Rac2在二烯丙基二硫诱导人白血病K562细胞凋亡中的作用

The role of Rac2 in diallyl disulfide inducing apoptosis in human leukemia K562 cells

目的研究二烯丙基二硫(diallyl disulfide,DADS)诱导人白血病K562细胞凋亡的分子机制。方法 Real-time PCR检测Rac2基因mRNA水平;Western blot检测Rac2、JNK、p38等蛋白的表达;基因沉默Rac2蛋白的表达;流式细胞术检测凋亡细胞百分率以及细胞内的活性氧(reactive oxygen species,ROS)水平;琼脂糖凝胶电泳检测晚期凋亡。结果随着DADS质量浓度的增加,Rac2基因mRNA水平明显上调(P<0.05)。与未干扰组相比,siRac2RNA组凋亡率明显降低(P<0.05)。Rac2siRNA干扰的DADS诱导的K562细胞并未出现梯状条带。与未干扰组相比,siRac2RNA组在5.0,10.0和15.0mg·L-1 DADS作用于K562细胞8h后,荧光强度显著降低(P<0.05)。Western blot分析结果显示:与对照组相比,10.0和15.0mg·L-1 DADS作用于K562细胞24h后,蛋白Rac2的表达水平明显上调(P<0.05);用siRNA抑制Rac2蛋白的表达后,JNK1的表达显著降低,p38和磷酸化的p38的表达在Rac2干扰前后没有明显变化。结论 Rac2与DADS诱导K562细胞凋亡、活性氧的产生密切相关。活化的Rac2选择性地激活JNK信号通路而不是p38信号通路导致细胞凋亡。

Objective To research the molecular mechanism of diallyl disulfide （DADS ）-induced apoptosis in human leukemia K562 cells .Methods mRNA levels of Rac2 were detected by real-time PCR ;Rac2 ,JNK and p38 levels were measured by Western blot .Treatment of K562 cells with siRNAs resulted in inhibition of Rac2 expression .Flow cytometry method was used to deter-mine the percentage of apoptosis cells and DNA agarose electrophoresis was used to determine the late stage of apoptosis .Levels of ROS were measured by 2′,7′-dichlorofluorescein diacetate （DCFH-DA） fluorescence .Results There was a significant up regu-lation of the mRNA level of Rac2 in K562 cells after the treatment with different concentration of DADS （P〈0 .05） .Compared with the control group ,there was a significant up regulation of Rac2 protein in K562 cells treated with 5 .0 and 10 .0 mg · L -1 DADS for 24 h （P〈0 .05） .Treatment of cells with DADS for 24 h markedly reduced the percentage of apoptotic cells after inhibi-tion of Rac2 with siRNA .SiRNA-treated groups showed normal bands on agarose gel electrophoresis .After suppression of Rac2 , ROS production was markedly reduced after DADS treatment （P〈0 .05） .The expression of JNK1 in K562 cells decreased when Rac2 expression was inhibited with siRNA ,while there was no change in p38 and p-p38 .Conclusion These results indicate Rac2 have a critical role in DADS-induced apoptosis and ROS production in DADS-induced K562 cells .Rac2 selectively activated the JNK pathway ,not the p38 pathway in DADS-induced apoptosis in K562 cells .