益气养阴方及其拆方对急性髓系白血病细胞细胞凋亡及Cyt-C,Apaf-1,Smac/Diablo,AIF表达的影响

Effect of Yiqi Yangyin Recipe and Its Separated Recipes on Apoptosis Rate and Expression of Cyt-C,Apaf-1,Smac / Diablo,AIF in Acute Myeloid Leukemic Mice

目的:研究益气养阴方及其拆方后的扶正方、祛邪方对人急性髓系白血病细胞凋亡及细胞色素C(Cytochrome C,Cyt-C),凋亡蛋白酶活化因子(apoptotic protease activating factor 1,Apaf-1),第二个线粒体衍生的半胱天冬蛋白酶激活因子(the second mitochondria-derived activator of caspase/direct IAP-blinding protein with low PI,Smac/Diablo),凋亡诱导因子(apoptosis-inducing factors,AIF)表达的影响,探讨益气养阴方治疗白血病的可能作用机制。方法:采用NOD/SCID小鼠,以KG-1a细胞株建立人急性髓系白血病模型,随机分为全方组、祛邪组、扶正组和空白组,取NOD/SCID小鼠脾细胞制成细胞悬液,应用流式细胞术检测NOD/SCID小鼠细胞凋亡率;采用免疫组化法检测NOD/SCID小鼠骨髓中线粒体相关凋亡因子CytC,Apaf-1,Smac/Diablo和AIF的表达。结果:用药后,用药组小鼠生存时间较空白组显著延长(P<0.01),全方组小鼠生存时间较扶正组与祛邪组显著延长(P<0.01),祛邪组小鼠生存时间较扶正组显著延长(P<0.01)。用药组细胞凋亡率较空白组明显升高(P<0.01),全方组较扶正组与祛邪组显著升高(P<0.01),祛邪组较扶正组显著升高(P<0.01)。用药组小鼠骨髓中Cyt-C,Apaf-1,Smac/Diablo,AIF的表达较空白组显著升高(P<0.05),其中Cyt-C与Apaf-1表达显著升高(P<0.01);全方组均高于扶正组与祛邪组(P<0.01);祛邪组较扶正组均升高(P<0.05),Cyt-C,Apaf-1和Smac/Diablo表达显著升高(P<0.01)。其中对于各指标的影响全方组均优于扶正组与祛邪组,祛邪组均优于扶正组。结论:益气养阴方能够提高NOD/SCID小鼠的细胞凋亡率,上调线粒体相关凋亡因子Cyt-C,Apaf-1,Smac/Diablo,AIF的表达,并诱导其凋亡,抑制白血病细胞增殖,其机制可能与线粒体凋亡通路有关。

Objective： To study the action mechanism of Yiqi Yangyin recipe with supplementing Qi and nourishing Yin） in treating leukemia by observing the effects of Yiqi Yangyin recipe and its separated recipes on expressions of Cytochrome C （Cyt-C）, apoptotic protease activating factor 1 （Apaf-1）, the second mitochondria-derived activator of caspase/direct IAP-blinding protein with low PI （Smac/Diablo）, apoptosis- inducing factors （AIF） in acute myeloid leukemic cells. Method： NOD/SCID mice were taken as the research platform and injected with KG-la cells through tail vein to establish the model of human acute myeloid leukemia. The model mice were randomly divided into Quanfang group, Fuzheng group, Quxie group and control group. The spleen cell suspensions of NOD/SCID mice were prepared to measure the cell apoptosis rates by flow cytometry. The immunohistochemistry was used to detect the expression of the mitochondrial-related apoptosis factors in bone marrow of mice, including Cyt-C, Apaf-1, Smac/Diablo and AIF. Result： After treatment, the survival time of treatment groups were significantly longer than that of control group （P 〈 0. 01 ）, the survival time of Quanfang group mice were longer than that of Fuzheng group and Quxie group （P 〈 0.01 ）, the survival time of Quxie group mice were longer than that of Fuzheng group mice （P 〈 0.01 ）. The cell apoptosis rate in all treatment groups was significantly higher than that of control group （P 〈 0.01 ） , the cell apoptosis rate of Quanfang group were higher than that of Fuzheng group and Quxie group （P 〈 0.01 ）, and the cell apoptosis rate of Quxie group were higher than that of Fuzheng group （P 〈 0.01 ）. With respect to the expressions of Cyt-C, Apaf-1, Smac/Diablo and AIF in bone marrow of mice, the treatment groups were higher than control group （P 〈 0.05） , particularly Cyt-C and Apaf-1 （ P 〈 0.01 ）. All of the above expressions of Quanfang group were higher than those of Fuzheng group and Quxie group （P 〈 O. 01 ）. Quxie group was higher than Fuzheng group （P 〈 0.05） , particularly Cyt-C, Apaf-1 and Smac/Diablo （P 〈0.01 ）. In terms of the effect on each tested indicator, Quanfang group was better than Fuzheng group and Quxie group, and Quxie group was better than Fuzheng group. Conclusion： Yiqi Yangyin recipe can increase the cell apoptosis rate in NOD/SCID, improve the expression of the mitochondrial-related apoptosis factors Cyt-C, Apaf-1, Smac/Diablo and AIF, induce apoptosis of leukemia cells and inhibit the proliferation of leukemia cells, which may be associated with the mitochondrial apoptosis pathway.