目的 探讨吴茱萸碱通过抑制组蛋白去乙酰化酶6（HDAC6）促进人白血病K562细胞周期阻滞以及凋亡的机制。方法 采用CCK-8法检测吴茱萸碱对K562细胞增殖的影响；流式细胞术检测K562细胞周期和凋亡；化学比色法检测K562细胞HDAC6活性；Western blotting法检测K562细胞中HDAC6、Cyclin D1、CDK4、Bcl-2、Bax、Cleaved Caspase-3、ERK、p-ERK、p38和p-p38蛋白的表达。结果 CCK-8法结果提示，吴茱萸碱在一定浓度范围内（1～16 μmol/L）可以有效抑制K562细胞增殖，并呈时间和浓度依赖性；流式细胞术结果显示，吴茱萸碱可将K562的细胞周期阻滞于G₀/G₁期；2、4、8 μmol/L吴茱萸碱诱导K562细胞48 h后，其凋亡率分别为（11.47±1.05）%、（12.77±0.79）%和（18.58±1.37）%，与对照组（2.79±1.01）%相比差异显著（P<0.01）；化学比色法结果显示，吴茱萸碱能有效抑制HDAC6的活性；Western blotting结果显示，吴茱萸碱能上调Bax、Cleaved Caspase-3、p38和p-p38蛋白的表达，下调CDK4、Cyclin D1、Bcl-2、HDAC6、ERK和p-ERK蛋白的表达。结论 吴茱萸碱可能是通过抑制HDAC6的活性，激活MAPK信号通路，进而上调促凋亡蛋白的表达，下调周期蛋白的表达，从而抑制K562细胞的增殖，诱导细胞周期阻滞和凋亡。

Objective To explore the mechanism of evodiamine (Evo) inducing cell cycle arrest and apoptosis in K562 cells. Methods The effect of Evo on proliferation of K562 cells was measured by Cell Counting Kit-8 assay (CCK-8 assay), and cell cycle distribution and apoptosis were determined by flow cytometry (FCM). Chemical colorimetry assay was used to examine the activity of histone modification enzymes. The expression levels of histone deacetylase 6 (HDAC6), Cyclin D1, CDK4, Bcl-2, Bax, Cleaved Caspase-3, ERK, p-ERK, p38, and p-p38 proteins were ascertained by Western blotting. Results The proliferation of K562 cells was inhibited by Evo (1-16 μmol/L) in a dose- and time-dependent manner. FCM analyses revealed that Evo induced cell-cycle arrest in G₀/G₁ phase in K562 cells. The apoptosis rates of K562 cells were (11.47 ±1.05)%, (12.77 ±0.79)%, and (18.58 ±1.37)% respectively after induced by Evo with different concentration (2, 4, and 8 μmol/L), which showed statistically significant difference compared with the control group (2.79 ±1.01)% (P<0.01). The activity of HDACs was reduced after treated with Evo (2, 4, and 8 μmol/L). Western blotting assay showed that the expression of Bax, Cleaved Caspase-3, p38, and p-p38 proteins increased, while CDK4, Cyclin D1, Bcl-2, HDAC6, ERK, and p-ERK proteins down-reguation after induced by Evo. Conclusion Evo can induce cell cycle arrest and apoptosis in K562 cells through the inhibition of HDAC6.

国家自然科学基金资助项目（31271368）；重庆市渝中区科技计划项目（20140123）