自噬在红景天甙诱导胃癌细胞凋亡中的作用及机制

Effect and mechanism of autophagy on apoptosis induced by salidroside in gastric cancer cells

目的:探讨自噬在红景天甙诱导人胃癌细胞凋亡中的作用及机制。方法:红景天甙处理人胃癌AGS细胞,Hoechst33342染色观察凋亡形态学变化,流式细胞术检测凋亡细胞数;透射电镜观察自噬体形成,mRFP-GFP-LC3转染观察荧光自噬斑点;Western blot观察凋亡蛋白、自噬蛋白及PI3K/AKT/mTOR通路蛋白表达;自噬抑制剂氯喹(CQ)预处理AGS细胞,流式细胞术检测凋亡细胞数,Western blot观察凋亡相关蛋白变化,Hoechst33342染色观察凋亡形态学变化;PI3K/AKT激动剂胰岛素样生长因子(IGF-1)预处理AGS细胞,mRFP-GFP-LC3转染和Western blot分别观察自噬变化。结果:红景天甙诱导人胃癌AGS细胞自噬体形成,荧光自噬斑点增多,抑制PI3K/AKT/mTOR通路蛋白活化;CQ预处理胃癌细胞后,细胞凋亡数量显著增加,细胞核固缩现象增强,抗凋亡蛋白Bcl-2表达下调,促凋亡蛋白Bax、cleaved-Caspase3、cleaved-Caspase9表达上调;IGF-1预处理胃癌细胞明显减弱红景天甙诱导的自噬现象。结论:中药红景天甙可诱导人胃癌AGS细胞自噬,与抑制PI3K/AKT/mTOR通路相关,抑制自噬可促进红景天甙诱导的细胞凋亡。

Objective:To investigate effect and mechanism of autophagy on apoptosis of human gastric cancer cells induced by salidroside.Methods:After exposure of human gastric cancer AGS cells to salidroside,apoptosis morphological changes were observed by Hoechst33342 nuclear staining,and number of apoptotic cells was detected by flow cytometry.Autophagosome was observed by transmission electron microscope,and fluorescence autophagy spots were observed by mRFP-GFP-LC3 transfection.Expressions of apoptotic proteins,autophagy proteins and PI3K/AKT/mTOR pathway proteins were observed by Western blot.After AGS cells were pretreated with autophagy inhibitor chloroquine(CQ),apoptotic cell number was detected by flow cytometry,changes of apoptotic related proteins were observed by Western blot,and apoptosis morphological changes were observed by Hoechst33342 nuclear staining.After AGS cells were pretreated with PI3K/AKT agonist insulin-like growth factor 1(IGF-1),autophagy was observed by mRFP-GFP-LC3 transfection and Western blot.Results:Salidroside induces formation of autophagosomes in human gastric cancer AGS cells,and increases number of fluorescent autophagy spots,inhibits activation of PI3K/AKT/mTOR pathway proteins.After CQ pretreatment,apoptosis of gastric cancer cells was significantly increased,nuclear shrinkage was enhanced,anti-apoptotic protein Bcl-2 was down-regulated,pro-apoptotic protein Bax,cleaved-Caspase3,cleaved-Caspase9 were up-regulated.Pretreatment of AGS cells with IGF-1 significantly reduced salidroside-induced autophagy.Conclusion:Salidroside could induce autophagy in human gastric cancer AGS cells,which is associated with inhibition of PI3K/AKT/mTOR pathway.Inhibition of autophagy could promote apoptosis mediated by salidroside.