黄曲霉毒素M_1与赭曲霉毒素A联合作用诱导分化Caco-2细胞凋亡的机制

Combined Effects of Aflatoxin M_1 and Ochratoxin A on the Apoptosis and Mechanism in Differentiated Caco-2 Cells

目的:研究了黄曲霉毒素M_1(Aflatoxin M_1,AFM_1)和赭曲霉毒素(ochratoxin A,OTA)交互作用诱导分化人结肠癌Caco-2细胞凋亡及其作用机制。方法:AFM_1和OTA单独及联合作用于分化的Caco-2细胞后,通过阿尔玛蓝(Alamar-Blue)法测量细胞存活率,研究AFM_1和OTA对细胞增殖的抑制作用;通过流式细胞术检测细胞凋亡率,活性氧荧光探针检测细胞活性氧(ROS)释放量,JC-1线粒体膜电位荧光探针检测细胞线粒体膜电位变化。蛋白免疫印迹法检测凋亡蛋白Caspase-3和Caspase-9的表达水平。结果:AFM_1和OTA可抑制分化的Caco-2细胞增殖并诱导其凋亡,单独使用12μmol/L AFM_1和6μmol/L OTA作用于细胞存活率分别为95.59%和93.14%,联合作用下细胞存活率为81.57%,提示联合作用具有协同作用;AFM_1和OTA单独处理细胞24 h后,细胞的凋亡率分别为25.68%和33.7%,联合作用细胞的凋亡率为37.4%;AFM_1和OTA单独作用下细胞ROS的释放量分别是空白对照组的1.37倍和1.79倍,交互作用是空白对照组的6.21倍,交互作用对活性氧的释放量明显增加。结论:线粒体膜电位和线粒体相关凋亡蛋白的表达水平变化,说明AFM_1和OTA可能通过线粒体途径诱导分化的Caco-2细胞凋亡。

Purpose: To study the combined mechanism of AFM1(aflatoxin M1, AFM1) and OTA(ochratoxin A,OTA) on the apoptosis and mechanism. Method: differentiated Caco-2(human colorectal adenocarcinoma) cells were cultured and treated in the following groups: AFM1(12 μmol/L), OTA(6 μmol/L), AFM1(12 μmol/L)+OTA(6 μmol/L). Cell viability and proliferation were detected by Alamar-Blue method. Cell apoptotic rate was quantified by AnnexinV-FITC/PI staining through flow cytometry, intracellular ROS(reactive oxygen species, ROS) release was detected by active oxygen fluorescent probe, and the mitochondrial membrane potential was detected by a JC-1 probe. Additionally, the protein of Caspase-3 and Caspase-9 were detected by Western blotting. Results: The results showed that AFM1 and OTA have a synergistic effect in inhibiting the proliferation of differentiated Caco-2 cells, and inducing cell apoptosis. Cell viability of the three group data was 95.59%, 93.14% and 81.57%, respectively. Apoptotic rate of the three group was 25.68%,33.7% and 37.4%, respectively. What’s more, the ROS release in AFM1 group was 1.37-fold of control group level, in OTA group was 1.79-fold of control group level, and the one in combined group was 6.21-fold of control group level.Conclusion: Expression levels of mitochondrial membrane potential and apoptotic proteins suggested apoptosis of differentiated Caco-2 cells was induced through mitochondrial pathways, moreover, this induction seemed to be more obvious in the combination group.