黄芩苷诱导小鼠结肠癌发生坏死性凋亡作用的体外研究

Effect of Baicalin on the Necroptosis of Mouse Colon Cancer in Vitro

目的探讨黄芩苷对小鼠结肠癌CT26.WT细胞的作用及细胞死亡形式。方法 CT26.WT细胞分为4组,(1)正常对照组,加入新鲜培养基,常规培养;(2)黄芩苷组,加入终浓度为100μmol·L-1黄芩苷;(3) z-VAD-fmk组,加入终浓度为20μmol·L-1z-VAD-fmk;(4)联合用药组,加入终浓度为20μmol·L-1z-VAD-fmk 1 h后加入100μmol·L-1黄芩苷。采用CCK8法检测黄芩苷对细胞增殖抑制作用和细胞存活率。DAPI染色检测细胞核的变化;透射电镜观察细胞超微结构; QPCR法和Western blotting法检测黄芩苷对细胞内RIP3基因和蛋白表达水平的影响。结果与正常对照组比较,黄芩苷组和联合用药组差异有统计学意义(P<0.05);正常对照组细胞死亡率为(10.54±0.19)%,黄芩苷组为(34.93±0.16)%,z-VAD组为(11.23±0.59)%,联合用药组为(23.27±1.20)%(P<0.01)。与正常对照组比较,黄芩苷组核浓染,出现碎裂现象;与黄芩苷组比较,联合用药组核浓染,有明显核碎裂现象。电镜结果显示黄芩苷组细胞发生坏死性凋亡,细胞肿胀,线粒体肿胀,内容物泄漏。黄芩苷组RIP3 mRNA的表达明显上调(P<0.01),RIP3蛋白的表达明显增强(P<0.05)。结论黄芩苷诱导CT26.WT细胞发生坏死性凋亡,可使RIP3的基因和蛋白表达明显上调。

Objective To investigate the effect of baicalin on CT26.WT cells of colon cancer in mice,and to discuss the cell death form.Methods CT26.WT cells were divided into four groups including of control group,routine cultured in fresh medium,the baicalin group,added with concentration of 100μmol·L^-1 baicalin,the z-VAD-fmk group,was added with final concentration of 20μmol·L^-1 z-VAD-fmk,and the combination group,added final concentration of 20μmol·L^-1 z-VAD-fmk,1 h before adding 100μmol·L-1 baicalin.Then the inhibitory effect of baicalin on cell proliferation and cell viability were detected by CCK-8 method.The changes of nucleus were detected by DAPI staining,the ultrastructure of cells was observed by TEM,and the effect of baicalin on the expression of RIP3 gene and protein in cells was detected by QPCR method and Western blotting.Results Compared with control group,the differences of baicalin group and combination group had statistically significance(P<0.05).cell death rate for control group was(10.54±0.19)%,for baicalin group was(34.93±0.16)%,for z-VAD group was(11.23±0.59)%,and combination group was(23.27±1.20)%(P<0.01).Compared with the normal control group,baicalin group showed nuclear concentration and fragmentation.there was obvious nuclear fragmentation in the combination group against baicalin group.The results of electron microscopy showed that the cells of baicalin were necrotic,cell swelling,mitochondria swelling and contents leaking.Baicalin group significantly up-regulated RIP3 mRNA expression(P<0.01)and enhanced RIP3 protein expression(P<0.05).Conclusion Baicalin induces the necrosis of ct26.WT cells,and can significantly increase the gene and protein expression of RIP3.