乳岩内消霜通过调控Nrf2/SLC7A11/GPX4信号通路促进乳腺癌前病变细胞铁死亡的机制  

Mechanism of Ruyan Neixiao Cream in Promoting Ferroptosis in Breast Precancerous Lesion Cells by Regulating Nrf2/SLC7A11/GPX4 Signaling Pathway

作　　者：张昊天 邱叶贝 苏燃 颜显欣 马民 ZHANG Haotian;QIU Yebei;SU Ran;YAN Xianxin;MA Min(School of Traditional Chinese Medicine,Jinan University,Guangzhou 510632,China;The First Affiliated Hospital,Jinan University,Guangzhou 510630,China)

机构地区：[1]暨南大学中医学院,广州510632 [2]暨南大学附属第一医院,广州510630

出　　处：《中国实验方剂学杂志》2025年第7期98-107,共10页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(82074430);中央高校基本科研业务费专项(21623122);广东省基础与应用研究基金项目(2022A1515011674,2024A1515012176,2024A1515011722);广州市科技计划项目(2024B03J1262,2024B03J1261);广东省中医药局科研项目(20241062)。

摘　　要：目的:探讨乳岩内消霜(RUC)诱导乳腺癌前病变(BPL)细胞铁死亡的作用机制,丰富RUC治疗BPL的理论基础。方法:通过细胞增殖与活性检测法(CCK-8)实验、克隆形成实验检测1%、2%和4%浓度RUC透皮液(RUT)对细胞增殖的抑制情况;使用DCFH-DA探针对活性氧(ROS)进行检测,使用相应试剂盒检测细胞内亚铁离子(Fe^(2+))、谷胱甘肽(GSH)和丙二醛(MDA)含量;采用脂质过氧化荧光探针C11-BODIPY^(581/591)检测脂质过氧化;通过蛋白免疫印迹法(Western blot)检测核因子E_(2)相关因子2(Nrf2)、溶质载体家族7成员11(SLC7A11)和谷胱甘肽过氧化物酶4(GPX4)蛋白的表达情况。使用二甲基苯并蒽(DMBA)联合雌孕激素构建BPL大鼠模型,同时使用RUC外用治疗,第12个周期后安乐死大鼠,通过苏木素-伊红(HE)染色观察乳腺组织病理学改变,使用相应试剂盒检测乳腺组织Fe^(2+)和MDA含量;通过免疫组化法(IHC)和Western blot检测BPL大鼠乳腺组织Nrf2、SLC7A11和GPX4蛋白的表达情况。结果:与基质组比较,1%、2%和4%RUT组MCF-10AT细胞活性明显降低(P<0.05),具有浓度依赖性,24 h半数抑制浓度(IC50)为2.23%;与4%RUT组比较,RUT+Fer-1组细胞活性明显提高(P<0.05)。与基质组比较,1%、2%和4%RUT组MCF-10AT细胞克隆形成率明显降低(P<0.05);与4%RUT组比较,RUT+Fer-1组细胞克隆形成率明显增高(P<0.05)。与基质组比较,1%、2%和4%RUT组细胞内ROS和Fe^(2+)水平明显增高(P<0.05),GSH水平明显降低(P<0.05),MDA和脂质过氧化水平明显增高(P<0.05);与4%RUT组比较,RUT+Fer-1组细胞内ROS和Fe^(2+)水平明显降低(P<0.05),GSH水平明显增高(P<0.05),MDA和脂质过氧化水平明显降低(P<0.05)。与基质组比较,1%、2%和4%RUT组MCF-10AT细胞内Nrf2、SLC7A11和GPX4蛋白表达水平明显降低(P<0.05);与4%RUT组比较,RUT+Fer-1组细胞内Nrf2、SLC7A11和GPX4蛋白表达水平明显增高(P<0.05)。在体内实验中,与基质组比较,RUC组BPL大鼠乳腺组织病理状态有效改�Objective:To explore the mechanism by which Ruyan Neixiao cream(RUC)induces ferroptosis in breast precancerous lesion(BPL)cells,and to enrich the theoretical foundation for its use in the treatment of BPL.Methods:The inhibition of cell proliferation by 1%,2%,and 4%concentrations of Ruyanneixiao Cream transdermal solution(RUT)was assessed using cell counting kit-8(CCK-8)and a colony formation assay.Reactive oxygen species(ROS)were measured using the DCFH-DA probe,and the levels of ferrous ions(Fe^(2+)),glutathione(GSH),and malondialdehyde(MDA)were determined using appropriate kits.Lipid peroxidation was detected with the C11-BODIPY^(581/591) fluorescent probe.The expression of nuclear factor E_(2)-related factor 2(Nrf2),solute carrier family 7 member 11(SLC7A11),and glutathione peroxidase 4(GPX4)proteins was analyzed by Western blot.The BPL rat model was constructed using 2,2′-bis(hydroxymethyl)butyric acid(DMBA)combined with estrogen and progesterone,and the rats were treated with RUC for external application.After the 12th cycle,the rats were euthanized,and histopathological changes in breast tissue were observed by hematoxylin-eosin(HE)staining.Fe^(2+)and MDA levels in breast tissue were measured using corresponding kits.The expression of Nrf2,SLC7A11,and GPX4 proteins in BPL rat breast tissue was detected by immunohistochemistry(IHC)and Western blot.Results:Compared with the matrix group,the cell viability of MCF-10AT cells in the 1%,2%,and 4%RUT groups was significantly reduced(P<0.05)in a concentration-dependent manner,with the 24-hour half inhibitory concentration(IC50)being 2.23%.Compared with the 4%RUT group,cell viability in the RUT+Fer-1 group was significantly increased(P<0.05).Compared with the matrix group,the colony formation rates of MCF-10AT cells in the 1%,2%,and 4%RUT groups were significantly decreased(P<0.05).Compared with the 4%RUT group,the cell colony formation rate of the RUT+Fer-1 group was significantly increased(P<0.05).Compared with the matrix group,the levels of ROS and Fe^(2+)in th

关 键 词：乳腺癌前病变 乳岩内消霜 铁死亡 脂质过氧化 核因子E_(2)相关因子2(Nrf2) 

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