马钱苷对肝癌细胞HepG2增殖与凋亡的影响及机制研究

Study on the Effects of Loganin on the Proliferation and Apoptosis of Liver Cancer HepG2 Cells and Its Mechanism

目的:研究马钱苷对肝癌细胞HepG2增殖及凋亡的影响,并探讨其作用机制。方法:运用CCK-8法检测不同质量浓度(10、25、50、100、150、200、300、400μg/mL)的马钱苷作用24、48 h对HepG2细胞增殖活性的影响。将HepG2细胞分为对照组和马钱苷低、中、高浓度组(50、100、150μg/mL),加药作用24 h后,采用Hoechst 33342荧光染色法检测细胞凋亡形态学变化;采用流式细胞术检测细胞凋亡和周期分布情况;采用Western blotting法检测各组细胞中G1/S-特异性周期蛋白D1(Cyclin D1)、增殖细胞核抗原(PCNA)、B淋巴细胞瘤2(Bcl-2)、胱天蛋白酶3(Caspase-3)、活化的Caspase-3(Cleaved-Caspase-3)、Caspase-9、Cleaved-Caspase-9蛋白的表达水平。结果:马钱苷对HepG2细胞增殖具有抑制作用,且呈浓度依赖趋势。与对照组比较,马钱苷低、中、高浓度组细胞出现核固缩、碎裂等凋亡现象,细胞凋亡率显著升高(P<0.01);细胞主要阻滞于S期;马钱苷低、中、高浓度组细胞中Cyclin D1、PCNA、Caspase-3蛋白的相对表达量均显著降低、Cleaved-Caspase-3蛋白的相对表达量均显著升高(P<0.05或P<0.01),马钱苷中、高浓度组细胞中Cleaved-Caspase-9蛋白的相对表达量均显著升高、Bcl-2和Caspase-9蛋白的相对表达量均显著降低(P<0.05或P<0.01)。结论:马钱苷对HepG2细胞的增殖具有明显的抑制作用,并可诱导其凋亡,其作用机制可能与抑制Bcl-2蛋白表达,促进Caspase-3、Caspase-9活化有关。

OBJECTIVE:To study the effects of loganin on the proliferation and apoptosis of liver cancer HepG2 cells,and to explore its mechanism.METHODS:CCK-8 assay was used to detect the effects of different concentrations(10,25,50,100,150,200,300,400μg/mL)of loganin on the proliferation activity of HepG2 cells for 24 and 48 h.HepG2 cells were divided into control group,loganin low-concentration,medium-concentration and high-concentration groups(50,100,150μg/mL).After treated for 24 h,morphological changes of apoptosis of cells were detected by Hoechst 33342 fluorescence staining.The apoptosis and cycle distribution of cells were detected by flow cytometry.Western blotting was used to detect protein expression of Cyclin D1,PCNA,Bcl-2,Caspase-3,Cleaved-Caspase-3,Caspase-9 and Cleaved-Caspase-9.RESULTS:Loganin inhibited the proliferation of HepG2 cells,in concentration-dependent trend.Compared with control group,apoptosis as pyknosis and fragmentation occurred,and the apoptosis rate increased significantly in loganin low-concentration,medium-concentration and high-concentration groups(P<0.01);the cell were mainly blocked in S phase;relative protein expression of Cyclin D1,PCNA and Caspase-3 were significantly decreased,while that of Cleaved-Caspase-3 were significantly increased in loganin lowconcentration,medium-concentration and high-concentration groups(P<0.05 or P<0.01);relative protein expression of Cleaved-Caspase-9 were increased significantly,while that of Bcl-2 and Caspase-9 were decreased significantly in loganin medium-concentration and high-concentration groups(P<0.05 or P<0.01).CONCLUSIONS:Loganin can significantly inhibit the proliferation and induce apoptosis of HepG2 cells,the mechanism of which may be associated with inhibiting Bcl-2 protein expression and promoting Caspase-3,Caspase-9 activation.