杠柳甙元对人肝癌Hep3B细胞增殖、凋亡、迁移的调控作用及其机制研究

Role of periplogenin on the regulation of proliferation,apoptosis and migration of hepatocellular carcinoma Hep3B cells and its mechanism

目的:观察杠柳甙元(Periplogenin,PPG)对人肝癌Hep3B细胞增殖、凋亡、迁移的调控作用,并探讨其相关机制。方法:将Hep3B细胞分为空白组、PPG组。空白组的Hep3B细胞未行PPG处理,PPG组的Hep3B细胞分别以2.5、5、12.5μmol/mL PPG干预48 h。使用CCK-8法检测细胞增殖;光镜观察细胞形态变化;DAPI/PI染色法观察细胞凋亡状态;平板克隆法检测细胞克隆形成能力;划痕法检测细胞迁移能力;Western Blot法检测细胞凋亡相关蛋白Caspase-3、Cleaved Caspase-3、Bcl-2以及细胞迁移相关蛋白基质金属蛋白酶-9(Matrix metalloproteina 9,MMP-9)。结果:与空白组比较,各PPG组细胞增殖水平降低,细胞数量减少且出现体积缩小、染色质固缩、核破裂等现象。PPG处理后,Hep3B细胞克隆形成水平降低,细胞迁移率降低。与空白组相比,Bcl-2蛋白水平显著降低,Cleaved Caspase-3/Caspase-3蛋白比例增高;MMP-9蛋白表达水平降低,差异均具有统计学意义(均P<0.05)。结论:杠柳甙元可有效抑制肝癌Hep3B细胞的增殖、迁移,促进其凋亡,并呈剂量依赖性,其作用机制可能与激活Caspase信号通路、抑制MMP-9活性相关。

Objective:To observe the effects of and explore the underlying mechanisms of Periplogenin(PPG)on the regulation of proliferation,apoptosis and migration of hepatocellular carcinoma Hep3B cell.Methods:Hep3B cells were divided into control group and PPG group.The Hep3B cells in the control group were not treated with PPG,and the other in the PPG group were intervened with 2.5,5 and 12.5μmol/mL PPG for 48 h.Cell viability was measured using the CCK-8 assay.Cell morphological changes was observed using by light microscope;The apoptotic state was observed by DAPI/PI staining.The colony-formation assay and scratch method was used for detecting the cell clone forming ability and cell migration ability,respectively.The cell apoptosis-related proteins Caspase-3,Cleaved Caspase-3,Bcl-2 and cell migration-related protein Matrix metalloproteinase-9(MMP-9)were detected by Western Blot.Results:Compared with the control group,the cell proliferation level and the cell number in each PPG group were decreased,accompanying by cell volume reduction,chromatin pyknosis,and nuclear rupture in Hep3B cells.After PPG treatment,the clone formation level and cell mobility of Hep3B cells were decreased.Compared with the control group,the level of Bcl-2 protein was decreased,while the proportion of Cleaved Caspase-3/Caspase-3 protein was increased.The expression of MMP-9 proteins was decreased,with significantly statistically differences(P<0.05).Conclusion:Periplogenin could inhibit the proliferation,and migration of Hep3B cells and also promote the cell apoptosis in a dose-dependent manner,which might be related to the activation of Caspase signaling pathway and inhibition of MMP-9 activity.