斑蝥酸钠维生素B6诱导人肝癌HepG2细胞凋亡及周期阻滞

Sodium Cantharidinate and Vitamin B6 Injection Promotes Apoptosis of Hepatocellular Carcinoma Cell Line HepG2 through Regulating PI3K/Akt Signaling Pathway

目的:研究斑蝥酸钠维生素B6(艾易舒)注射液对人肝癌HepG2细胞增殖、细胞周期与凋亡的影响,探讨其分子机制。方法:将不同浓度的斑蝥酸钠维生素B6注射液(0,1,5,10 mg·L-1)作用于人肝癌HepG2细胞12,24,48 h,MTT法检测细胞增殖抑制率,流式细胞学技术检测细胞凋亡率及细胞周期的变化,Western blot检测蛋白激酶B(Akt),磷酸化蛋白激酶B(pAkt),B细胞淋巴瘤-2基因相关X蛋白(Bax),B细胞淋巴瘤-2基因(Bcl-2)及细胞周期依赖性激酶抑制因子p21蛋白表达变化,RT-PCR检测Bax,Bcl-2及p21 mRNA表达变化。结果:斑蝥酸钠维生素B6可显著抑制HepG2细胞的增殖,诱导细胞凋亡,具有显著的时间和浓度效应(P<0.05);同时G0/G1期细胞比例显著升高,S期细胞比例显著降低(P<0.05)。Western blot结果显示斑蝥酸钠维生素B6可下调HepG2细胞中pAkt和Bcl-2蛋白的表达,上调Bax和p21蛋白的表达(P<0.05)。RT-PCR结果显示Bax,p21 mRNA的表达水平明显升高,而Bcl-2 mRNA的表达水平下降(P<0.05)。结论:斑蝥酸钠维生素B6可抑制人肝癌HepG2细胞的增殖,诱导其凋亡并引发G0/G1期阻滞,其机制可能与斑蝥酸钠维生素B6参与调控磷脂酰肌醇-3-激酶/蛋白激酶B(PI3K/Akt)通路有关。

Objective： To investigate the effects of Sodium Cantharidinate and Vitamin B6 injection （SCV） on proliferation and apoptosis of human hepatocellular carcinoma cell line HepG2,and to explore its underlying mechanism. Method： After treatment with different concentrations of SCV （0,1,5,10 mg L-1） for different periods （12,24,48 h）,the proliferation of HepG2 cells was analyzed by MTT. The cell cycle and apoptosis rate were detected by flow cytometry （FCM）. The protein levels of protein kinase B（Akt）,phosphate Akt（pAkt）,Bcl-2 associated x protein（Bax）,B cell lymphoma/leuremia-2（Bcl-2） and p21 were detected by Western blot. The mRNA levels of Bax,Bcl-2 and p21 were detected by reverse transcription-polymerase chain reaction （RT-PCR）. Result： SCV significantly inhibited cell proliferation and induced apoptosis of HepG2 cells. Those effects were in a time and dose-dependent manners. SCV induced an increase in the percentage of G0/G1 phase cells and a decrease in the percentage of S phase cells accompanied by the change in DNA ploidy（P〈0.05）. The levels of Bax and p21 mRNA expression in HepG2 cells was significantly up-regulated after treatment with SCV （P〈0.05）,while the expression level of Bcl-2 mRNA was down-regulated. The expression levels of pAkt and Bcl-2 proteins were down-regulated,while that of Bax and p21 protein was up-regulated（P〈0.05）. Conclusion： SCV could inhibit cell proliferation and induce apoptosis in human hepatocellular carcinoma cell line HepG2.PI3K/Akt signaling pathway and the related downstream targets are involved in those processes.