基于PI3K/Akt信号通路的汉黄芩素抗骨肉瘤作用机制研究

Effect of Wogonin on osteosarcoma cells and its mechanism based on a PI3K/Akt signaling pathway

目的本实验旨在探讨汉黄芩素对人骨肉瘤MG-63细胞株增殖、凋亡及PI3K/Akt信号通路的影响。方法人骨肉瘤MG-63细胞株培养成功后,取单细胞悬液,用不同浓度(0、50、100及200μmol/L)汉黄芩素分组,进行干预后,MTT法检测24 h、48 h及72 h时各组人骨肉瘤MG-63细胞的增殖活性;流式细胞术检测各组人骨肉瘤MG-63细胞的凋亡率;细胞划痕实验检测细胞迁移能力变化;透射电镜检测MG-63细胞超微结构改变;Western blot法检测COX-2、Caspase-3和p-Akt蛋白表达变化。结果汉黄芩素对MG-63细胞的增殖能力具有抑制作用,且该作用与药物浓度及作用时间呈正相关,差异有统计学意义(P<0.05);MG-63细胞凋亡率与汉黄芩素浓度呈正相关,差异有统计学意义(P<0.05);划痕实验显示汉黄芩素可明显抑制MG-63细胞的迁移能力,呈剂量依赖性;透射电镜观察细胞超微结构,可见汉黄芩素对MG-63超微结构有明显的促凋亡作用;Western blot结果显示,与对照组相比,实验组COX-2和p-Akt蛋白的表达水平显著降低,而实验组Caspase-3表达水平显著高于对照组,差异有统计学意义(P<0.05)。结论汉黄芩素可抑制MG-63细胞增殖,促进细胞凋亡,其机制可能与下调PI3K/Akt信号通路有关。

Objective The aims of this study were to explore effects of Wogonin on proliferation,apoptosis and the PI3 K/Akt signaling pathway in human osteosarcoma MG-63 cells. Methods MG-63 cells were treated with different concentrations of Wogonin（ 0,50,100 and 200 μmol/L） for 24,48 and 72 h,MTT assay was used to determine the cell proliferation,and the rates of apoptosis of MG-63 cells were assessed by the flow cytometry; Cell scratch assay was used to detect cell migration and TEM to detect morphological changes of MG-63 cells; Western blot were employed to examine the expression of COX-2,Caspase-3 and P-Akt protein in MG-63 cells. Results Wogonin significantly inhibited proliferation of MG-63 cells in a dose-or time-dependent manner,and this effect was positively correlated with drug concentration and duration of action（ P〈 0. 05）. The apoptotic rate of MG-63 cells was positively correlated to the concentrations of Wogonin. Their differences were statistically significant（ P〈 0. 05）. Wogonin also significantly inhibited the migration of MG-63 cells in a dose-dependent manner（ P〈 0. 05）. Wogonin significantly down-regulated the levels of COX-2 and p-Akt protein expression and significantly up-regulated the level of caspase-3 protein expression in MG-63 cells when compared to the negative control group（ P〈 0. 05）. Conclusion Wogonin can inhibit the proliferation of MG-63 cells and promote apoptosis,which may be related to down-regulation of PI3 K/Akt signaling pathway.