线粒体途径在大黄素促人混合培养淋巴细胞凋亡中的作用

摘要

目的：探讨大黄素促进人混合培养淋巴细胞凋亡的作用机制，为进一步研究大黄素的免疫抑制作用奠定实验基础。方法：建立混合淋巴细胞培养模型，给予不同浓度大黄素（1μmol/L、10μmol/L、100μmol/L）培养24、48、72h后检测各组淋巴细胞凋亡、细胞内活性氧及线粒体膜电位情况。Western blot法检测共培养48h后淋巴细胞中细胞色素C、caspase-3、caspase-9、Bcl-2/Bax蛋白表达变化。给予还原性谷胱甘肽（ GSH）清除细胞内活性氧后，检测其对线粒体膜电位及淋巴细胞凋亡的影响。结果：随着培养时间和药物浓度的增加，大黄素对淋巴细胞增殖抑制和促凋亡作用逐渐增强，呈现浓度和时间依赖性。高浓度大黄素提高细胞内活性氧、降低线粒体膜电位水平，激活线粒体凋亡通路。使用还原性谷胱甘肽清除细胞内活性氧后，能够降低线粒体膜电位和淋巴细胞凋亡率。结论：线粒体信号途径的激活在大黄素促淋巴细胞凋亡中发挥重要作用。%Objective:To investigate the effect of mitochondrial signaling pathway on Emodin-induced apoptosis in human mixed lymphocyte culture model. Methods:The model of human mixed lymphocyte culture was established and lymphocytes were isolated from the peripheral blood samples of different healthy adult volunteers. To investigate the immunosuppressive effect of Emodin. Emodin was administered with different concentrations at 1μmol/L,10μmol/L,100μmol/L for 24,48,72h. At the end of culture,the apoptotic rate of lymphocytes and the level of reactive oxygen species( ROS)and mitochondrial membrane potential(ΔΨm)were evaluated with flow cytometry. Protein expres-sions of cytosolic cytochrome C,caspase-3 and caspase-9 were analyzed by Western blot. In the high concentration Emodin group,lymphocytes were cultured with reduced glutathione(GSH)and Emodin for 48h,GSH can remove in-tracellular ROS. The apoptotic rate of lymphocytes and the levels of ROS andΔΨm were also evaluated by flow cytom-etry. Protein expressions of Bcl-2 and Bax were analyzed by Western blot. Results:With the enhanced concentration of Emodin ,the inhibition of proliferation and increase of apoptotic rate were observed . The results further showed generation of ROS,disruption ofΔΨm,decrease of the Bcl-2/Bax ratio and cytosolic cytochrome C. Caspase-3 and caspase-9 levels also increased after the Emodin treatment. The decline in the Bcl-2/Bax ratio and the increase of ROS,cytosolic cytochrome C and caspase-3 and caspase-9 levels were consistent with the increase of the lympho-cyte apoptotic ratio. In the GSH group,the level of ROS declined,and the apoptotic rate also reduced compared with the high concentration Emodin group(p﹤0. 05),however,the ΔΨm enhanced compared with control group( p ﹤0. 05). Conclusion:Our results strongly suggested that the mitochondrial signaling pathway was involved in Emodin-induced apoptosis in human mixed lymphocyte culture.