黄芩苷对人皮肤恶性黑素瘤A875细胞增殖、凋亡和细胞周期的影响

Effects of baicalin on proliferation,apoptosis and cycle of human skin malignant melanoma A875 cells

目的探讨黄芩苷对人皮肤恶性黑素瘤A875细胞增殖、凋亡和细胞周期的影响。方法体外培养A875细胞,将细胞分为0μg/mL(对照组)、80μg/mL、160μg/mL、320μg/mL黄芩苷组,每组分别给予相应浓度黄芩苷作用A875细胞,干预48 h、72 h后采用四甲基偶氮唑盐法检测各组细胞存活率,干预7 d后,采用细胞克隆形成实验检测细胞增殖能力,干预72 h后,采用流式细胞术测算各组细胞凋亡率及细胞周期百分比,并采用蛋白质印迹法检测各组细胞半胱氨酸天冬氨酸蛋白酶(Caspase)-3、Caspase-9、细胞周期蛋白(Cyclin)D1蛋白表达水平。结果黄芩苷作用于A875细胞48 h、72 h后,随着黄芩苷药物浓度的升高和作用时间的延长,A875细胞存活率逐渐下降(P<0.05);干预72 h后,随着黄芩苷浓度升高,G 0/G 1期细胞比例均升高,S期细胞比例减少,与对照组比较,320μg/mL黄芩苷组细胞Caspase-3、Caspase-9蛋白表达水平升高,CyclinD1蛋白表达水平降低(P<0.05);干预7 d后,随着黄芩苷浓度升高,细胞克隆数减少(均P<0.05)。结论黄芩苷能抑制A875细胞增殖,并诱导其凋亡,将A875细胞阻滞于G 0/G 1期,抑制细胞分裂,其机制可能与黄芩苷升高Caspase-3、Caspase-9蛋白表达水平,降低CyclinD1蛋白表达水平有关。

Objective To investigate the effects of baicalin on proliferation,apoptosis and cycle of human skin malignant melanoma A875 cells.Methods A875 cells were cultured in vitro,and were divided into 0μg/mL(control group),80μg/mL,160μg/mL,and 320μg/mL baicalin groups,each group was given corresponding concentration of baicalin to treat A875 cells.The survival rate of each group was detected by methyl thiazolyl tetrazolium(MTT)assay after 48 and 72 hours of intervention.The proliferation ability of the cells was detected by colony forming assay after seven days of intervention.The apoptosis rate and the percentage of cell cycle in each group were measured by flow cytometry after 72 hours of intervention.In addition,the expression levels of cysteinyl aspartate specific proteinase(Caspase)-3,Caspase-9 and CyclinD1 proteins were detected in each group by Western blotting.Results After A875 cells were treated with baicalin for 48 and 72 hours,the survival rate of A875 cells gradually decreased with the increase of baicalin concentration and the prolongation of action time(P<0.05);after 72 hours of intervention,with the increasing concentration of baicalin,the proportion of the cells in G 0/G 1 phase rose while the proportion of the cells in S phase declined,the 320μg/mL baicalin group exhibited elevated expression levels of Caspase-3 and Caspase-9 proteins and decreased expression level of CyclinD1 protein as compared with the control group(P<0.05);after seven days of intervention,the number of cell clone declined with the increasing baicalin concentration(all P<0.05).Conclusion Baicalin can not only suppress proliferation and induce apoptosis of A875 cells,but also arrest A875 cells in G 0/G 1 phase and inhibit cell division.The mechanism may be related to baicalin′s raising the expression levels of Caspase-3 and Caspase-9 proteins and reducing the expression level of CyclinD1 protein.