抑制JNK通路对染料木黄酮诱导肝癌MHCC97-L细胞凋亡及caspase-3和caspase-9活性的影响

Effect of Genistein inducing liver cancer MHCC97-L cells apoptosis and activity of caspase-3 and caspase-9 through inhibiting JNK pathway

目的探讨抑制JNK通路对染料木黄酮(Genistein)诱导肝癌MHCC97-L细胞凋亡及caspase-3和caspase-9活性的影响。方法实验分为对照组、SP600125(SP)10、20、30μmol/L组。蛋白质印迹法筛选SP的最适工作浓度。实验分对照组、Gen组、SP组和联用组,倒置显微镜、荧光显微镜和分光光度法分别检测细胞凋亡形态、细胞凋亡指数、caspase-3和caspase-9活化水平。结果SP能明显阻断P-JNK蛋白表达(P<0.01),其最小工作浓度为20μmol/L。倒置显微镜下,各药物组细胞均出现染色质着色变深或局部凝集的凋亡特征性改变,以联用组细胞变化最为明显。荧光显微镜下,各药物组均可观察到早期凋亡和晚期凋亡细胞。与对照组比较,所有药物组凋亡指数均明显升高(P<0.01),且联用组高于Gen组和SP组(P<0.01)。与对照组比较,所有药物组的caspase-3和caspase-9活性均明显升高(P<0.01)。与Gen组、SP组比较,联用组caspase-3活性最高(P<0.01),SP组caspase-3活性高于Gen组(P<0.01);联用组caspase-9活性高于Gen组(P<0.01),SP组与Gen组、联用组与SP组caspase-9活性差异均无统计学意义(P>0.05)。结论抑制JNK通路可通过激活caspase依赖的线粒体途径来促进Genistein诱导MHCC97-L细胞凋亡。

Objective To explore the effect of Genistein inducing liver cancer MHCC97-L cells apoptosis and activity of caspase-3 and caspase-9 through inhibiting JNK pathway.Methods The experiment was divided into control group and SP600125(SP)10,20,30μmol/L groups.Western blotting was carried out to search the optimum working concen-tration of SP.The experiment was divided into control,Gen,SP and combined groups.Apoptosis morphology,apoptosis index,activity of caspase-3 and caspase-9 were detected by inverted microscope,fluorescence microscope and spec-trophotometry respectively.Results SP obviously blocked the expression of P-JNK protein at a minimum working con-centration of 20μmol/L(P<0.01).Under inverted microscope,chromatin became darker or condensate in each drug group,and cell changes most obvious in combined group.Early apoptotic cells and late apoptotic cells were observed under fluorescence microscope.Compared with control group,apoptotic index of all drug groups was significantly in-creased(P<0.01),among which apoptotic index of combined group was highest and pro-apoptotic effect was stronger than that of Gen group and SP group(P<0.01).Compared with control group,the activity of cas-pase-3 and caspase-9 in all drug groups were signifi-cantly increased(P<0.01).Compared with Gen group and SP group,the activity of caspase-3 in combination group was the highest(P<0.01),and the activity of caspase-3 in SP group was higher than that in Gen group(P<0.01).The activity of caspase-9 in the combination group was high-er than that in the Gen group(P<0.01),while the activity of caspase-9 in the SP group and Gen group and the com-bined group and the SP group have no significant differences(P>0.05).Conclusions These results suggest that inhibi-tion of JNK pathway can promote the apoptosis of Gen induced MHCC97-L cells by activating the caspase-dependent mitochondrial pathway.