土贝母苷乙通过诱导铁自噬抑制非小细胞肺癌细胞增殖  

Tubeimoside II inhibits proliferation of non-small-cell lung cancer cells by inducing ferritinophagy

作　　者：杨翘伊 张春云 孙硕 李文敏 黄鑫 梁艳[1] 张伟伟[1,2] 李怀永 杨清竹[1,2] YANG Qiaoyi;ZHANG Chunyun;SUN Shuo;LI Wenmin;HUANG Xin;LIANG Yan;ZHANG Weiwei;LI Huaiyong;YANG Qingzhu(College of Life Science and Agroforestry,Qiqihar University,Qiqihar 161006,China;Heilongjiang Provincial Key Laboratory of Resistance Gene Engineering and Protection of Biodiversity in Cold Areas,Qiqihar University,Qiqihar 161006,China;Department of Radiotherapy,The First Hospital of Qiqihar,Qiqihar 161000,China)

机构地区：[1]齐齐哈尔大学生命科学与农林学院,黑龙江齐齐哈尔161006 [2]抗性基因工程与寒地生物多样性保护黑龙江省重点实验室,黑龙江齐齐哈尔161006 [3]齐齐哈尔市第一医院放疗科,黑龙江齐齐哈尔161000

出　　处：《中国病理生理杂志》2024年第10期1834-1843,共10页Chinese Journal of Pathophysiology

基　　金：黑龙江省自然科学基金(联合引导项目)(No.LH2023H104);黑龙江省省属高等学校基本科研业务费科研项目(No.135509222);2023年齐齐哈尔大学研究生创新科研项目(No.QUZLYS_CX2023010)。

摘　　要：目的:探讨土贝母苷乙(tubeimosideⅡ,TBMSⅡ)诱导非小细胞肺癌细胞铁死亡的作用及潜在的分子机制。方法:体外培养非小细胞肺癌细胞H460,MTT法检测不同TBMSⅡ剂量处理的H460细胞的存活率,根据药物对细胞的抑制率的计算结果,选择细胞存活率50%的TBMSⅡ剂量为参照进行后续实验;Transwell实验检测细胞的迁移能力,克隆形成实验检测TBMSⅡ对H460细胞增殖的影响。流式细胞仪及荧光显微镜检测H460细胞中脂质活性氧(lipid reactive oxygen species,lipid ROS)水平的变化;试剂盒检测细胞中谷胱甘肽(glutathione,GSH)、总抗氧能力(T-AOC)、丙二醛(MDA)和Fe2+水平;Western blot法检测细胞中谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)、溶质载体家族7成员11(solute carrier family 7 membrane 11,SLC7A11)、铁蛋白重链1(ferritin heavy chain 1,FTH1)、核受体辅激活因子4(nuclear receptor coactivator 4,NCOA4)、自噬蛋白P62和微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)的表达;透射电镜检测细胞中线粒体结构的变化;免疫荧光实验检测细胞中LC3分别与FTH1和NCOA4的共定位,同时检测LC3和NCOA4与溶酶体的共定位。结果:与对照组比较,随着TBMSⅡ药物浓度增大,H460细胞的存活率、迁移能力以及克隆形成数量逐渐降低且细胞内部出现空泡;H460细胞中加入TBMSⅡ作用后,细胞中GSH和T-AOC水平降低而MDA水平升高,并且细胞中抗氧化蛋白SLC7A11和GPX4蛋白表达下降;H460细胞中的lipid ROS水平以及Fe2+随着TBMSⅡ浓度的升高而升高,而TBMSⅡ诱导的细胞死亡能够被铁死亡抑制剂Fer-1所逆转;自噬标志蛋白LC3Ⅱ/LC3 I和P62的水平随着TBMSⅡ浓度的增加而升高,且TBMSⅡ对H460细胞的线粒体形态产生影响,并且随着TBMSⅡ浓度的增加细胞中线粒体荧光强度降低;H460细胞中NCOA4蛋白的表达随着TBMSⅡ浓度增加而增加,但FTH1蛋白表达则随着TBMSⅡ浓度的增加而降低AIM:This study aimed to explore the induction of ferroptosis in non-small-cell lung cancer(NSCLC)cells by tubeimoside II(TBMS II)and to elucidate the underlying molecular mechanisms.METHODS:H460 NSCLC cells were cultured in vitro.Cell survival rates were assessed by using MTT assays,and doses of TBMS II resulting in below 50%survival were selected for further experimentation.Cell migration was evaluated using Transwell assays and the effects of TBMS II on H460 cell proliferation were assessed by colony formation assays.Flow cytometry and fluorescence microscopy were used to assess changes in lipid peroxidation(lipid ROS),and the levels of GSH,T-AOC,MDA,and Fe2+were measured using commercial kits.Protein levels of GPX4,SLC7A11,FTH1,NCOA4,P62,and LC3 were examined using Western blot.Changes in mitochondrial structure were detected by transmission electron microscopy,and immunofluorescence was used to assess LC3 co-localization of FTH1 and NCOA4,as well as co-localization of LC3 and NCOA4 with lysosomes.RESULTS:Compared with the control group,TBMS II dose-dependently reduced H460 cell viability,migration,and clone formation,accompanied by the appearance of vacuoles within the cells.TBMS II treatment also led to decreased GSH and T-AOC levels,while increasing the cellular contents of MDA,indicating oxidative stress.Additionally,there was a decrease in the expression of the antioxidant proteins SLC7A11 and GPX4 in the cells,while lipid ROS and Fe2+levels were increased in proportion to the TBMS II concentration.The ferroptosis inhibitor ferrostatin-1 reversed cell death caused by TBMS II,suggesting ferroptosis induction.Furthermore,increasing the TBMS II concentration resulted in an upregulation of the autophagy marker proteins LC3 II/LC3 I and P62,indicative of increased autophagy.TBMS II also affected mitochondrial morphology in the cells,as seen in reduced mitochondrial fluorescence intensity.Protein expression of NCOA4 increased with higher TBMS II concentrations,while that of FTH1 decreased.Co-localization of LC3

关 键 词：土贝母苷乙 非小细胞肺癌 核受体辅激活因子4 铁自噬 铁死亡 

分 类 号：R363.2[医药卫生—病理学] R285[医药卫生—基础医学] R730.23