Objective: To observe the change levels of nuclear factor-kappa B (NF-κB) p65 protein in cytoplasm and nuclear, phosphorylation of inhibitor of kappa B (p-IκB) protein and cytochrome C (Cyt-c) , cleaved cysteinyl aspartate specific proteinase-3 (Cleaved caspase-3) , B-cell lymphoma/leukemia-2 (Bcl-2) in cytoplasm in the process of N, N-dimethylformamide (DMF) -induced apoptosis in H9c2 cardiomyocytes, and explore the tentative mechanism of apoptosis. Methods: H9c2 cardiomyocytes were exposed to 200 mmol/L DMF. Western blotting was used to detect the protein expression levels of p65 in cytoplasm and nuclear, p-IκB after exposure for 0, 2, 4, 6, 8, 12 h, and the protein expression levels of Cyt-c, Cleaved caspase-3, Bcl-2 in cytoplasm after exposure for 0, 2, 4, 8, 12, 24 h. Immunofluorescencecytochemistry (IFC) was used to observe the location of Cyt-c after 200 mmol/L DMF exposure for different times. Results: The levels of p65 in cytoplasm and nuclear and p-IκB among groups were statistically significant (F were 7.79, 33.11, 90.25, respectively, all P<0.01) . Compared with the control group, the levels of p65 in cytoplasm of 2, 4, 6 h group were significantly decreased (all P<0.01) ; the levels of p65 in nuclear of 2, 4, 6, 8 h were significantly increased (all P<0.01) ; the levels of p-IκB of 2, 4, 6 h group were significantly increased (all P<0.01) . The levels of Cyt-c, Cleaved caspase-3 and Bcl-2 among groups were statistically significant (F were 51.42, 503.68, 73.37, respectively, all P<0.01) . Compared with the control group, the levels of Cyt-c of 8, 12 h group were significantly increased (both P<0.01) ; the levels of Cleaved caspase-3 of 2, 4, 8, 12, 24 h were significantly increased (all P<0.01) ; the levels of Bcl-2 of 2, 4, 8, 12, 24 h group were significantly decreased (all P<0.01) . IFC showed that Cyt-c was released from the mitochondria to the cytoplasm gradually as the extension of the exposure time. Conclusion: NF-κB signaling pathway and mitochondrial pathway are involved in the mechanism of DMF-induced apoptosis in H9c2 cardiomyocytes.
目的： 观察二甲基甲酰胺（DMF）致H9c2心肌细胞凋亡过程中核因子-κB（NF-κB）信号通路相关蛋白胞质p65蛋白、核内p65蛋白、磷酸化核因子-κB抑制（p-IκB）蛋白以及线粒体通路相关蛋白胞质细胞色素C（Cyt-c）、活化的半胱氨酸天冬氨酸蛋白酶-3（Cleaved caspase-3）、B细胞淋巴瘤/白血病-2（Bcl-2）水平的变化，探讨DMF引起H9c2心肌细胞凋亡的可能机制。 方法： 给予体外培养的H9c2心肌细胞200 mmol/L DMF染毒，蛋白免疫印迹（Western blotting）法检测染毒0、2、4、6、8、12 h后胞质p65、核内p65、p-IκB的蛋白表达水平以及染毒0、2、4、8、12、24 h后胞质Cyt-c、Cleaved caspase-3、Bcl-2的蛋白表达水平，免疫荧光细胞化学（IFC）法对染毒不同时间后的细胞内Cyt-c进行定位。 结果： 各组胞质p65、核内p65、p-IκB蛋白表达水平组间差异均有统计学意义（F=7.79、33.11、90.25，均P<0.01）；与对照组比较，2、4、6 h染毒组胞质p65蛋白表达水平均降低，2、4、6、8 h染毒组核内p65蛋白表达水平均升高，2、4、6 h染毒组p-IκB蛋白表达水平均升高，差异均有统计学意义（均P<0.01）。各组胞质Cyt-c、Cleaved caspase-3、Bcl-2蛋白表达水平组间差异均有统计学意义（F=51.42、503.68、73.37，均P<0.01）；与对照组比较，8、12 h染毒组Cyt-c蛋白表达水平均升高，2、4、8、12、24 h染毒组Cleaved caspase-3蛋白表达水平均升高、Bcl-2蛋白表达水平均降低，差异均有统计学意义（均P<0.01）。IFC法对细胞内Cyt-c检测显示，随着DMF染毒时间的延长，Cyt-c逐渐从线粒体释放到胞质内。 结论： NF-κB信号通路与线粒体通路可能参与了DMF诱导的H9c2心肌细胞凋亡。.
Keywords: Apoptosis; Dimethylformamide; H9c2 cardiomyocytes; Mitochondria; NF-kappa B.