摘要
目的 探讨紫檀芪对人结肠癌细胞LoVo的作用,并研究核转录因子E2相关因子2(Nrf2)在紫檀芪作用LoVo细胞过程中的调控机制.方法 应用不同浓度的紫檀芪(5、10、20、40、60、80、100 pmol/L)处理LoVo细胞.CCK-8检测细胞活力,划痕实验检测细胞迁移,Transwell实验检测细胞侵袭,TUNEL染色检测细胞凋亡,JC-1检测线粒体膜电位水平,2',7'-二氯荧光素二乙酸酯检测活性氧水平,Western blot检测细胞内Nrf2、磷酸化的Nrf2、血红素加氧酶-1及凋亡蛋白(Bcl2、Bax)的蛋白表达.此外,联合应用Nrf2特异性激动剂萝卜硫素后,重复检测细胞活力、细胞凋亡率及Nrf2的蛋白表达.结果 与对照组相比,40、60、80、100 μmol/L紫檀芪均可显著降低细胞活性(P=0.014,P＜0.001,P＜0.001,P＜0.001).5、10、20 pmol/L紫檀芪对LoVo细胞活性无影响,但可显著抑制细胞迁移(P=0.008,P＜0.001,P＜0.001)和侵袭(P均＜0.001).TUNEL染色、JC-1、2',7'-二氯荧光素二乙酸酯染色结果显示40、60、80 μmol/L紫檀芪增加LoVo细胞凋亡(P=0.014,P＜0.001,P＜0.001),使线粒体膜电位去极化(P=0.026,P＜0.001,P＜0.001)并增加细胞内活性氧聚积(P均＜0.001).40、60、80 μmol/L紫檀芪下调了LoVo细胞中磷酸化的Nrf2(P=0.030,P＜0.001,P＜0.001)、血红素加氧酶-1(P=0.015,P＜0.001,P＜0.001)、Bc12(P=0.039,P＜0.001,P＜0.001)的蛋白表达;60、80 µmol/L紫檀芪降低了Nrf2的蛋白表达(P=0.001,P＜0.001),增加了Bax的蛋白表达(P均＜0.001).应用萝卜硫素联合处理后,紫檀芪抑制细胞活性(P＜0.001)、增加细胞凋亡(P＜0.001)及下调Nrf2表达(P=0.022)的作用明显减弱.结论 紫檀芪是一种有效抑制结肠癌细胞的化合物,其抗癌机制与抑制Nrf2通路有关.
Abstract
Objective To investigate the effects of pterostilbene on human colon cancer LoVo cells and study the regulatory mechanism of nuclear factor E2-related factor 2(Nrf2)in the process of pterostilbene acting on LoVo cells.Methods LoVo cells were treated with different concentrations(5,10,20,40,60,80,100 panol/L)of pterostilbene.Cell viability,migration,invasion,and apoptosis were examined by CCK-8,scratch,Tran-swell,and TUNEL assays,respectively.The mitochondrial membrane potential was measured by the mitochon-drial membrane potential assay kit with JC-1.The reactive oxygen species level was measured by 2',7'-dichlo-rofluorescein diacetate.The protein levels of Nrf2,phosphorylated Nrf2,heme oxygenase 1,and apoptotic pro-teins(Bcl2 and Bax)were determined by Western blotting.In addition,cell viability,Nrf2 expression,and ap-optosis rate were determined after co-application of the Nrf2-specific agonist sulforaphane.Results Compared with the control group,40,60,80,100 µmol/L pterostilbene reduced the viability of LoVo cells(P=0.014,P＜0.001,P＜0.001,P＜0.001).Pterostilbene at 5,10,20 µmol/L did not show effects on cell viability but inhibited cell migration(P=0.008,P＜0.001,P＜0.001)and invasion(all P＜0.001).Pterostilbene at 40,60,80 μmol/L increased apoptosis(P=0.014,P＜0.001,P＜0.001),promoted mitochondrial membrane potential depolarization(P=0.026,P＜0.001,P＜0.001)and reactive oxygen species accumula-tion(all P＜0.001),and down-regulated the expression of phosphorylated Nrf2(P=0.030,P＜0.001,P＜0.001),heme oxygenase 1(P=0.015,P＜0.001,P＜0.001),and Bc12(P=0.039,P＜0.001,P＜0.001)in LoVo cells.Pterostilbene at 60,80 µmol/L down-regulated Nrf2 expression(P=0.001,P＜0.001)and up-regulated Bax expression(both P＜0.001).The application of sulforaphane reversed the effects of pterostilbene on cell viability(P＜0.001),apoptosis(P＜0.001),and Nrf2 expression(P=0.022).Conclusion Pterostilbene is a compound that can effectively inhibit colon cancer cells by inhibiting the Nrf2 pathway.
基金项目
首都临床特色诊疗技术研究及转化应用(Z221100007422061)
空军特色医学中心博士助推基金(21ZT16)
2021年空军军医大学临床研究基金(2021LC2201)
国家科技期刊平台
NSTL
万方数据