冬凌草甲素诱导人多发性骨髓瘤ARH-77细胞凋亡及其可能机制

Oridonin induces apoptosis of human multiple myeloma ARH-77 cells and its possible mechanism

目的:探讨冬凌草甲素(oridonin,Ori)对人多发性骨髓瘤ARH-77细胞的致凋亡作用及其可能的机制。方法:不同浓度Ori(2.5、5、10μmol/L)作用于ARH-77细胞,MTT法检测ARH-77细胞的增殖,相差显微镜和Hoechst33258染色观察细胞凋亡的形态学改变,流式细胞术检测ARH-77细胞的早期凋亡及线粒体膜电位(Δψm)的变化,caspase8凋亡试剂盒检测ARH-77细胞caspase8的活化。结果:Ori对ARH-77细胞的生长有明显的抑制作用,且呈时间和剂量依赖性。10μmol/LOri作用于ARH-77细胞24h后可见细胞变小、胞质中出现空泡,并可见凋亡小体。流式细胞术结果显示,经不同浓度Ori(2.5、5、10μmol/L)处理后,细胞早期凋亡率分别为(15.07±0.78)%、(21.00±1.49)%和(27.45±2.47)%,均高于对照组的(5.27±1.46)%(P<0.01)。不同浓度Ori(2.5、5、10μmol/L)处理后,反映Δψm的绿色荧光细胞百分率分别为(26.80±0.75)%、(36.81±2.27)%和(49.48±1.10)%,均高于对照组的(16.96±0.50)%(P<0.01),提示ARH-77细胞凋亡有显著增加。同时,Ori处理可上调ARH-77细胞caspase8的活性(P<0.01)。结论:冬凌草甲素能明显诱导多发性骨髓瘤ARH-77细胞的凋亡,其机制可能与激活内、外源凋亡通路有关。

Objective ：To investigate the apoptosis induction effect of oridonin （Ori） on human multiple myeloma cell line ARH-77 and the possible mechanism. Methods： ARH-77 cells were treated with different concentrations of oridonin （2.5, 5 and 10 μmol/L） , and then the proliferation of ARH-77 cells was detected by MTT assay. The morphological change of apoptotic ARH-77 cells was observed under phase contrast microscope and Hoechst 33258 staining. The apoptosis and the change of mitoehondrial membrane potential （Aψm） of ARH-77 cells were examined by flow cytometry. The caspase 8 activity was measured by caspase-8 apoptosis kit. Results： Ori significantly inhibited the growth of ARH-77 cells in a time- and dose-dependent manner. After treatment with 10μmol/L Ori for 24 h, ARH-77 cells became shrunk, with cytoplasm vacuoles and apoptotic bodies. Flow cytometry results revealed that the apoptotic rates of ARH-77 cells after Ori treatments （2.5,5 and 10 μmol/L） were significantly higher than that of control group, with apoptotic rates being （ 15.07 ±0.78） %, （21.00±1.49） % , （27.45 ± 2.47） % vs （5.27 ± 1.46） % （ P 〈 0. 01 ）. Green fluorescence percentages of Aψm of ARH-77 cells were significantly increased in Ori-treated groups （ 2.5, 5 and 10 μmol/L） compared with that in control group, with percentages being （26.80 ± 0. 75 ） %, （ 36.81± 2.27 ） %, （49.48± 1.10） % vs （ 16.96 ± 0.50） % （ P 〈 0.01 ）. Meanwhile, caspase 8 activity of ARH-77 cells was significantly up-regulated by Ori treatment （P 〈 0.01 ）. Conclusion： Oridonin can markedly induce apoptosis of human multiple myeloma ARH-77 cells, which might be related to extrinsic and intrinsic pathways.