白花蛇舌草注射液抑制白血病细胞增殖及对线粒体跨膜电位的影响

The Inhibition Effect and the Level of Mitochondrial Trans-membrane Potential of Hedyotic Diffusa Willd Injection on Leukemia Cells

目的：通过对白花蛇舌草注射液（HDI）在体外抑制人白血病细胞株（HL-60、K562）增殖作用的观察,来探讨HDI用于治疗白血病患者的作用机制。方法：以白血病HL-60、K562细胞株为靶细胞,采用MTT法观察不同浓度的HDI对HL-60、K562细胞增殖的影响;流式细胞术检测两细胞在药物处理前后细胞内线粒体跨膜电位（ΔΨm）的水平;用激光共聚焦显微镜观察药物处理前后细胞内线粒体的荧光探针5,5’,6,6’四氯1,1’,3,3’四乙基苯并咪唑基羰化青碘（JC-1）在细胞内的荧光颜色变化。结果：低浓度的HDI（1.56mL/L）对HL-60、K562细胞增殖均无明显抑制作用（P〉0.05）,但当HDI浓度提高至3.12-25mL/L时,对HL-60细胞则出现明显的增殖抑制作用,且随浓度增加抑制作用增强（P〈0.05或P〈0.01）;而明显抑制K562细胞增殖,则浓度提高至6.25mL/L,且随浓度增加抑制增强（P〈0.05或P〈0.01）。通过流式细胞术对JC-1检测发现HDI能降低HL-60、K562细胞内线粒体跨膜电位（ΔΨm）水平,且随浓度增加降低越明显;激光共聚焦显微镜观察发现经药物处理过的凋亡早期细胞内JC-1不能聚集在线粒体的基质中，为单体，细胞则发绿色荧光，而未经药物处理过的活细胞内JC-1聚集在线粒体的基质中形成聚合物（J—aggregates），细胞则发桔黄色荧光，两细胞产生的荧光颜色变化也提示药物处理前后细胞内线粒体膜电位的变化。结论：白花蛇舌苹注射液在一定浓度下，能够直接抑制HL-60、K562白血病细胞增殖，其作用机制有可能通过降低线粒体跨膜电位，触发细胞凋亡过程，从而抑制细胞增殖。

Objective： To explore the inhibition effect and mechanism of Hedyotic diffusa Willd Injection（HDI） on leukemia cell line in vitro.Methods： The leukemia cell line of HL-60 and K562 were used as target cells.The inhibition effects of different concentration HDI were observed by MTT assays.The level of mitochondrial transmembrane potential using the J-aggregate forming lipophilic cation 5,5 ,6,6 -Tetrachloro-1,1 ,3,3 -tetraethyl-benzimidazolcarbocyanine Iodide（JC-1） was measured by flow cytometry.The change of fluorescence colours in leukemia cell was observed by confocal microscopy. Results ： The results showed that the growth of HL - 60 cells was inhibited by higher concentration of HDI （ 3.12 - 25mL/L） and inhibited obviously by HDI in the manner of dose dependence （ P 〈 0. 05 or P 〈 0. 01 ）, but not suppressed by low concentration of HDI （1.56mL/L） in liquid culture system （ P 〉 0. 05 ）. However the growth of K562 cells was inhibited by higher concentration of HDI （6.25 - 25mL/L） and inhibited obviously by HDI in the manner of dose dependence （ P 〈 0.05 or P 〈 0. 01 ）. The FCM results showed that the level of mitochondrial transmembrane potential on leukemia cell （ HL - 60, K562） was reduced and leveled down obviously by HDI in the manner of dose dependence. We also observed that JC - 1 was able of forming J - aggregates that are associated with a large shift in emission （590 nm） depending on the transmembrane potential. The colors of the dye changed reversibly from greenish orange to green as the mitochondrial transmembrane become more depolarized when the leukemia cells were treated with HDI. Conclusion ： It is concluded that HDI could inhibit HL - 60 and K562 cells in the presence of its higher concentrations. The mechanisms of HDI may reduce the mitochondrial transmembrane potential in leukemia cells, and trigger the cell to apoptosis, inhibit the growth of HL - 60 and K562 cells.