槐果碱对肝癌细胞SMMC-7721和肝细胞L-O2生长的影响

The Effects of Sophocarpine on the Survival of SMMC-7721 and LO2 cells

目的:探讨槐果碱对肝癌细胞SMMC-7721和肝细胞LO2增殖、凋亡及线粒体膜电位的影响及其机制。方法:选取SMMC-7721和LO2,利用不同浓度梯度的槐果碱处理SMMC-7721,根据IC50筛选出两个适宜的浓度同时处理SMMC-7721和LO2细胞,镜下观察,并利用MTT实验、流式细胞凋亡实验及线粒体膜电位检测分别分析增殖、凋亡及线粒体膜电位改变情况;利用Western blot法检测凋亡相关蛋白表达水平。结果:槐果碱作用SMMC-7721的IC50为536.3 mg/L(95%CI:412.7,697.0)。选取500 mg/L及1000 mg/L处理细胞后,细胞分布密度降低并出现不同程度的细胞皱缩。MTT结果显示随着药物浓度的升高,两种细胞OD值均明显下降(P<0.001);而同浓度下,增殖能力比较,差异无统计学意义(P>0.05)。槐果碱作用后总凋亡率均随着浓度的升高而增加,但高浓度下对肝细胞的促凋亡作用明显高于肝癌细胞(P<0.05);线粒体膜电位水平均随着浓度的升高而降低(P<0.05);随着槐果碱浓度的升高,两种细胞内Bcl-2水平均减少,而Bax及Cleaved-Caspase-3蛋白水平均有所升高。结论:槐果碱通过上调肝癌细胞SMMC-7721和肝细胞LO2细胞内Bax和Cleaved-Caspase-3表达并抑制Bcl-2,抑制其增殖,降低线粒体膜电位水平,并促进细胞凋亡,提示槐果碱在抑制肝癌生长的同时有着不可避免的肝细胞损伤等毒副作用。

Objective： To investigate the effect of sophocarpine on proliferation, apoptosis and mitochondrial membrane potential of SMMC-7721 and LO2 hepatocellular carcinoma cells and its mechanism. Methods：SMMC-7721 and LO2 cells were treated with sophocarpine at different concentration gradients. Two suitable concentrations were selected according to IC50 to treat SMMC-7721 and LO2 cells simultaneously. The condition of proliferation, apoptosis and mitochondrial membrane potential was analyzed by MTT assay, flow cytometry and mitochondrial membrane potential test. The expression of Bcl-2, Bax and Cleaved-Caspase3 was detected by Western blot. Results： The IC50 of the SMMC-7721 was 536.3 mg/L（95% CI： 412.7-697.0）. After 500 mg/L and1,000 mg/L were selected to treat the cells, the cell density was decreased and the cells were shrunk after treated with sophocarpine. The results of MTT showed that the OD values of both cells were significantly decreased with the increase of the concentration of sophocarpine（P0.001）. In the same concentration, there was no significant difference of proliferation between the two cells（P〈0.05）. The total apoptosis rate was increased as the concentration of sophocarpine was increased, but the apoptosis increasing rate of high concentration in LO2 was significantly higher than that in SMMC-7721（P〈0.05）. The mitochondrial membrane potential was also decreased with the increase of the concentrat ion of sophocarpine（P〈0.05）. In addition, the levels of bcl-2 in these two cells were both decreased, while the levels of Bax and Cleaved-Caspase3 were increased, with the increase of sophocarpine. Conclusions： Sophocarpine could suppressed the cell proliferation, decreased the mitochondrial membrane potential, and promoted the cell apoptosis of SMMC-7721 and LO2 through up-regulating levels of Bax and Cleaved-Caspase3 and down regulating the level of Bcl-2, which implied that sophocarpine caused liver injury inevitably when killing liver carcinoma cells.