二氢杨梅素对胶质瘤细胞株U87增值凋亡及PI3K/Akt通路的影响

Effects of Dihydromyricetin on the Apoptosis and PI3K/Akt Pathway of Glioma Cell Line U87

目的:探讨二氢杨梅素(DMY)对胶质瘤细胞株U87增殖、凋亡、自噬及磷脂酰肌醇-3激酶/丝-苏氨酸蛋白激酶(PI3K/Akt)信号通路的影响。方法:体外培养胶质瘤U87细胞,U87细胞分为4组:空白对照组、DMY低(25μmol·L^-1)、中(50μmol·L^-1)、高(100μmol·L^-1)剂量组。采用MTT法检测U87细胞增殖情况,Hoechst染色法检测U87细胞凋亡,透射电镜下观察自噬体形成,蛋白印迹(WB)法检测凋亡蛋白B细胞淋巴瘤-2(Bcl-2)及其相关蛋白(Bax)、裂解的半胱氨酸天冬氨酸蛋白酶-3(cleaved caspase-3)、自噬相关蛋白Beclin-1、微管相关蛋白1轻链3(LC3)、P62及PI3K/Akt信号通路相关蛋白PI3K及其磷酸化激酶(p-PI3K)、Akt及其磷酸化蛋白(p-Akt)表达。结果:与空白对照组相比,DMY处理后U87细胞存活率、P62、Bcl-2、p-PI3K及p-Akt蛋白表达量均显著降低(P<0.05),且高剂量组显著低于低、中剂量组(P<0.05);与空白对照组相比,DMY处理后U87细胞凋亡率、自噬体数量、自噬泡/胞质总面积、LC3-Ⅱ/LC3-Ⅰ比值、LC3-Ⅱ、Beclin-1、Bax及cleaved caspase-3蛋白表达量均显著升高(P<0.05),且高剂量组显著高于低、中剂量组(P<0.05)。结论:二氢杨梅素可有效抑制胶质瘤U87细胞增殖,并可诱导自噬发生促使细胞凋亡,其作用机制可能与PI3K/Akt信号通路有关。

Objective:To investigate the effects of dihydromyricetin(DMY)on the proliferation,apoptosis,autophagy and phosphatidylinositol-3 kinase/serine threonine protein kinase(PI3K/Akt)signaling pathway of glioma cell line U87.Methods:U87 cells were cultured in vitro,and divided into four groups:the blank control group,DMY low(25μmol·L^-1),medium(50μmol·L^-1)and high(100μmol·L^-1)dose groups.MTT assay was used to detect the proliferation of U87 cells;the apoptosis of U87 cells was detected by Hoechst staining;the formation of autophages was observed under a transmission electron microscope;the expressions of apoptotic protein B cell lymphoma-2(Bcl-2)and its related protein(Bax),cleaved caspase-3,autophagy-related protein Beclin-1,microtubule-related protein 1 light chain 3(LC3),P62 and PI3K/Akt signal pathway-related protein PI3K and its phosphorylated kinase(p-PI3K),Akt and its phosphorylated protein(p-Akt)were detected by Western blot(WB).Results:Compared with those in the blank control group,the survival rate,P62,Bcl-2,p-PI3K and p-Akt protein expressions in U87 cells after DMY treatment were significantly decreased(P<0.05),and those in the high dose group were significantly lower than those in the low and middle dose groups(P<0.05).Compared with those in the blank control group,the apoptotic rate of U87 cells,number of autophages,total area of autophagic vesicles/cytoplasm,LC3-Ⅱ/LC3-I value and expressions of LC3-Ⅱ,Beclin-1,Bax and cleaved caspase-3 proteins were significantly increased after DMY treatment(P<0.05),and those in the high dose group were significantly higher than those in the low and middle dose groups(P<0.05).Conclusion:Dihydromyricetin can effectively inhibit the proliferation,induce the autophagy and promote the apoptosis of glioma U87 cells,and its mechanism may be related to PI3K/Akt signaling pathway.