淫羊藿素对KG-1a细胞增殖和凋亡的影响及其相关作用机制

Effects of Epihopin on Proliferation and Apoptosis of KG-1a cells and Its Related Mechanism

目的:分析淫羊藿素抑制Wnt/β-catenin信号通路干预急性髓系白血病KG-1a细胞增殖的可能机制,为开发治疗急性髓系白血病药物提供实验依据。方法:50只c57BL/6小鼠编号后随机分为空白对照、模型对照和淫羊藿素高、中、低剂量共5组(n=10只),除空白对照组外均采用腹腔注射人急性髓系白血病细胞KG-1a的方法建模。淫羊藿素高、中、低剂量组分别按80、40和20 mg/kg剂量肌肉注射淫羊藿素治疗3周,模型对照组和空白对照组肌肉注射生理盐水。采用蛋白免疫印迹法(Western blot)检测S期激酶相关蛋白2(SKP2)、β-连环蛋白(β-catenin)、E-钙黏蛋白(E-cadherin)及多聚腺素二磷酸核糖多聚酶[poly(ADP-ribose)polymerase;PARP]蛋白表达;采用分光光度法检测细胞中半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)和天冬酶-3酶原(Procaspase-3)活性;流式细胞术分别检测经淫羊藿素处理后的细胞周期时相分布和细胞凋亡率情况。结果:淫羊藿素可增强Caspase-3活性,降低Procaspase-3的水平,还可上调E-cadherin蛋白的表达,下调SKP2和β-catenin蛋白的表达,呈剂量依赖性增加PARP蛋白表达,经淫羊藿素处理后细胞凋亡明显,KG-1a细胞被阻滞于G_0/G_1期,KG-1a细胞生长明显受到抑制。结论:淫羊藿素能够剂量依赖性地裂解PARP,以诱导人急性髓系白血病细胞株KG-1a细胞凋亡,其作用机制可能与抑制Wnt/β-catenin信号通路及其下游相关基因表达有关。

Objective： To explore the possible mechanism underlaying interference of epihopin on the proliferation of AML KG-1a cells by inhibiting the Wnt/β-catenin signaling pathway,so as to prvide the experimental basis for development of drug to treat the AML.Methods： A total of 50 c57BL/6 mice were randomy divided into 5 group：blank control,model control,high,medium and low dose of epihopin.Except the blank control group,the KG-1a cells were injected in abdominal cavity of 4 groups for the establishment of model.The mice in high,middle and low dose groups were injected intramuscularly with 80,40 and 20 mg/kg of epihopin respectively,while the mice in blank control and model control group were injected intramuscularly with saline.The Western blot was used to detect the expression of S phase kinase-related protein 2（ SKP-2）,β-catenin,E-cadherin and poly-（ ADP ribose） polymerase（ PARP）; the spectrophotometry was used to detect the activity of caspase 3 and procaspase-3,the flow cytometry was used to detect the cell cycle distribution and the apoptotic rate of KG-1a cells treated with epihopin.Results： The epihopin could enhance the activity of caspase 3,decrease the level of procaspase 3; also could up-regulate the expression of Eacadherin and down-regulate the expression of SKP-2 and β-catenin; and could increase the expression of PARP in dose-dependent manner.After KG-1a cels were treated with epihopin,the apoptosis rate of cells significantly increased,the KG-1a cells were arrested in G0/G1 phase,therefore the growth of KG-1a cells was significantly inhibited.Conclusion：The epihopin can dose-dependently split PARP to induce the apoptosis of KG-1a cells,its mechanism may relate with inhibition of Wnt/β-catenin signaling pathway and its down-stream-related gene expression.