葫芦素B诱导细胞铁死亡抑制乳腺癌4T1细胞增殖作用及机制  

Role and Mechanism of Cucurbitacin B in Suppressing Proliferation of Breast Cancer 4T1 Cells via Inducing Ferroptosis

作　　者：阮意丹 张慧中 黄华婷 张萍芝 姚爱娜 张永强[1] 徐晓涵 李诗曼 倪健[1] 董晓旭[1] RUAN Yidan;ZHANG Huizhong;HUANG Huating;ZHANG Pingzhi;YAO Aina;ZHANG Yongqiang;XU Xiaohan;LI Shiman;NI Jian;DONG Xiaoxu(School of Chinese Material Medica,Beijing University of Chinese Medicine,Beijing 102488,China)

机构地区：[1]北京中医药大学中药学院,北京102488

出　　处：《中国实验方剂学杂志》2025年第7期91-97,共7页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家中医药管理局高水平建设学科项目(zyyzdxk-2023272)。

摘　　要：目的:探讨葫芦素B(CuB)诱导4T1细胞发生铁死亡作用及其机制。方法:采用噻唑蓝(MTT)比色法检测CuB(0.2、0.4、0.8μmol·L^(-1))对4T1细胞体外增殖能力的影响;平板克隆实验检测4T1细胞克隆形成能力;利用试剂盒检测4T1细胞中乳酸脱氢酶(LDH)的含量;流式细胞仪检测4T1细胞中线粒体膜电位和活性氧(ROS)水平;透射电镜观察4T1细胞的线粒体超微结构;采用蛋白免疫印迹法检测4T1细胞中铁死亡相关蛋白p53、溶质载体家族7成员11(SCL7A11)、谷胱甘肽过氧化物酶4(GPX4)、长链脂酰辅酶A合成酶4(ACSL4)、转铁蛋白受体1(TFR1)、铁蛋白重链1(FTH1)表达的情况。结果:与空白组比较,CuB各给药组4T1细胞存活率明显下降(P<0.05),细胞克隆数显著减少(P<0.01),细胞LDH的泄漏显著增多(P<0.01),细胞线粒体膜电位显著下降(P<0.01),细胞ROS水平显著提升(P<0.01)。与空白组比较,CuB各给药组细胞线粒体明显皱缩,线粒体嵴减少甚至消失。与空白组比较,CuB各给药组细胞p53、ACSL4、TFR1蛋白表达明显上调(P<0.05),SLC7A11、GPX4、FTH1蛋白表达明显下调(P<0.05)。结论:CuB可能通过上调p53表达,抑制SLC7A11和GPX4表达,进而调控p53/SLC7A11/GPX4信号通路轴;通过上调ACSL4表达,加速脂质过氧化底物的产生,同时上调TFR1表达促进细胞摄取Fe^(3+),下调FTH1的表达,降低储铁能力,使细胞内的游离Fe^(2+)水平上升,催化芬顿反应,产生过量的ROS,使抗氧化系统和铁代谢失衡,进而诱导4T1细胞铁死亡。Objective:To explore the role of cucurbitacin B(CuB)in inducing ferroptosis in 4T1 cells and its mechanism.Methods:The effects of CuB(0.2,0.4,0.8μmol·L^(-1))on the proliferation ability of 4T1 cells in vitro were detected using the methyl thiazolyl tetrazolium(MTT)assay.The clonogenic ability of 4T1 cells was detected by the plate cloning assay,and the levels of lactate dehydrogenase(LDH)in 4T1 cells were detected by the use of a kit.The mitochondrial membrane potential and reactive oxygen species(ROS)levels in 4T1 cells were detected by flow cytometry,and the mitochondrial ultrastructure of 4T1 cells was observed by transmission electron microscopy.The western blot was used to detect the expression of ferroptosis-related protein p53 in 4T1 cells,solute carrier family 7 member 11(SCL7A11),glutathione peroxidase 4(GPX4),long-chain acyl-CoA synthetase 4(ACSL4),transferrin receptor protein 1(TFR1),and ferritin heavy chain 1(FTH1).Results:Compared with that in the blank group,the survival rate of 4T1 cells in CuB groups was significantly decreased(P<0.05),and the number of cell clones in CuB groups was significantly reduced(P<0.01).In addition,compared with that in the blank group,the leakage of LDH in cells in CuB groups was significantly increased(P<0.01),and the mitochondrial membrane potential of cells in CuB groups decreased significantly(P<0.01).Cellular ROS levels were significantly elevated in CuB groups(P<0.01).The mitochondria of cells in CuB groups were obviously wrinkled,and the mitochondrial cristae were reduced or even disappeared.Compared with that in the blank group,the protein expression of p53,ACSL4,and TFR1 were significantly up-regulated in CuB groups(P<0.05),and that of SLC7A11,GPX4,and FTH1 were significantly down-regulated(P<0.05).Conclusion:CuB may inhibit SLC7A11 and GPX4 expression by up-regulating the expression of p53,which in turn regulates the p53/SLC7A11/GPX4 signaling pathway axis and accelerates the generation of lipid peroxidation substrate by up-regulating the expression of ACSL4.

关 键 词：乳腺癌 葫芦素B 铁死亡 机制 P53 

分 类 号：R2-031[医药卫生—中西医结合] R285.5[医药卫生—中医学] R737.9