高良姜素通过抑制Wnt／β-链蛋白信号通路诱导胶质瘤细胞凋亡

Galangin induces apoptosis of glioma cells through Wnt/β--Catenin signal pathway

目的探讨高良姜素对胶质瘤细胞体外增殖和凋亡的影响。方法（1）将胶质瘤细胞U251和U87分为空白对照组、DMSO组及高良姜素100、200、300、400μmol／L组，使用甲基噻唑基四唑（MTT）比色法观察对胶质瘤细胞增殖情况；（2）通过Hoechest染色观察在不同浓度（0、100和200μmol／L）高良姜素作用下胶质瘤细胞U251和U87凋亡情况；（3）使用流式细胞仪检测胶质瘤细胞U251和U87在不同浓度（100和200μmol／L）高良姜素作用下凋亡情况；（4）应用Westernblotting技术检测凋亡相关蛋白B．链蛋白、B淋巴细胞瘤．2基因（Bcl-2）、Bcl-2相关蛋白基因（Bax）、活化含半胱氨酸的天冬氨酸蛋白水解酶（活化caspase-3、活化caspase-9）、多聚腺苷二磷酸核糖基聚合酶（PARP）蛋白等在胶质瘤细胞U87和U251的表达。结果（1）在100、200、300、400μmol／L浓度的高良姜素作用下，体外培养的胶质瘤细胞U251和U87的增殖被明显抑制，并呈现一种量效关系。100、200、300、400μmol／L高良姜素作用于胶质瘤细胞U251和U8724h时，其半数抑制浓度分别为281、321、276、229μmol／L及289．4、261．1、247．4、225．3μmol／L。（2）胶质瘤细胞U251和U87随着高良姜素浓度增加（0、100和200μmol／L1，凋亡率也相应增加，差异具有统计学意义（P〈0．05）。（3）流式细胞仪检测细胞凋亡发现了同样的结果。（4）0、100、200μmol／L浓度高良姜素作用下，胶质瘤细胞U87和U251的Wnt／β-链蛋白表达依次减少，Bcl-2依次减少，Bax依次增加，活化caspase-3、活化caspase-9表达依次增多,活化PARP表达依次增加，差异具有统计学意义（P〈0．05）。结论高良姜素能抑制胶质瘤细胞U251和U87增殖，并通过Wnt／β-链蛋白信号通路及线粒体途径诱导其凋亡。

Objective To investigate the effect of galangin on proliferation and apoptosis of glioma cells in vitro. Methods （1） The gliorna ceils U87 and U251were divided into blank control group, DMSO group, 100, 200, 300 and 400 μmol/L galangin treatment groups. MTT was used to study the effects of drugs on the proliferation of U251 and U87 cells. （2） Hoechest staining was used to observe cell apoptosis in the presence of different concentrations of galangin （0, 100 and 200μmol/L）. （3） Flow cytometry was employed to detect the apoptosis of U251 and U87 cells in the presence of different concentrations ofgalangin （100 and 200 μmol/L）. （4） Western blotting was employed to detect the expressions of apoptosis-related protein β--Catenin, B-cell lymphoma-2 （Bcl-2）, Bcl-2 related protein gene （Bax）, cleaved-caspase-3, cleaved-caspase-9 and poly （ADP-ribose） polymerase （PARP） in the presence of different concentrations of galangin. Results （1） The proliferation of U251 and U87 cells was obviously inhibited after 100, 200, 300 and 400 μmol/L galangin treatments, and dose-effect relation was noted. The concentrations of galangin at half rate of inhibition （IC50） were 281, 321,276 and 229 μmol/L in U251 cells, and 289.4, 261.1,247.4 and 225.3μmol/L in the U87 cells after 100, 200, 300 and 400 μmol/L galangin treatments for 24 h. （2） Under the action of galangin, corresponding increase in apoptosis rates of U251 and U87 cells was noted following the increase of galangin concentrations （0, 100 and 200 μmol/L）, with significant differences （P〈0.05）. （3） The detection of cell apoptosis by flow cytometry found similar changes. （4） Western blotting results indicated that galangin at the concentration of 0, 100 and 200μmol/L could significantly decrease the expressions of apoptosis-related protein β--Catenin and Bcl-2, and increase the Bax, cleaved-caspase-3 and cleaved-caspase-9, and cleaved-PARP expressions; significant differences were noted between each two concentrations （P〈0.05）. Conclusion Galangin can inhibit proliferation of glioma cells U251 and U87, and induce mitochondrial pathway of apoptosis via Wnt/β--Catenin signaling.