圣草酚调控Akt/mTOR通路对胶质瘤细胞自噬的影响

Effect of eriodictyol on autophagy in glioma cells via Akt/mTOR signaling pathway

为研究圣草酚对胶质瘤细胞自噬的影响,探讨其通过调控Akt/mTOR通路在胶质瘤自噬过程中的作用。本研究采用CCK8和细胞克隆形成实验检测圣草酚对胶质瘤细胞增殖的影响。构建GFP-mRFP-LC3B稳定表达的细胞株,圣草酚(100μmol/L)处理48 h,观察自噬小体的形成情况;然后分别用圣草酚(100μmol/L)、雷帕霉素(200 nmol/L)以及圣草酚(100μmol/L)+雷帕霉素(200 nmol/L)处理48 h后观察自噬流的情况。利用蛋白免疫印迹法检测圣草酚对自噬及Akt/mTOR通路相关蛋白表达水平的影响。建立裸鼠皮下移植瘤模型,观察圣草酚在体内对胶质瘤自噬过程的影响。实验结果显示,圣草酚能够显著抑制胶质瘤细胞的增殖,且呈时间—剂量依赖性(P<0.05)。圣草酚使胶质瘤细胞中绿色自噬小体明显增多,LC3Ⅱ、Beclin 1、ATG5蛋白表达上调。圣草酚处理GFP-mRFP-LC3B稳转细胞株48 h后呈亮黄色,且减弱了雷帕霉素对自噬流的促进作用,并上调P62蛋白的表达。此外,圣草酚可抑制p-Akt、p-mTOR的表达,但该作用被Akt激动剂740 Y-P逆转。体内实验结果同体外一致。说明圣草酚能够抑制胶质瘤细胞的增殖和自噬流,其影响自噬过程的作用机制可能与下调Akt/mTOR通路有关。

This study was designed to investigate the effect of eriodictyol on glioma cell autophagy and its role in glioma autophagy by regulating the Akt/mTOR pathway.In this essay,CCK-8 assay and colony formation were conducted to evaluate the effects of eriodictyol on proliferation.The glioma cell lines with stable GFP-mRFP-LC3B expression were constructed to observe the autophagic flux.After treatment of eriodictyol(100μmol/L)for 48 h,autophagosomes were observed by fluorescent microscopy.Then,respectively treated with eriodictyol(100μmol/L),rapamycin(200 nmol/L),or eriodictyol(100μmol/L)+rapamycin(200 nmol/L)for 48 h,glioma cell lines were observed using fluorescent microscopy to ensure autophagic flux.The autophagy and AKT/mTOR pathway-related proteins were detected by immunoblotting.Moreover,A model of glioma subcutaneously transplant in nude mice was constructed to study the effects of eriodictyol on the autophagy of glioma in vivo.The results showed that the proliferation of glioma cells was significantly inhibited by eriodictyol in a dose and time-dependent manner(P<0.05).The formation of autophagosome and the expressions of LC3Ⅱ,Beclin 1,ATG5 proteins were enhanced by eriodictyol.After treatment with eriodictyol,the glioma cells expressing GFP-mRFP-LC3B appeared bright yellow.Eriodictyol weakened the effect of rapamycin on autophagic flux and increased P62 expression in glioma cells.Furthermore,the inhibition of p-Akt and p-mTOR proteins by eriodictyol in glioma cells was rescued by Akt agonist 740 Y-P.In vivo,similar results were obtained as in vitro.In summary,the proliferation and autophagic flux in glioma cells was inhibited by eriodictyol.The mechanism of eriodictyol affecting autophagy might be through the downregulation of Akt/mTOR pathway.