白头翁总皂苷碱水解产物通过线粒体通路促进肝癌细胞凋亡研究

Alkali hydrolysate of total saponins from Pulsatilla chinensis inhibits hepatic carcinoma cell growth by inducing apoptosis through mitochondrial pathway

目的研究白头翁总皂苷碱水解产物(PAHS)抗肝癌作用及其相关机制。方法采用MTT法检测PAHS对于人肝癌细胞SMMC-7721增殖的影响,并用Giemsa染色法观察细胞形态;采用Hoechst 33258染色法以及流式细胞术检测PAHS对细胞凋亡、细胞周期及线粒体膜电位的影响;采用Western blotting检测凋亡相关蛋白Cytochrome C、Caspase-3、cleaved Caspase-3和Bcl-2的表达。采用ICR小鼠腋下sc肝癌细胞H22建立体内肝癌模型,测定肿瘤生长抑制率,并通过HE染色和透射电镜观察组织形态。结果体外结果显示,PAHS能够以剂量和时间依赖的方式抑制SMMC-7721细胞的增殖,能够将肿瘤细胞的生长周期阻滞在S期,降低线粒体膜电位。PAHS可以上调Cytochrome C、cleaved Caspase-3的表达和下调Bcl-2、Caspase-3的表达。体内实验结果显示,PAHS(50、100、200 mg/kg)可以抑制小鼠肝癌细胞的生长,具有剂量依赖性。组织学观察显示PAHS组的小鼠肿瘤组织均有大面积坏死及凋亡细胞的出现。结论 PAHS可通过诱导细胞凋亡发挥抗肝癌作用,其作用机制与线粒体凋亡途径有关。

Objective To investigate the anti-hepatocarcinoma effect and mechanisms of the alkali hydrolysate of total saponins from Pulsatilla chinensis(PAHS).Methods MTT assay was used to evaluate the effect of PAHS on proliferation of human liver cancer cell line SMMC-7721 in vitro;Cell morphology was observed by Giemsa staining.The effects of PAHS on apoptosis,cell cycle,and mitochondrial membrane potential of SMMC-7721 were detected by Hoechst 33258 staining and flow cytometry assay;Western blotting was employed to detect the protein expression of Cytochrome C,Caspase-3,cleaved Caspase-3,and Bcl-2 in SMMC-7721 cells.An in vivo liver cancer model was established using ICR mice subcutaneously received H22 hepatoma carcinoma cells to detect tumor growth inhibitory rate.Morphological changes of the tumor samples were observed by HE staining and transmission electron microscope.Results MTT assay showed that PAHS could inhibit proliferation and increase apoptosis of SMMC-7721 cells in dose-and time-dependent manners in vitro,block the cell cycle in S phase,and decrease mitochondria membrane potential;PAHS could significantly increase the expression of Cytochrome C and cleaved Caspase-3 and decrease the expression of Bcl-2 and Caspase-3.PAHS dramatically decreased the weight of tumor tissue in a dose-dependent manner.Histopathological examination showed that large necrosis area and apoptotic cells were found in tumor tissues of mice in PAHS administrated group.Conclusion PAHS exerts antitumor activity in vitro and in vivo by inducing apoptosis,and its mechanism is related to regulation of the mitochondrial pathway.