黄芪甲苷联合BMSCs-Exos对PC12细胞铁死亡的影响  

Effects of astragaloside IV combined with BMSCs-Exos on ferroptosis in PC12 cells

作　　者：刘恩旭 聂颖[1,2] 段嘉豪 孙飞[1,2] 孙钰 杨少锋 杨雷[1,2] LIU Enxu;NIE Ying;DUAN Jiahao;SUN Fei;SUN Yu;YANG Shaofeng;YANG Lei(Hunan University of Chinese Medicine,Changsha 410208,China;The First Affiliated Hospital of Hunan University of Chinese Medicine,Changsha 410007,China)

机构地区：[1]湖南中医药大学,湖南长沙410208 [2]湖南中医药大学第一附属医院,湖南长沙410007

出　　处：《中草药》2024年第23期8056-8066,共11页Chinese Traditional and Herbal Drugs

基　　金：湖南省自然科学基金面上项目(2023JJ30472);湖南省教育厅重点课题(22A0261);湖南省卫生健康委科研项目(B202304078103);湖南省中医药管理局课题(B2023028)。

摘　　要：目的探讨黄芪甲苷联合骨髓间充质干细胞外泌体(bone marrow mesenchymal stem cells exosomes,BMSCs-Exos)对脊髓损伤细胞模型PC12细胞铁死亡的影响。方法采用过氧化氢(hydrogen peroxide,H_(2)O_(2))诱导PC12细胞构建脊髓损伤细胞模型,使用黄芪甲苷联合BMSCs-Exos进行干预。采用激光扫描共聚焦显微镜观察细胞对外泌体的摄取情况;流式细胞术检测细胞内活性氧(reactive oxygen species,ROS)水平;试剂盒测定丙二醛(malondialdehyde,MDA)、谷胱甘肽(glutathione,GSH)、亚铁离子(ferrous ion,Fe^(2+))和谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)水平;JC-1荧光染色检测线粒体膜电位与透射电镜观察线粒体形态;运用qRT-PCR和Western blotting检测铁死亡相关因子GPX4、铁蛋白重链1(ferritin heavy chain 1,FTH1)和铁转运蛋白(ferroportin,FPN)的表达情况。结果H2O2处理增加了细胞内ROS、MDA和Fe^(2+)水平(P<0.05),降低了GSH、GPX4、FTH1和线粒体膜电位(P<0.05),诱发线粒体形态改变和铁死亡特征。BMSCs-Exos和黄芪甲苷单独及联合干预均显著降低细胞内ROS、MDA和Fe^(2+)水平(P<0.05),提升FPN、GSH、GPX4和线粒体膜电位(P<0.05),改善线粒体形态、调节铁代谢及增强抗氧化能力。其中黄芪甲苷联合BMSCs-Exos处理效果更显著(P<0.05)。结论黄芪甲苷和BMSCs-Exos均能有效抑制脊髓损伤细胞模型中的细胞铁死亡,并显示出显著的神经保护作用。黄芪甲苷联合BMSCs-Exos不仅具有治疗脊髓损伤的潜力,还可能在涉及氧化应激和铁死亡的其他神经疾病和损伤中发挥治疗作用。Objective To explores the effects of astragalosideⅣcombined with bone marrow mesenchymal stem cells exosomes(BMSCs-Exos)on ferroptosis in PC12 cell model of spinal cord injury.Methods Hydrogen peroxide(H_(2)O_(2))was used to induce PC12 cells to construct an spinal cord injury cell model,with interventions by astragalosideⅣcombined with BMSCs-Exos.Laser scanning confocal microscopy was employed to observe the uptake of exosomes by the cells.Flow cytometry was used to detect intracellular reactive oxygen species(ROS)levels.Kits were utilized to measure levels of malondialdehyde(MDA),glutathione(GSH),ferrous ion(Fe^(2+)),and glutathione peroxidase 4(GPX4).JC-1 fluorescence staining was applied to detect mitochondrial membrane potential,and transmission electron microscopy was used to observe mitochondrial morphology.The expression levels of ferroptosis-related factors,including GPX4,ferritin heavy chain 1(FTH1),and ferroportin(FPN),were analyzed using qRT-PCR and Western blotting.Results H₂O₂treatment increased the levels of ROS,MDA,and Fe^(2+)(P<0.05),while decreasing GSH,GPX4,FTH1,and mitochondrial membrane potential(P<0.05),leading to mitochondrial morphological changes and characteristics of ferroptosis.Both BMSCs-Exos and astragalosideⅣ,individually and in combination,significantly reduced ROS,MDA,and Fe^(2+)levels(P<0.05),increased FPN,GSH,GPX4,and mitochondrial membrane potential(P<0.05),improved mitochondrial morphology,regulated iron metabolism,and enhanced antioxidant capacity.The combined treatment of astragalosideⅣand BMSCs-Exos showed a more significant effect(P<0.05).Conclusion AstragalosideⅣand BMSCs-Exos can effectively inhibit ferroptosis in the spinal cord injury cell model and demonstrate significant neuroprotective effects.The combination of astragalosideⅣand BMSCs-Exos not only holds potential for the treatment of spinal cord injury but may also exhibit similar therapeutic effects in other neurological diseases and injuries involving oxidative stress and ferroptosis.

关 键 词：黄芪甲苷 BMSCs-Exos 脊髓损伤 铁死亡 氧化应激 

分 类 号：R285.5[医药卫生—中药学]