蝙蝠葛碱对肝癌Huh7细胞增殖和凋亡的作用及机制研究

Effect and mechanism of dauricine on proliferation and apoptosis of hepatoma Huh7 cells

目的研究蝙蝠葛碱对肝癌Huh7细胞增殖和凋亡的作用,探索其抗肿瘤的作用机制和与Hedgehog信号通路的关系。方法不同质量浓度(2、4、8μg/mL)的蝙蝠葛碱处理Huh7细胞,采用四甲基偶氮唑蓝(MTT)法检测Huh7细胞增殖能力的变化;流式细胞术分析细胞凋亡;real-timePCR、Westernblotting法检测Hedgehog信号通路相关基因及蛋白表达水平。结果随着蝙蝠葛碱质量浓度及作用时间的增加,其对Huh7细胞增殖的抑制率升高。其中蝙蝠葛碱8μg/mL作用48h对细胞增殖抑制率最高(48.8%)。蝙蝠葛碱可明显诱导Huh7细胞发生凋亡。随着蝙蝠葛碱质量浓度的增大,细胞的凋亡率显著升高(P<0.05、0.01)。与对照组比较,蝙蝠葛碱各质量浓度组细胞Hedgehog信号通路中PTCH1、GLi1、SMO、SHHm RNA和蛋白表达水平显著降低,同时cleavedCaspase-3蛋白水平显著升高,而Bcl-2表达水平降低(P<0.05、0.01),并呈质量浓度依赖性。结论蝙蝠葛碱可明显抑制Huh7细胞的增殖,促进其凋亡,其可能通过抑制Hedgehog信号通路发挥作用。

Objective To study the effect of dauricine on the proliferation and apoptosis of hepatoma Huh7 cells,and explore its anti-tumor mechanism and its relationship with Hedgehog signaling pathway.Methods The effects of different concentrations of dauricine(2,4,8μg/mL)on the proliferation of Huh7 cells were detected by MTT assay.Apoptosis of Huh7 cells was analyzed by flow cytometry.Real-time PCR and Western blotting were used to detect the levels of Hedgehog signaling pathway-related genes and proteins.Results With the increase of the concentration of dauricine and the duration of action,the inhibition rate of Huh7 cell proliferation was increased.Among them,8μg/mL dauricine had the highest inhibition rate(48.8%)at 48 h.Dauricine induced the apoptosis in Huh7 cells.With the increase of the concentration of dauricine,the apoptotic rate of cells was increased significantly(P<0.05,0.01).The mRNA and protein expression levels of PTCH1,GLi1,SMO and SHH genes in Hedgehog signaling pathway were significantly decreased,while the level of cleaved Caspase-3 protein was significantly increased,accompany with the decreased expression of Bcl-2 in dauricine concentration-dependent pattern(P<0.05,0.01)in dauricine group compared with the control group.Conclusion Dauricine could significantly inhibit the proliferation and promote apoptosis of Huh7 cells,which may play a role by blocking Hedgehog signaling pathway.