柴胡皂苷D通过调控mTORC信号通路诱发肝癌细胞自噬的作用研究

Effect and Mechanism of Saikosaponin D on Autophagy by Regulating mTORC Pathway in Human Hepatocellular Carcinoma Cells

目的本实验旨在研究和探讨柴胡皂苷D(SSD)诱发人肝癌细胞凋亡和自噬的作用以及可能的分子机制。方法采用四甲基偶氮唑蓝(MTT)法检测SSD对肝癌细胞增殖活性的抑制作用;采用GFP荧光法检测肝癌细胞自噬体的形成;采用流式细胞术检测SSD对肝癌细胞凋亡的影响;采用Western blot的方法检测SSD对肝癌细胞中LC3Ⅱ、Beclin 1、p-S6K1蛋白表达的影响。结果人肝癌细胞PLC、MHCC-97H、SMMC-7721以及Huh7经SSD干预48 h后,增殖活性明显受到抑制,且具有剂量依赖性。IC50分别为51. 48,46. 19,39. 87和48. 95μmol·L^(-1)。经SSD处理后,肝癌细胞出现自噬体的数量明显多于未处理的细胞(P <0. 05)。经SSD+3-MA和SSD+z-DEVD-fmk处理后,肝癌细胞死亡率明显低于仅用SSD处理的细胞死亡率(P <0. 05)。经SSD作用后,肝癌细胞LC3Ⅱ和Beclin 1的表达量较未作处理的细胞明显上调,而p-S6K1蛋白表达量明显降低(P <0. 05)。结论 SSD可以通过负调控mTORC信号传导通路诱发肝癌细胞自噬性死亡,从而发挥抗肿瘤活性。

OBJECTIVE To discuss the effect and mechanisms of saikosaponin D （SSD） on apoptosis and autophagy of human hepatocellular carcinoma ceils. METHODS The proliferation of cells in treatment of SSD was detected by MTT. The autophagosome was detected by GFP fluorescence staining. The apoptosis of hepatoma cells were detected by flow cytometry. The expressions of LC3- Ⅱ, p-S6K1 and Beelin 1 in human hepatocellular carcinoma cells was detected by Western blot. RESULTS The proliferations of PLC, MHCC-97H, SMMC-7721 and Huh7 cells with SSD intervention for 48 h were dose-independent inhibited. The IC50 of PLC, MHCC-97H, SMMC-7721 and Huh7 cells were 51.48, 46. 19, 39.87, 48. 95 μmol · L^-1, respectively. After SSD treatment, the number of autophagosome of PLC, MHCC-97H, SMMC-7721 and Huh7 was significantly more than the normal control group （P 〈 0. 05 ）. The apoptosis rate of PLC, MHCC-97H, SMMC-7721 and Huh7 cells induced by SSD ＋ 3-MA or SSD ＋ z-DEVD-fmk was less than the SSD treated cells （P 〈0. 05）. The expressions of LC3-Ⅱ and phosphorylated S6K1 protein （p-S6KI） in PLC, MHCC-97H, SMMC-7721 and Huh7 cells induced by SSD were more than the normal cells, while the expression of Beclin 1 protein decreased. CONCLUSION It＇s suggested that SSD increase the autophagy and apoptosis of human hepatoma cells by regulating the mTORC sig- naling pathway.