熊果酸对胃癌细胞株MGC-803 凋亡和自噬的调控及其作用机制

摘要] 目的:观察熊果酸(UA)对胃癌细胞株MGC-803 自噬和凋亡的调控作用，并探讨UA基于PI3K/AKT/mTOR信号通路诱发MGC-803 细胞自噬的机制。方法:体外培养人胃癌细胞株MGC-803，分为空白对照组、UA干预组和UA+3-MA组。采用流式细胞术检测各组细胞凋亡水平，双荧光mRFP-eGFP-LC3 质粒转染细胞法检测各组细胞自噬发生情况，qPCR实验检测各组细胞LC3B、BAX、Bcl-2 mRNA的表达水平,WB实验检测各组细胞Ⅰ型PI3K、p-AKT、p-mTOR、ULK1、LC3B、BAX、Bcl-2 蛋白的表达水平。结果: 与空白对照组相比，UA干预组细胞凋亡率显著增加（P<0.05）；与UA干预组相比，UA+3-MA组细胞凋亡率显著降低（P<0.05）。双荧光mRFP-eGFP-LC3 质粒转染法显示，与空白对照组相比，UA 干预组绿色和红色荧光亮点数均显著增加（P<0.05）；与UA干预组相比，UA+3-MA组绿色和红色荧光亮点数均显著减少（P<0.05）。qPCR和WB实验结果显示，与空白对照组相比，UA干预组BAX和LC3B mRNA和蛋白以及ULK1 蛋白表达水平均显著上调，Bcl-2 基因和蛋白表达水平以及Ⅰ型PI3K、p-AKT、p-mTOR蛋白表达水平均显著下调（均P<0.05）；与UA干预组相比，UA+3-MA组BAX、LC3B mRNA和蛋白表达水平显著下调，Bcl-2 基因和蛋白表达水平显著上调（均P<0.05），Ⅰ型PI3K、p-AKT、p-mTOR 和ULK1 蛋白表达水平无显著差异（P>0.05）。结论: UA诱导的自噬可以促进胃癌细胞MGC-803 的凋亡，其机制可能与UA参与调控PI3K/AKT/mTOR信号通路相关蛋白表达有关。

Ursolic acid regulates apoptosis and autophagy of gastric cancer cell line MGC-803 and its mechanism

Abstract] Objective: To observe the effect of ursolic acid (UA) on autophagy and apoptosis of gastric cancer cell line MGC-803, and to explore the mechanism of UA-induced autophagy of MGC-803 cells based on PI3K/AKT/mTOR signaling pathway. Methods: Human gastric cancer cell line MGC-803 was cultured in vitro and divided into blank control group, UA intervention group and UA+3-MA group. The cell apoptosis in each group was detected by flow cytometry. Cell autophagy was detected by double fluorescence mRFPeGFP-LC3 plasmid transfection method. The mRNA expression levels of LC3B, BAX and Bcl-2 were detected by qPCR. The protein expression levels of PI3K type I, p-AKT, p-mTOR, ULK1, LC3B, BAX and Bcl-2 were detected by WB. Results: Flow cytometry showed that the cell apoptotic rate of UA intervention group was significantly higher than that of blank control group (P<0.05). Compared with UA intervention group, the apoptotic rate in UA + 3-MA group was significantly reduced (P<0.05). The double fluorescence mRFP-eGFP-LC3 plasmid transfection method showed that the green and red fluorescent bright spots in UA intervention group increased significantly compared with the blank control group (P<0.05), and the green and red fluorescent bright spots in UA+3-MA group were significantly reduced compared with UA intervention group (P<0.05). Real-time quantitative PCR and WB method showed that compared with the blank control group, the mRNA and protein expressions of BAX and LC3B, and ULK1 protein were significantly increased in UA intervention group, while the mRNA and protein expressions of Bcl-2, and the protein expressions of PI3K, p-AKT and p-mTOR were significantly decreased in UA intervention group (all P<0.05); Compared with UA intervention group, mRNA and protein expressions of BAX and LC3B were significantly down-regulated and the mRNA and protein expressions of Bcl-2 were significantly up-regulated in UA+3-MA group (all P<0.05), while protein levels of PI3K, p-AKT, p-mTOR and ULK1 were not significantly changed in UA+3-MA group (P>0.05). Conclusion: UA can promote apoptosis of MGC-803 cells via inducing autophagy, which may be related to UA''s involvement in regulating the expressions of PI3K/AKT/mTOR signaling pathway-related proteins.