摘要
目的观察隐丹参酮在人肝癌HepG2细胞铁死亡中的作用。方法体外培养HepG2细胞,采用CCK-8法检测细胞活力并计算半数抑制浓度(IC50),倒置相差显微镜观察细胞形态,DCFH-DA探针检测活性氧(ROS)水平变化,谷胱甘肽(GSH)测定试剂盒检测GSH水平变化,Western blot法检测铁死亡相关蛋白胱氨酸谷氨酸逆转运体轻链蛋白(xCT)和谷胱甘肽过氧化物酶4(GPX4)蛋白表达。以铁死亡抑制剂Ferrostain-1(Fer-1)、铁螯合剂去铁胺(DFO)、ROS清除剂乙酰半胱氨酸(NAC)进行干预,同样检测细胞活力、ROS水平、GSH水平及xCT和GPX4表达。结果隐丹参酮可显著抑制HepG2细胞活力,并引起HepG2细胞形态学变化和死亡,IC50为93.73μmol/L。隐丹参酮可显著诱导HepG2细胞ROS累积,降低GSH水平,并下调xCT和GPX4表达。Fer-1、DFO、NAC均可不同程度恢复隐丹参酮引起的HepG2细胞活力下降。Fer-1可抑制隐丹参酮诱导的ROS累积,并恢复GSH水平及xCT和GPX4表达。结论隐丹参酮可能通过抑制GPX4和xCT表达使HepG2细胞中ROS累积,导致细胞发生铁死亡。
Objective To observe the effect of cryptotanshinone on the ferroptosis of human liver cancer HepG2 cells.Methods The viability of the HepG2 cells cultured in vitro was determined using the Cell Counting Kit-8(CCK-8),and the half maximal inhibitory concentration(IC50)was calculated.The cell morphology was observed using an inverted microscope.The reactive oxygen species(ROS)level was detected with the 2’,7’-dichlorodihydrofluorescein diacetate(DCFH-DA)probe.The glutathione(GSH)assay kit was used to determine the GSH level.Western blot analysis was employed to detect the expression of cystine/glutamate antiporter system light chain(xCT)and glutathione peroxidase 4(GPX4),two marker proteins in ferroptosis.Additionally,the cell viability,ROS level,GSH level,and the expression levels of xCT and GPX4 were detected for the cells treated with the ferroptosis inhibitor ferrostain-1(Fer-1),the iron chelator deferoxamine(DFO),and the ROS scavenger N-acetylcysteine(NAC).Results Cryptotanshinone significantly inhibited the cell viability of HepG2 cells with an IC50 of 93.73μmol/L,and caused the morphological changes and death of the cells.It could significantly induce ROS accumulation,reduce GSH level,and down-regulate the expression of xCT and GPX4 in HepG2 cells.Fer-1,DFO,and NAC can remedy the cryptotanshinone-caused decrease in the cell viability of HepG2 cells.Fer-1 could inhibit cryptotanshinone-induced ROS accumulation,restore GSH level,and recover the expression of xCT and GPX4.Conclusion Cryptotanshinone may increase the accumulation of ROS by inhibiting the expression of xCT and GPX4 to induce the ferroptosis of HepG2 cells.
作者
刘金丽
佟雷
罗烨
高月娟
LIU Jinli;TONG Lei;LUO Ye;GAO Yuejuan(Department of Liver Diseases and Infectious Diseases,Affiliated Hongqi Hospital,Affiliated Hongqi Hospital,Mudanjiang Medical University,Mudanjiang,Heilongjiang 157011,China;College of Pharmacy,Affiliated Hongqi Hospital,Mudanjiang Medical University,Mudanjiang,Heilongjiang 157011,China;Department of Clinical Pharmacy,Affiliated Hongqi Hospital,Mudanjiang Medical University,Mudanjiang,Heilongjiang 157011,China)
出处
《中国医学科学院学报》
CAS
CSCD
北大核心
2021年第3期366-370,共5页
Acta Academiae Medicinae Sinicae
基金
黑龙江省卫生计生委科研基金(2019-00810)。
关键词
隐丹参酮
肝癌
铁死亡
细胞活力
cryptotanshinone
liver cancer
ferroptosis
cell viability
