蟾酥对肝癌HepG2细胞增殖和凋亡的影响及其机制

Influence of Venenum Bufonis on proliferation and apoptosis in liver cancer cell HepG2 and relevant mechanisms

目的研究蟾酥对肝癌Hep G2细胞增殖和凋亡的影响及其作用机制。方法采用细胞活性CCK-8法检测蟾酥对肝癌细胞增殖的影响;采用Hoechst 33258染色技术观察蟾酥对细胞核型的影响,并用流式双染法检测其凋亡情况;采用Rhodamine 123染色法在流式细胞仪中检测蟾酥对线粒体膜电位的影响;利用凋亡因子caspase 8/9/3的特异性抑制剂预处理细胞,然后检测蟾酥对细胞活性的影响。结果细胞活性检测结果发现,蟾酥对肝癌细胞的杀伤作用具有明显的时间和浓度依赖性。共聚焦显微镜观察发现蟾酥能够明显引起细胞核皱缩,并形成凋亡小体。20μg/m L蟾酥处理24 h后导致56.4%的细胞凋亡,并引起细胞线粒体膜电位下降51.9%。Caspase 9和caspase 3的抑制剂预处理细胞后,相比单独蟾酥处理的细胞,明显引起细胞活性的上升。结论蟾酥能明显抑制肝癌Hep G2细胞增殖,并促进其凋亡。凋亡机制与诱发线粒体膜电位下降及活化caspase 9/3分子有关。

Objective To investigate the influence of Venenum Bufonis on proliferation and apoptosis in liver cancer cell HepG2 and relevant mechanisms.Methods Cell viability CCK-8 method was used to detect the influence of Venenum Bufonis on proliferation in liver cancer cells.Hochest 33258 staining technique was used to observe the influence of Venenum Bufonis on cellular karyotype and the apoptosis was detected by Annexin V-FITC/PI kit and flow cytometry.Rhodamine 123 staining method was used to detect the effect of Venenum Bufonis on mitochondrial membrane potential on flow cytometry.Cells were pretreated with apoptotic factor caspase8/9/3 specific inhibitors and the effect of Venenum Bufonis on cell viability was detected by CCK-8method.Results Cell viability detection showed that Venenum Bufonis killed cells in an obvious concentrationand time-dependent manner.Confocal microscope observation found that Venenum Bufonis significantly caused nucleus shrivel,which resulted in forming apoptotic body.Treatment with 20 μg/mL Venenum Bufonis for 24 h caused apoptosis in 56.4%of cells and a decrease in mitochondrial membrane potential by 51.9%.Pretreatment with caspase9/3 inhibitors induced more increase in cell viability than treatment with Venenum Bufonis alone.Conclusion Venenum Bufonis can significantly inhibit proliferation in liver cancer cell HepG2 and induce apoptosis.Apoptosis mechanisms may be associated with mitochondrial membrane potential decrease and caspase 9/3 activation.