory role in this process.