赤芍总苷诱导K562细胞凋亡及对线粒体膜电位和Ca^2＋的影响

Changes in intracellular mitochondrial membrane potential and Ca^(2+) levels in process of K562 cells apoptosis induced by the total glucosides of radix paeoniae rubra

目的:赤芍总苷能诱导鼠HepA和S180细胞凋亡,但赤芍总苷能否抑制K562细胞增殖及其诱导K562细胞凋亡的机制尚不明确。观察赤芍总苷诱导人红白血病细胞株K562细胞凋亡及线粒体膜电位与Ca2+水平的变化,以探讨凋亡机制。方法:实验于2007-03-02/2007-10-20在北京中医药大学细胞生化实验室,国家重点实验完成。①实验材料:赤芍总苷由西安奥晶科技发展有限公司提供,批号:060417,纯度>95%。人红白血病K562细胞株购自上海中科院细胞库。②实验方法:取对数生长期的K562细胞,培养24h后加入50mg/L赤芍总苷进行干预,光镜下观察细胞形态。MTT显色法检测25,50,75,100,150,200,400mg/L赤芍总苷对K562细胞增殖的抑制作用,以仅加入无血清培养基培养的为对照。③实验评估:采用AnnexinV/PI双标记法检测凋亡效应,罗丹明123(Rh123)染色流式检测线粒体膜电位和荧光染料Fluo-3AM流式检测K562细胞内游离Ca2+浓度。结果:①50mg/L赤芍总苷作用K562细胞24h后,出现核固缩、核碎裂和凋亡小体等形态学改变。②与对照组相比,不同质量浓度赤芍总苷能明显抑制K562细胞的增殖(P<0.01),并具有量效关系,呈时间和剂量依赖性。③细胞线粒体经Rh123染色呈现明亮绿,不同质量浓度赤芍总苷干预组荧光强度均较对照组明显减弱,膜电位水平较高。④赤芍总苷可引起细胞线粒体膜电位下降。不同质量浓度赤芍总苷组细胞内游离Ca2+浓度均升高,线粒体膜电位下降,与对照组比较差异显著(P<0.01)。结论:赤芍总苷诱导K562细胞凋亡的可能机制是通过减低细胞内线粒体膜电位及提高游离Ca2+水平,从而导致细胞凋亡。

AIM： Total glucosides of radix paeoniae rubra （TGC） can induce the apoptosis of mouse HepA and S180 cells. However, whether TGC can inhibit K562 cell proliferation, and its induction mechanism are unclear. This study investigated the effects of its mitochondrial membrane potential （MMP） and Ca^2＋ levels in K562 cells induced by TGC to predict the mechanism of apoptosis. METHODS： Experiments were performed at the Laboratory of Cell Biochemical Laboratory, Beijing University of Traditional Chinese Medicine from March 2^nd to October 20^th, 2007. （1）TGC was provided by Xi＇an Aojing Science and Technology Developing Co., Ltd, batch number 060417 with the purity 〉95%. K562 cells strain was purchased from Cell Bank of Shanghai Zhongkeyuan. （2） K562 cells in logarithmic growth phase were treated with 50 mg/L of TGC. Morphology change was observed under light microscope after 24 hours. Inhibitory effects of TGC （25, 50, 75, 100, 150, 200, 400 mg/L） on K562 cells was assessed by the MTT assay. Cells cultured in serum-free medium were served as control. （3）Annexin V-FITC/PI was used to detect the apoptotic ratio of K562 cells; mitochondrial membrane potential （MMP） by Rh123 staining; Flow cytometry assay was used to detect the Ca^2＋ levels. RESULTS： （1）24 hours after K562 cells had been treated by 50 mg/L TGC, there were some morphological changes in the cells such as karyopyknosis, nuclear fragmentation and apoptotic body emerged. （2）TGC had markedly inhibited the proliferation on k562 cell in a time-dependent and dosage：dependent （P 〈 0.01）. （3）Rh123 staining showed light green mitochondria. Fluorescence intensity in the TOC groups was weaker than in the control group, with high level of membrane potential. （4）TGC could reduce the mitochondria membrane potential. Different concentrations of TGC could markedly increase the levels of Ca^2＋ and decrease mitochondrial membrane potential compared with the control group （P 〈 0.01）. CONCLUSION： TGC might induce apoptosis of K562 cells by decreasing intracellular mitochondrial membrane potential and increasing Ca^2＋ level.