维生素E琥珀酸酯处理人胃癌细胞过程中自噬与活性氧蓄积的交互作用

目的：探讨维生素E琥珀酸酯（VES）处理人胃癌SGC-7901细胞过程中自噬与活性氧（ROS）蓄积是否存在交互作用。方法：不同剂量（0、5、10、15、20 μg/mL）VES处理人胃癌SGC-7901细胞24 h，采用免疫荧光染色观察细胞内自噬标志蛋白微管相关蛋白l轻链3（LC3）的荧光强度和分布情况，Western blot检测自噬标志蛋白LC3和Beclin-1的表达情况，流式细胞术检测细胞内ROS水平；用ROS淬灭剂N-乙酰半胱氨酸（NAC）预处理2 h后以20 μg/mL VES处理人胃癌SGC-7901细胞24 h，免疫荧光染色观察细胞内LC3荧光强度和分布情况，Western blot检测LC3和Beclin-1的表达情况；RNA瞬时干扰阻断BECN-1基因的表达后，以20 μg/mL VES处理人胃癌SGC-7901细胞24 h，流式细胞术检测细胞内ROS水平。结果：与对照组相比，随着VES作用剂量的增加，LC3的荧光强度逐渐增强；LC3和Beclin-1蛋白表达均逐渐升高；流式细胞术检测结果显示胞内ROS水平逐渐增强；淬灭ROS之后LC3荧光强度以及LC3和Beclin-1蛋白表达水平均明显低于VES单独处理组（P<0.05）；阻断自噬关键基因BECN-1后细胞内ROS水平高于VES单独处理组（P<0.05）。结论：VES可诱导人胃癌细胞发生自噬及ROS蓄积，并且VES处理人胃癌细胞过程中自噬与ROS水平可以相互调节，具有交互作用。

Induction of autophagy and reactive oxygen by vitamin E succinate in human gastric carcinoma cells

OBJECTIVE: To investigate the expression of autophagy and accumulation of reactive oxygen species (ROS) after human gastric cancer SGC-7901 cells were treated with vitamin E succinate (VES). METHODS: Human gastric cancer SGC-7901 cells were treated with VES at different doses (0,5,10,15,20 μg/mL) for 24 h. Then,laser confocal microscopy was used to observe fluorescence intensity and distribution of the autophagy marker protein:microtubule-associated protein 1 light chain 3 (LC3). Western blot was used to evaluate the expression levels of LC3 and Beclin-1. Flow cytometry was used to detect the level of intracellular ROS. In addition,cells exposed to 20 μg/mL VES for 24 h with or without antioxidant N-acetyl-L-cysteine (NAC,20 mmol/L),were observed for fluorescence intensity and distribution of LC3. Western blot was used to evaluate levels of LC3 and Beclin-1. Furthermore,cells with BECN-1 knocked-down by RNA interference were treated with 20 μg/mL VES for 24 h. Then,flow cytometry was used to detect the level of intracellular ROS. RESULTS: Compared with the control group,VES treatment increased the amount of intracellular LC3 punctate fluorescence and the number of autophagic vacuoles,and increased the expression of endogenous autophagy marker protein LC3 and Beclin-1. Flow cytometry showed that the level of intracellular ROS was gradually increased. When ROS production was reduced by the antioxidant NAC,the fluorescence intensity of LC3,the expression of LC3 and Beclin-1 were significantly lower compared with VES-treatment alone. After the cells were transfected with BECN-1 siRNA,the level of intracellular ROS was higher compared with VES alone. CONCLUSION: VES induced autophagy and accumulation of ROS in human gastric cancer SGC-7901 cells. In addition,there were interactions between autophagy and ROS level in VES-treated cells.