VPA增强ORI诱导HL-60细胞凋亡及机制研究

VPA enhancement ORI induction HL-60 cells apoptosis and mechanism study

目的：探讨VPA与冬凌草甲素（oridonin，ORI）联合诱导HL-60细凋亡的作用及其机制。方法：CCK-8法选取ORI和VPA最佳协同作用剂量；VPA和ORI单独或联合作用于HL-60细胞后，Hoechst33342染色后荧光显微镜下观察细胞核形态学变化；Western blot检测凋亡相关基因caspase-3剪切片段的蛋白表达；预先加入或不加入caspase抑制剂处理HL-60细胞，采用Annexin V／PI流式试剂盒检测两药联合后细胞凋亡的变化。结果：Hoechst33342染色后荧光显微镜下，ORI＋VPA组比单独用药纽核碎裂数目明显增多；ORI和VPA单独作用组caspase-3活性片段表达较对照组高但均不如ORI＋VPA组明显；ORI＋VPA组细胞凋亡率（16．7±2．0）％。与对照组（1．2±1．5）％相比，具有显著差异（P〈0．05）；预先加入caspase抑制剂组细胞几乎没有凋亡，与对照组相比，不具有显著差异（P〉0．05）。结论：VPA确实能增强冬凌草甲素诱导HL-60细胞凋亡，且凋亡机制与caspase凋亡通路密切相关。

Objective ： Oridonin（ORI） obtained from the traditional Chinese herbal medicine rabdosia rubescens exerts potent antitumor activities in cancer ceils. Valproic acid（VPA）, as a potent histone deaeetylase inhibitor（HDACI）, possesses significant antileukemic activitites. But no research shows the anti-leukemic effect of oridonin eotreated with VPA. In this study, we demonstrated that oridonin in combination with VPA synergistically inhibited the proliferation of HL-60 cells. Methods ： In our study, we select optimal doses of ORI and VPA by CCK-8, and four groups were derided： control, ORI, VPA and ORI＋VPA group; HL-60 cells were treated with ORI and/or VPA for 24 h, then stained by Hoechst33342 and observed the morphological changes of nucleus; Also, we detected expression of cleaved caspase-3 by western blot; Moreover, we used caspase inhibitor 30min before treatment of the two drugs on HL-60 cells to further validate our test.Results ： Significant apoptosis was observed, we find shrinking cell body, nuclear pyrosis, nuclear fragmentation and the formation apoptotic body, and ORI＋VPA group was the most obvious; After pretreatment with caspase inhibitor, almost no eel1 apoptosis was found, compared with the cotreatment group（16.7 ± 2.0）%,（P 〈 0.05）, the cotreatment triggered apoptosis depending on caspase-3 activation. Conclusions ： Together, the novel combination of oridonin and VPA may be a promising treatment for APL.