冬凌草甲素对急性单核细胞白血病细胞株THP-1的作用研究

Anti-leukemia Effect of Oridonin on Acute Monoblastic Leukemia

目的探讨冬凌草甲素对急性单核细胞白血病细胞株THP-1的抗白血病效应及其作用机制。方法以急性单核细胞白血病细胞株THP-1为研究对象,应用改良四甲基偶氮唑盐法测定4、6、8μmol/L浓度冬凌草甲素处理72 h后THP-1细胞的增殖抑制率,以及计算2、4、6、8、10μmol/L冬凌草甲素处理24、72、120 h后的细胞生存率并绘制生长曲线;显微镜下观察4、6、8μmol/L冬凌草甲素处理24 h后THP-1细胞的形态学变化;采用流式细胞术检测0、4、6、8μmol/L冬凌草甲素处理24 h后THP-1细胞凋亡率;采用Western blot法检测6μmol/L冬凌草甲素处理THP-1细胞0、12、24 h后Akt/m TOR、Raf/MEK/ERK细胞信号转导通路的变化,以及凋亡调节蛋白Bcl-2和Bax的变化。结果 14、6、8μmol/L冬凌草甲素处理72 h后细胞增殖抑制率分别为(20.02±11.15)%、(45.99±12.76)%、(86.39±9.18)%,与4μmol/L冬凌草甲素比较,6、8μmol/L冬凌草甲素处理后细胞增殖抑制率均升高,差异均有统计学意义(P<0.05或P<0.01);冬凌草甲素处理72 h后细胞半抑制浓度为(6.12±1.48)μmol/L;细胞生长曲线显示,冬凌草甲素对THP-1细胞的生长抑制作用呈时间和浓度依赖性。2冬凌草甲素处理24 h后,THP-1细胞出现凋亡,形成凋亡小体,浓度越高细胞凋亡小体形成越多。30、4、6、8μmol/L冬凌草甲素(分别设为对照组及4、6、8μmol/L组)处理24 h后,THP-1细胞凋亡率分别为(3.7±1.1)%、(16.2±3.3)%、(30.1±4.3)%、(49.5±6.7)%,对照组与各浓度组凋亡率比较差异均有统计学意义(P<0.05或P<0.01);与4μmol/L组比较,6、8μmol/L组凋亡率均升高,差异均有统计学意义(P<0.05)。4经Western blot法检测6μmol/L冬凌草甲素处理THP-1细胞24 h后可抑制细胞内Akt/m TOR、Raf/MEK/ERK信号转导通路的活化,并下调Bcl-2、上调Bax的表达。结论冬凌草甲素通过抑制Akt/m TOR、Raf/MEK/ERK信号转导通路的活化,以及调节凋亡调节蛋白Bax和Bcl-2的表达而发挥抗白血病效应。

Objective To investigate the anti-leukemia effect of oridonin on acute monoblastic leukemia cell line THP-1. Methods Acute monoblastic leukemia cell line THP-1 was cultured in vitro. Cell proliferation Inhibition rate after72 h of treatment of 4,6,8 μmol / L oridonin was examined using modified MTT assay,cell survival rate after 24,72,120 h of treatment of 2,4,6,8,10 μmol / L oridonin was calculated and the growth curve was drawn. The cellular morphologic changes after 24 h of treatment of 4,6,8 μmol / L oridonin were observed under a light microscope. The percentage of apoptosis of THP-1 cells after 24 h of treatment of 0,4,6,8 μmol / L oridonin was evaluated using flow cytometric analysis. The activation levels of Akt / m TOR,Raf / MEK / ERK signaling pathways and the expression levels of Bcl-2 and Bax after 0,12,24 h of treatment of 6 μmol / L oridonin were examined by Western blot. Results 1 Cell proliferation inhibition rate after 72 h of treatment of 4,6,8 μmol / L oridonin was（ 20. 02 ± 11. 15） %,（ 45. 99 ± 12. 76） %,（ 86. 39 ± 9. 18） % respectively,compared with 4 μmol / L oridonin treatment group,cell proliferation inhibition rate after 6,8 μmol / L oridonin treatment were higher,with statistically significant differences（ P〈0. 05 or P〈0. 01）. Cell growth curve showed that oridonin inhibited the growth of THP-1 cells in time- and dose- dependent manner and the IC50 of oridonin was（ 6. 12 ± 1. 48） μmol / L after 72 h of treatment. 2 After 24 h of treatment of oridonin,THP-1 cells apoptosis occurred and apoptotic bodies were formed,the higher the concentration of oridonin,the more apoptotic bodies were. 3 The percentage of apoptosis rate after 24 h of treatment of0,4,6,8 μmol / L oridonin were（ 3. 7 ± 1. 1） %,（ 16. 2 ± 3. 3） %,（ 30. 1 ± 4. 3） % and（ 49. 5 ± 6. 7） % respectively.The differences of apoptosis rate between various oridonin concentration groups and control group were statistically significant（ P〈0. 05 or P〈0. 01）. The differences between various oridonin concentration groups were also statistically significant（ P〈0. 05）. 4 6 μmol / L oridonin treatment inhibited the activation of Akt / m TOR and Raf / MEK / ERK signaling pathways in THP-1 cells,and down-regulated the level of anti-apoptotic protein Bcl-2 and up-regulated the expressions of pro-apoptotic protein Bax. Conclusion Oridonin exerts anti-leukemia effect in THP-1 cells by inhibiting the activation of Akt / m TOR and Raf / MEK / ERK signaling pathways,regulating the expression of Bcl-2 and Bax apoptosis-modulating proteins.