厚朴酚对HL-60细胞增殖和凋亡的作用及分子机制研究

Effect of Magnolol on Proliferation and Apoptosis of HL-60 Cells and Its Molecular Mechanism

目的:探讨厚朴酚对人急性髓系白血病HL-60细胞增殖和凋亡的影响及其作用机制。方法:采用MTT法检测不同浓度厚朴酚(5、10、20、40、80和160μg/ml)作用于HL-60细胞24 h和48 h后的细胞增殖情况;应用光学显微镜检观察细胞形态学的变化;DAPI染色后在荧光显微镜下观察细胞核形态学变化;用Annexin V/PI双染流式细胞术检测细胞凋亡的改变;用逆转录-聚合酶链式反应(RT-PCR)检测BCL-2和BAX的mRNA水平;用Western blot检测caspase家族蛋白表达变化。结果:厚朴酚能够明显抑制HL-60细胞的增殖,并随时间延长及剂量增加,细胞增殖抑制率也明显升高(P<0.05);HL-60细胞经厚朴酚处理后体积明显变小、且有凋亡小体产生,细胞核固缩、碎裂,呈现典型的凋亡形态学改变;40μg/ml厚朴酚处理HL-60细胞24 h后细胞早期凋亡率为(11.7±2.4)%,与对照组(1.4±1.1)%相比具有显著统计学差异(P<0.05);RT-PCR检测结果表明,厚朴酚可上调BAX、下调BCL-2的mRNA表达;Western blot检测结果表明,厚朴酚可提高caspase-3、caspase-8和caspase-9的剪切片段表达。结论:厚朴酚对人急性髓系白血病HL-60细胞增殖具有较明显的抑制作用,并可诱导HL-60细胞凋亡,其机制与caspase激活、促进BAX蛋白表达以及抑制BCL-2蛋白表达相关。

Objective:To investigate the effect of magnolol on proliferation and apoptosis of HL-60 cells and its mechanism.Methods:MTT assay was used to measure the proliferation of HL-60 cells after treatment with different concentration of magnolol(5,10,20,40,80 and 160 μg/ml).The morphological changes of HL-60 cells were examined by light microscopy,and DAPI staining was performed to observe the nuclear morphology of HL-60 cells.The early cell apoptosis was detected by flow cytometry with Annexin V-FITC/PI double-staining.RT-PCR was carried out to examine the mRNA expression of BAX and BCL-2.Western blot was performed to detect the protein expression of caspase family.Results:The magnolol inhibited HL-60 cell proliferation,and the inhibitory rate of cell proliferation increased significantly in a dose-and time-dependent manner(P <0.05).HL-60 cells became small,even apoptotic bodies appeared after treatment with magnolol.In addition,nuclear condensation or fragmentation could be observed,which is the typical morphological features of apoptosis.When HL-60 cells were treated with 40 μg/ml of magnolol for 24 h,the ratio of early apoptotic cells reached to(11.7 ±2.4)%,which was significant different from control(1.4±1.1)%(P<0.05).RT-PCR results showed that treatment of HL-60 cells with magnolol up-regulated the expression of BAX,whereas downregulated the expression of BCL-2.Western blot results showed that the cleavages of caspase-3,-8 and-9 were significantly enhanced by magnolol.Conclusion:The magnolol can significantly inhibit the proliferation of HL-60 cells and induce the apoptosis of HL-60 cells,which may occur through up-regulation of BAX,down-regulation of BCL-2 and the activation of caspases.