铁过载诱导成骨前体细胞铁死亡并抑制成骨分化  

Iron overload induces ferroptosis in osteoblast precursor cells and inhibits osteogenic differentiation

作　　者：潘玉 赵刃峰 李兴平 张成栋 匙峰 蒲超[2] 罗栩伟[2] 肖东琴[1,2] Pan Yu;Zhao Renfeng;Li Xingping;Zhang Chengdong;Shi Feng;Pu Chao;Luo Xuwei;Xiao Dongqin(Institute of Basic Medicine and Forensic Medicine,North Sichuan Medical College,Nanchong 637000,Sichuan Province,China;Research Institute of Tissue Engineering and Stem Cells,Nanchong Central Hospital,Second Clinical College of North Sichuan Medical College,Nanchong 637000,Sichuan Province,China;Department of Orthopedics,Tongyong Medical Chengfei Hospital,Chengdu 610091,Sichuan Province,China;Collaboration Innovation Center for Tissue Repair Material Engineering Technology,China West Normal University,Nanchong 637002,Sichuan Province,China)

机构地区：[1]川北医学院基础医学与法医研究所,四川省南充市637000 [2]川北医学院第二临床学院,南充市中心医院组织工程与干细胞研究所,四川省南充市637000 [3]通用医疗成飞医院骨科,四川省成都市610091 [4]西华师范大学组织修复材料工程技术协同创新中心,四川省南充市637002

出　　处：《中国组织工程研究》2025年第30期6381-6390,共10页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金(82002289),项目负责人:肖东琴;四川省自然科学基金(2023NSFSC1740),项目负责人:张成栋;南充市市校合作科研项目(22SXJCQN0002),项目负责人:肖东琴;四川省医学科研课题计划(Q22061),项目负责人:蒲超;四川省医学科研课题计划(Q22034),项目负责人:李兴平;通用医疗科研基金项目(TYYLKYJJ-2022-051),项目负责人:李兴平。

摘　　要：背景:铁过载作为诱发骨质疏松的独立因素,其作用机制目前尚不清楚,因此探讨铁过载对成骨相关细胞的影响,有助于深入理解骨质疏松发病机制并为骨质疏松治疗提供潜在策略。目的:探讨铁过载环境对成骨前体细胞活性、铁死亡及成骨分化的影响。方法:①将成骨前体细胞(MC3T3-E1细胞)分为空白组、铁过载组、fer-1组和去铁胺组,铁过载组在培养基中添加300μmol/L柠檬酸铁铵作用48 h模拟铁过载微环境,fer-1组和去铁胺组细胞采用5μmol/L抗氧化剂fer-1与5μmol/L去铁胺分别预处理8 h,然后加入300μmol/L柠檬酸铁铵作用48 h,采用CCK-8法测定细胞活力,采用活性氧荧光探针检测细胞内活性氧水平,采用线粒体膜电位荧光探针检测细胞内线粒体膜电位变化,使用透射电镜观察线粒体形态变化,采用还原型谷胱甘肽比色法试剂盒检测细胞谷胱甘肽水平,采用丙二醛比色法试剂盒检测细胞内脂质氧化水平,采用亚铁离子比色法试剂盒检测细胞亚铁离子水平,采用碱性磷酸酶染色、茜素红染色验证细胞成骨及矿化能力,采用天狼星红染色检测胶原分泌能力,采用RT-qPCR及Western blot检测成骨/铁死亡相关基因及蛋白的表达。结果与结论:①铁过载环境下,细胞线粒体膜电位下降且结构受损,细胞内脂质过氧化水平升高,铁死亡抵抗相关基因与蛋白表达均降低;而fer-1与去铁胺预处理后,细胞内线粒体膜电位升高且形态部分恢复,细胞内脂质过氧化水平降低,铁死亡抵抗相关基因与蛋白表达均升高。②铁过载环境下,细胞碱性磷酸酶含量、矿化结节形成与胶原纤维生成均降低,fer-1与去铁胺预处理均能逆转此现象。③综上所述,铁过载会上调细胞内氧化应激水平,介导MC3T3-E1细胞发生铁死亡并抑制成骨分化,进而诱导骨质疏松。因此,维持铁稳态及抑制成骨相关细胞铁死亡可能是预防或治疗骨质疏松�BACKGROUND:Iron overload is an independent factor inducing osteoporosis,but the action mechanism is currently unclear.Therefore,exploring the effects of iron overload on osteoblast-related cells will help to deeply understand the pathogenesis of osteoporosis and provide potential strategies for osteoporosis treatment.OBJECTIVE:To explore the effects of iron overload environment on osteoblast precursor cell activity,ferroptosis,and osteogenic differentiation.METHODS:Osteoblast precursor cells(MC3T3-E1 cells)were divided into blank group,iron overload group,fer-1 group,and deferoxamine group.The iron overload group was treated with 300μmol/L ammonium ferric citrate in the culture medium for 48 hours to simulate the iron overload microenvironment.The cells in fer-1 group and deferoxamine group were pretreated with 5μmol/L antioxidant fer-1 and 5μmol/L deferoxamine for 8 hours,respectively,and then added with 300μmol/L ammonium ferric citrate for 48 hours.CCK-8 assay was used to determine the cell viability.Intracellular reactive oxygen species levels were detected employing a reactive oxygen species fluorescent probe.Changes in mitochondrial membrane potential were monitored with a mitochondrial membrane potential fluorescent probe.Mitochondrial morphology was observed employing transmission electron microscopy.Cellular glutathione levels were measured with a reduced glutathione colorimetric assay kit.Lipid peroxidation levels were assessed with a malondialdehyde colorimetric assay kit.Cellular ferrous ion levels were determined with a ferrous ion colorimetric assay kit.The osteogenic and mineralization capabilities of the cells were verified by alkaline phosphatase staining and alizarin red staining.Collagen secretion ability was detected using Sirius Red staining.The expression of osteogenic/ferroptosisrelated genes and proteins was examined through reverse transcription quantitative polymerase chain reaction and western blot analysis.RESULTS AND CONCLUSION:(1)In an iron-overload environment,the mitochondrial

关 键 词：铁过载 骨质疏松 铁死亡 柠檬酸铁铵 成骨前体细胞 线粒体功能:成骨分化 脂质过氧化 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R580