紫草素通过氧化胁迫诱导K562细胞凋亡

Shikonin Induce K562 Cell Apoptosis through Oxidative Stress

目的:研究紫草素(shikonin)诱导人红白血病细胞(K562)凋亡作用并初步探讨其机制。方法:噻唑蓝(thiazolylblue,MTT)染色法检测紫草素对K562细胞的增殖抑制作用;Hoechst 33258和吖啶橙(acridine orange,AO)/溴化乙啶(ethidiumbromide,EB)染色法观察细胞凋亡形态;反转录酶-聚合酶链锁反应(reverse transcription-polymerase chain reaction,RT-PCR)法检测与凋亡相关基因:B细胞淋巴因子基因2相关X蛋白(B-cell lymphoma gene 2 associated X protein,Bax)、B细胞淋巴因子基因2同源3结构域相互作用基团死亡促进因子(BH3 interacting domain death agonist,Bid)、B细胞淋巴因子基因xL(B-celllymphoma gene xL,Bcl-xL)、p53上调凋亡调控因子(p53 up-regulated modulator of apoptosis,PUMA)、B细胞淋巴因子基因2同源拮抗因子(B-cell lymphoma gene 2 homologous antagonist,Bak)、B细胞淋巴因子-w(B-cell lymphoma-w,Bcl-w)、B细胞淋巴因子基因2相关死亡促进因子(B-cell lymphoma gene 2 associated death promoter,Bad)信使核糖核酸(Messenger RNA,mRNA)表达的变化;荧光染料二乙酸-2',7'-二氯荧光素盐(oxalic acid-2',7'-dichlorofluorescein,DCFHDA)分析细胞内总活性氧(ReactiveOxygen Species,ROS)的变化;荧光染料5-氯甲基二已酸荧光素(5-chloromethylfluorescein diacetate,CMFDA)分析细胞内还原型谷胱甘肽(reduced glutathione hormone,GSH)含量的变化。结果:①0.2～1.6 mg.L-1的紫草素对K562细胞增殖呈浓度依赖性抑制;②0.2～0.5 mg.L-1的紫草素处理组细胞出现明显凋亡特征;③0.2～0.5 mg.L-1的紫草素处理组促凋亡蛋白Bid,Bad,Bak,PUMA,Bax的mRNA表达显著增加,抑制凋亡蛋白Bcl-w和Bcl-xL的mRNA表达明显下调;④0.2～0.5 mg.L-1的紫草素处理组,细胞内总活性氧含量升高,还原型谷胱甘肽含量降低。结论:紫草素有较强的抑制K562细胞增殖并诱导其凋亡的作用,该过程伴随细胞内氧化还原状态的改变,紫草素诱导的K562细胞的凋亡与细胞内氧化胁迫有关。

Objective： Tostudy the mechanism of shikonin inducing K562 cells apoptosis.Method： MTT colorimetric assay was used to examine the proliferation inhibition of shikonin on K562 cells.Hoechst 33258 and acridine orange（AO） / ethidium bromide（EB） fluorescent staining were used to observe the morphological changes of apoptotic cell.The mRNA expression levels of B-cell lymphoma gene 2 associated X protein（Bax）,BH3 interacting domain death agonist（Bid）,B-cell lymphoma gene xL（Bcl-xL）,p53 up-regulated modulator of apoptosis（PUMA）,B-cell lymphoma gene 2 homologous antagonist（Bak）,B-cell lymphoma-w（Bcl-w） and Bcell lymphoma gene 2 associated death promoter（Bad） were detected using RT-PCR analysis.The changes of reactive oxygen species（ROS） and reduced GSH were detected using molecular probe DCFHDA and CMFDA： CMFDA,separately.Result： Shikonin（0.2-1.6 mg.L-1） inhibited K562 cell proliferation in concentrationdependent manner,and induced cell apoptosis.Typical apoptotic changes were observed in K562 cells under the shikonin（0.2-0.5 mg.L-1） treatment groups.The expression of proapoptotic protein（Bid,Bad,Bak,PUMA,Bax） was increased and antiapoptotic protein（Bcl-w,Bcl-xL） decreased.ROS formation increased,but reduced glutathione level was decreased.Conclusion： Shikonin can inhibite K562 cell proliferation and induce apoptosis through inducing oxidative stress.