柴胡皂苷d通过AKT/mTOR信号通路调控胶质瘤C6细胞自噬

Saikosaponin d regulates autophagy in glioma C6 cells through the AKT/mTOR signaling pathway

目的 探讨柴胡皂苷d在胶质瘤C6细胞自噬中的作用及其机制。方法 分别以终浓度为0μmol/L(Control组)、2μmol/L、4μmol/L、6μmol/L、8μmol/L、10μmol/L、12μmol/L的柴胡皂苷d作用胶质瘤C6细胞后,用CCK-8检测细胞增殖能力,后续分别采用克隆形成实验、划痕实验检测柴胡皂苷d(8μmol/L)对胶质瘤C6细胞增殖能力和细胞迁移能力的影响,Western blot和qRT-PCR检测胶质瘤C6细胞中自噬相关蛋白Beclin1、LC3和AKT/mTOR信号通路相关蛋白及基因的表达情况。结果 与Control组比较,各浓度柴胡皂苷d均能抑制胶质瘤C6细胞增殖且增殖抑制率随着药物浓度的升高而升高(均P<0.01)。8μmol/L柴胡皂苷d作用24 h后,与Control组比较,胶质瘤C6细胞的克隆形成数目显著减少[(479.33±30.66)个vs(258.66±73.35)个,P<0.01],细胞迁移率显著降低[(70.83±4.29)%vs(19.47±1.71)%,P<0.001],自噬相关蛋白Beclin1和LC3的蛋白及mRNA表达水平均显著升高(均P<0.01),而AKT和mTOR信号蛋白及mRNA表达水平均降低(均P<0.01)。结论 柴胡皂苷d可能通过抑制AKT/mTOR信号通路诱导胶质瘤C6细胞发生自噬并抑制其增殖与迁移。

ObjectiveTo investigate the role of saikosaponin d in the autophagy of glioma C6 cells and its mechanism.MethodCCK-8was used to detect the cell proliferation ability of the glioma C6 cells treated by saikosaponin d with final concentrations of 0μmol/L(Control group), 2 μmol/L, 4 μmol/L, 6 μmol/L, 8 μmol/L, 10 μmol/L and 12 μmol/L, respectively. The effects of saikosaponin d with concentrations of 8 μmol/L on the proliferation and migration of glioma C6 cells were detected by clonal formation experiments and scratch experiments, respectively. Western blot and qRT-PCR were used to detect the expression of autophagy-related proteins Beclin1,LC3 and AKT/mTOR signaling pathway-related proteins and genes in glioma C6 cells.ResultsCompared with the Control group, all the other concentrations of saikosaponin d could inhibit the proliferation of glioma C6 cells, and the proliferation inhibition rate increased with the increase of drug concentration(all P<0.01). After the treatment of 8 μmol/L saikosaponin d for 24 h, compared with the Control group, the clone number of glioma C6 cells was significantly reduced(479.33±30.66 vs 258.66±73.35, P<0.01), the cell migration rate was significantly decreased[(70.83±4.29) % vs(19.47±1.71) %, P<0.001], and the protein and mRNA expression levels of autophagy-related proteins Beclin1 and LC3 were significantly increased(all P<0.01), while the protein and mRNA expression levels of AKT and mTOR were decreased(all P<0.01).ConclusionsSaikosaponin d may induce autophagy, and inhibit the proliferation and migration of glioma C6 cells by inhibiting the AKT/mTOR signaling pathway.