内质网应激在维生素E琥珀酸酯诱导的胃癌细胞凋亡和自噬中作用的研究  

Effects of endoplasmic reticulum stress on apoptosis and autophagy of gastric cancer cells induced by vitamin E succinate

作　　者：赵海霞 段宵阳 卓翠竹 闻新奕 侯丽颖 ZHAO Hai-xia;DUAN Xiao-yang;ZHUO Cui-zhu;WEN Xin-yi;HOU Li-ying(North China University of Science and Technology,Tangshan,Hebei 063210,China)

机构地区：[1]华北理工大学公共卫生学院,河北唐山063210

出　　处：《现代预防医学》2023年第22期4170-4176,4200,共8页Modern Preventive Medicine

基　　金：河北省自然科学基金(H2019209453);中央引导地方科技发展资金项目(236Z7705G);华北理工大学大学生创新创业训练计划(X2022199);华北理工大学公共卫生学院青年人才托举计划(QNRC202305)。

摘　　要：目的明确内质网应激(endoplasmic reticulum stress,ERS)在维生素E琥珀酸酯(vitamin E succinate,VES)激活人胃癌MKN28细胞自噬和凋亡中发挥的作用。方法体外培养人胃癌MKN28细胞,不同剂量(5,10,15,20μg/ml)VES处理细胞24小时后,Western blot检测ERS标志物GRP78,自噬标志物LC3、Beclin-1,凋亡相关蛋白Caspase-3和ERS凋亡通路关键信号分子CHOP的表达情况,流式细胞仪检测不同剂量组凋亡率。选择ERS抑制剂4-苯基丁酸(4-phenylbutyric acid,4-PBA),ERS激活剂衣霉素(Tunicamycin,TM)自噬激活剂雷帕霉素(Rapamycin,RAPA),设置对照组、4-PBA组(3 mmol/L)、TM组(5μg/ml)、VES组(20μg/ml)、VES+4-PBA组、RAPA组(100 nmol/L),检测GRP78、LC3、Beclin-1、Caspase-3和CHOP蛋白的表达情况,同时检测不同组细胞凋亡率或在电子显微镜下观察自噬体形态。结果与对照组相比,随着VES作用剂量的上升,ERS标志蛋白GRP78、自噬相关蛋白Beclin-1、LC3Ⅱ和凋亡相关蛋白C-Caspase-3均有所增加(F=61.579,P<0.001;F=15.344,P<0.001;F=18.291,P<0.001;F=4.207,P<0.05)。凋亡率检测结果显示,与对照组相比,随着VES剂量的增加,各组凋亡率均上升(F=242.461,P<0.001)。透射电镜图像显示20μg/ml VES组、RAPA组以及TM组出现大量处于不同阶段的自噬体形态。抑制ERS后,VES+4-PBA组GRP78、Beclin-1、LC3Ⅱ/LC3I、C-Caspase-3/Pro Caspase-3较VES组均降低(F=18.799,P<0.001;F=6.643,P<0.01;F=3.492,P<0.05;F=9.436,P<0.01);凋亡率检测结果显示,与VES组相比,VES+4-PBA组的细胞凋亡率降低(F=193.546,P<0.001)。检测ERS凋亡通路关键蛋白CHOP,与对照组相比,随着VES作用剂量的上升,CHOP的表达增加(F=19.720,P<0.001);抑制ERS后,CHOP的表达较VES组降低(F=9.703,P<0.01)。结论VES可激活人胃癌MKN28细胞发生ERS,诱导胃癌细胞发生自噬和凋亡,ERS能促进细胞自噬的发生且可能通过激活CHOP凋亡途径促进VES诱导的细胞凋亡发生。Objective To determine whether vitamin E succinate(VES)can activate endoplasmic reticulum stress(ERS)in human gastric cancer MKN28 cells and induce autophagy and apoptosis,and to explore the role of endoplasmic reticulum stress in VES-induced autophagy and apoptosis.Methods Human gastric cancer MKN28 cells were cultured in vitro,and treated with VES at different doses(5,10,15,20μg/ml)for 24 hours.Western blot was used to detect the expression of ERS marker GRP78,autophagy markers LC3,Beclin-1,apoptosis related protein Caspase-3,and the key signaling molecule CHOP of ERS apoptosis pathway.Flow cytometry was used to detect the apoptosis rate of different dose groups.We selected the ERS inhibitor 4-phenylbutyric acid(4-PBA),ERS activator Tunicamycin(TM),and autophagy activator Rapamycin(RAPA),and set the control group,4-PBA group(3 mmol/L),TM group(5μg/ml),VES group(20μg/ml),VES+4-PBA group,RAPA group(100 nmol/L),and then detect the expression of GRP78,LC3,Beclin-1,Caspase-3,and CHOP proteins.Simultaneously we detected the apoptosis rate of different groups of cells or observe the morphology of autophagosomes under an electron microscope.Results In the dose group,the ERS marker protein GRP78,autophagy related protein Beclin-1,LC3 II,apoptosis related protein C-Caspase-3 and a key signal molecule in ERS apoptosis pathway CHOP all increased with the increase of VES dose(F=61.579,P<0.001;F=15.344,P<0.001;F=18.291,P<0.001;F=4.207,P<0.05;F=19.720,P<0.001).The apoptosis rate test results showed that with the increase of VES dose,compared with the control group,the apoptosis rate of each group increased(F=242.461,P<0.001).Transmission electron microscopy images showed that the 20μg/ml VES group,RAPA group and TM group had many different sizes and different stages of autophagosome morphology.After inhibiting ERS,GRP78,Beclin-1,LC3Ⅱ/LC3Ⅰ,C-Caspase-3/Pro Caspase-3 and CHOP in VES+4-PBA group expression level was significantly lower than VES group(F=18.799,P<0.001;F=6.643,P<0.01;F=3.492,P<0.05;F=9.436,P<0.01;F=9.703,P<

关 键 词：维生素E琥珀酸酯 内质网应激 凋亡 自噬 胃癌细胞 

分 类 号：R113[医药卫生—公共卫生与预防医学]