胡桃醌经由活性氧介导JNK、p38通路诱导SGC-7901细胞凋亡

Juglone Induces Apoptosis of Human Gastric Cancer SGC-7901 Cells through Reactive Oxygen Species Mediated JNK and p38 Pathway

目的研究胡桃醌诱导人胃癌SGC-7901细胞凋亡的作用及机制。方法噻唑蓝法检测胡桃醌对SGC-7901细胞的增殖抑制作用;流式细胞仪检测细胞凋亡率以活性氧(ROS)水平;Western blot检测JNK,p-JNK,p38,p-p38和caspase-3蛋白表达情况。各实验均设胡桃醌联合活性氧抑制剂N-乙酰半胱氨酸组,以阐明活性氧在胡桃醌诱导SGC-7901细胞凋亡中的作用。结果胡桃醌能明显抑制SGC-7901细胞的增殖,72 h的IC_(50)为24.16μmol·L^(-1),联合N-乙酰半胱氨酸后IC_(50)上升到36.91μmol·L^(-1)。胡桃醌作用24 h,5~20μmol·L^(-1)各组细胞随着药物浓度的升高,凋亡率逐渐升高,各组联合应用N-乙酰半胱氨酸后凋亡率均有所下降,其中20μmol·L^(-1)组凋亡率由32.06%下降到11.56%。胡桃醌作用24 h,细胞内活性氧水平升高、线粒体膜电位降低,N-乙酰半胱氨酸的加入对此有一定程度的拮抗作用。胡桃醌作用48 h,能上调p-P38、p-JNK和caspase-3蛋白的表达,N-乙酰半胱氨酸对此有一定的抑制作用。各给药组P38和JNK蛋白表达并无明显变化。结论胡桃醌通过活性氧介导的JNK、p38信号通路诱导人胃癌SGC-7901细胞凋亡。

OBJECTIVE To investigate the effect and mechanism of juglone on the apoptosis of human gastric cancer SGC-7901 cells. METHODS The antiproliferative effect of juglone on SGC-7901 cells was tested by the MTT assay. The apoptosis rate and intracellular reactive oxygen species（ ROS） level were detected by flow cytometry（ FCM）. The expression of JNK,p-JNK,p38,and pp38 proteins were examined by Western blot. In order to clarify the role of ROS in the apoptosis induced by juglone on SGC-7901 cells,the combination of the juglone and ROS inhibitor NAC groups were set up in each experiment above. RESULTS Juglone could effectively inhibit the proliferation of SGC-7901 cells（ IC_（50） for 72 h was 24. 16 μmol·L（-1））. When combination juglone with NAC,the IC_（50） raised to 36. 91 μmol·L（-1）. After 72 h of exposure to 5- 20 μmol·L（-1）of juglone,the cell apoptosis rate increased gradually with the increase of juglone concentration. After adding NAC,the apoptosis rates declined and the apoptosis rate of 20 μmol · L（-1）group decreased from 32. 06% to 11. 56%. After SGC-7901 cells were treated with juglone for 24 h,the ROS level increased and mitochondrial transmembrane potential decreased which were inhibited by the pretreatment of NAC. After 48 h of exposure to different concentration of juglone,the expressions of p-p38 and p-JNK proteins were up-regulated which could also be inhibited by the adding of NAC. Meanwhile,there were no significant changes in p38 and JNK protein expression in all groups. CONCLUSION Juglone can induce apoptosis of human gastric cancer SGC-7901 cells by JNK and P38 pathway mediated by reactive oxygen species.