去甲斑蝥素和吴茱萸碱联合处理对HepG2人肝癌细胞生长的抑制作用

Inhibitory effect of norcantharidin combined with evodiamine on the growth of human hepatic carcinoma cell line HepG2 in vitro

目的研究去甲斑蝥素(NCTD)与吴茱萸碱(EVO)联合处理对肝癌HepG2细胞增殖和凋亡的影响。方法以不同浓度的NCTD和不同浓度的EVO分别组成单药处理组和NCTD、EVO联合处理组,另设不加药的对照组,作用于HepG2细胞。MTT法检测NCTD、EVO单药及联用对HepG2细胞的增殖抑制作用,流式细胞术检测细胞周期,annexinⅤ-FITC/PI双染色结合流式细胞术检测药物对HepG2细胞凋亡的影响。Western blot法检测Bak、Bcl-2蛋白的表达。结果 MTT法证明NCTD和EVO对HepG2细胞的生长有抑制效果,两药联合处理具有明显的协同抑制作用(合用指数CI<1);与单一处理组相比,NCTD联合EVO上调G2/M期阻滞率(P<0.05),G2/M期阻滞率分别为EVO(36.13±1.63)%、NCTD(10.67±0.89)%、联合处理(73.42±1.92)%;此外,两药联合处理的肝癌细胞凋亡率由EVO(15.78±3.08)%、NCTD(11.47±1.60)%,显著上升到联合处理的(21.86±2.70)%(P<0.05);Western blot结果显示,与单独处理相比较,联合处理组Bax蛋白表达水平增高,Bcl-2表达降低(P<0.05)。结论 NCTD和EVO联合处理抑制肝癌细胞增殖并促进细胞凋亡。

Objective To investigate the combined effect of evodiamine （EVO） and norcantharidin （NCTD） on HepG2 cells. Methods HepG2 cells were treated with EVO or/and NCTD at different concentrations in vitro, with blank culture medium as the negative control. The inhibitory effect of EVO or/and NCTD was determined by MTT assay. Cell cycle and cell apoptosis were assessed by flow cytometry （FCM） combined with PI staining and annexin V-FITC/PI, respectively. The expressions of Bax and Bcl-2 were examined by Western blotting. Results M＇I-I- assay showed that NCTD and EVO had restraining effect on the growth of HepG2 cells. The combination of NCTD and EVO had significant synergistic inhibition effect （ CI 〈 1 ）. The arrest rate of G2/M phase was （36.13 ± 1.63） % in the EVO induced group and （ 10.67 ± 0.89） % in the NCTD induced group, and the combination of EVO and NCTD up-regulated the rate to （73.42 ± l. 92）% （P 〈 0.05）. In addition, the apoptosis rate of HepG2 cells treated by two-drug combination increased significantly from （ t5.78 ± 3.08） % in the EVO group and （ 1 l. 47 ± 1.60） % in the NCTD group to （21.86 ± 2.70） % in the EVO combined with NCTD group （ P 〈 0.05）. Western blotting demonstrated that the expression level of Bax protein went up significantly （ P 〈 0.05 ）, meanwhile Bcl-2 protein dropped significantly （ P 〈 0.05 ） in the combined induced group. Conclusion EVO combined with NCTD showed synergetic effect on anti-proliferation and pro-apoptosis in HepG2 cells.