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目的:确定活性氧(ROS)生成在Ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic-acid(5F)所诱导的HepG2细胞凋亡中的作用。方法:采用MTT分析检测5F对HepG2细胞增殖的影响,再以Hoechst/PI法分析凋亡细胞的形态变化。为了评价细胞内ROS水平,采用了GENMED试剂盒。HepG2细胞经5F处理24 h,或1 mmol.L-1GSH预处理1 h再经5F处理24 h,然后以Cell Death Detection ELISA试剂盒检测胞浆核小体片段。结果:通过细胞活性分析证实,5F对HepG2的细胞毒作用随着5F浓度升高而增强。而凋亡变化,如染色质浓缩,则被Hoechst/PI染色所证实。经5F处理的HepG2细胞内ROS生成减少被观察到。5F诱导的胞浆核小体片段并不由于GSH降低ROS介导的信号转导水平而发生变化。并且,顺铂(CDDP)诱导的ROS生成可被5F清除,同时5F还显示了与CDDP协同杀伤细胞作用。结论:5F不仅能通过非ROS依赖途径诱导细胞凋亡,还可缓解氧化应激。
AIM: To determine the role of reactive oxygen species (ROS) production in HepG2 cell apoptosis induced by Ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic-acid (5F). Methods: The effect of 5F on the proliferation of HepG2 cells was detected by MTT assay. The morphological changes of apoptotic cells were analyzed by Hoechst / PI. To evaluate intracellular ROS levels, a GENMED kit was used. HepG2 cells were treated with 5F for 24 h or pretreated with 1 mmol.L-1 GSH for 1 h and then treated with 5F for 24 h. Then the cytoplasmic nucleosome fragments were detected by Cell Death Detection ELISA kit. Results: The cytotoxicity of 5F to HepG2 was confirmed by the cell viability assay as the concentration of 5F increased. Changes in apoptosis, such as chromatin condensation, were confirmed by Hoechst / PI staining. Decreased ROS production in HepG2 cells treated with 5F was observed. 5F-induced cytoplasmic nucleosome fragments did not change due to GSH lowering ROS-mediated signal transduction levels. Moreover, cisplatin (CDDP) -induced ROS production can be cleared by 5F, while 5F also shows synergistic cytotoxic effects with CDDP. Conclusion: 5F can not only induce apoptosis through non-ROS-dependent pathway, but also relieve oxidative stress.