摘要
目的探讨黄芪甲苷预处理脐带间充质干细胞源性外泌体(hUCMSCs-Exos)对软骨细胞铁死亡的影响。方法2024年1-12月于新疆医科大学动物实验中心进行实验。应用黄芪甲苷(AS-Ⅳ)对hUCMSCs预处理后提取hUCMSCs-Exos,采用透射电子显微镜(TEM)和纳米粒子跟踪分析仪(NTA)对其形态和尺寸分布进行鉴定。大鼠软骨细胞根据不同的干预方式,分组为对照组(正常软骨细胞培养)、模型组(IL-1β干预组)、外泌体组(IL-1β+hUCMSCs-Exos干预)、黄芪甲苷联合外泌体组(IL-1β+AS-Ⅳ预干预hUCMSCs-Exos),电镜观察各组细胞内线粒体的形态变化。通过离心收集细胞,提取细胞样本中的谷胱甘肽(GSH)、丙二醛(MDA)、活性氧(ROS),并进行定量分析。RT-qPCR和蛋白质印迹法分别检测软骨细胞中Nrf2、GPX4的mRNA和蛋白表达水平。结果AS-Ⅳ预处理后的外泌体与未预处理囊泡结构、直径相似。与对照组比较,模型组线粒体明显萎缩、膜破裂;与模型组比较,外泌体组和黄芪甲苷联合外泌体组线粒体形态有所改善,且黄芪甲苷联合外泌体组改善更为显著。与对照组比较,模型组GSH含量下降,MDA、ROS含量升高(P<0.01);与模型组比较,外泌体组、黄芪甲苷联合外泌体组GSH含量依次升高,MDA、ROS依次降低(P<0.01),且黄芪甲苷联合外泌体组GSH含量高于外泌体组,MDA、ROS含量低于外泌体组(P<0.01)。与对照组比较,模型组软骨细胞中Nrf2、GPX4 mRNA及蛋白表达水平均降低(P<0.01);与模型组比较,外泌体组、黄芪甲苷联合外泌体组软骨细胞Nrf2、GPX4 mRNA及蛋白表达水平依次升高(P<0.01);黄芪甲苷联合外泌体组软骨细胞中Nrf2、GPX4 mRNA及蛋白表达高于外泌体组(P<0.05)。结论AS-Ⅳ预干预hUCMSCs-Exos对软骨细胞铁死亡的调控作用可能与其上调Nrf2/GPX4信号通路的表达有关,进而抑制软骨细胞铁死亡反应,减少软骨细胞的损伤。
Objective To investigate the impact of AstragalosideⅣ(AS-Ⅳ)pretreatment of human umbilical cord mesenchymal stem cell-derived exosomes(hUCMSCs-Exos)on ferroptosis in chondrocytes.Methods Experiments were conducted at the Animal Experimental center of Xinjiang Medical University from January 2024 to December 2024,hUCMSCs were pretreated with AS-Ⅳ,followed by the extraction of hUCMSCs-Exos,which were characterized for morphology and size distribution using transmission electron microscopy(TEM)and nanoparticle tracking analysis(NTA).Rat chondrocytes were divided into four groups based on intervention methods:control group(normal chondrocyte culture),model group(IL-1βintervention),exosome group(IL-1β+hUCMSCs-Exos intervention),and AS-Ⅳcombined exosome group(IL-1β+AS-Ⅳpretreated hUCMSCs-Exos).Mitochondrial morphology in each group was observed using electron microscopy.Cells were collected by centrifugation,and glutathione(GSH),malondialdehyde(MDA),and reactive oxygen species(ROS)were extracted and quantitatively analyzed.mRNA and protein expression levels of Nrf2 and GPX4 in chondrocytes were detected using RT-qPCR and Western blotting,respectively.Results Exosomes pretreated with AS-Ⅳexhibited similar vesicular structures and diameters compared to untreated exosomes.Compared to the control group,the model group showed significant mitochondrial atrophy and membrane rupture.Both the exosome group and the AS-Ⅳcombined exosome group demonstrated improved mitochondrial morphology compared to the model group,with the AS-Ⅳcombined exosome group showing more pronounced improvements.The model group exhibited decreased GSH content and increased MDA and ROS levels compared to the control group(t/P=34.851/<0.001).The exosome group and AS-Ⅳcombined exosome group showed sequentially decreased GSH content and increased MDA and ROS levels compared to the model group(F/P=6.977/<0.001;17.249/<0.001),with the AS-Ⅳcombined exosome group having higher GSH content and lower MDA and ROS levels than the exosome group(t/P=7.744/<0.001).The model group showed reduced mRNA and protein expression levels of Nrf2 and GPX4 in chondrocytes compared to the control group(t/P=18.871/<0.001).The exosome group and AS-Ⅳcombined exosome group exhibited sequentially increased mRNA and protein expression levels of Nrf2 and GPX4 compared to the model group(F/P=8.390/<0.001;4.143/<0.05),with the AS-Ⅳcombined exosome group showing higher expression levels than the exosome group(t/P=6.912/<0.05).Conclusion The regulatory effects of AS-Ⅳpretreated hUCMSCs-Exos on ferroptosis in chondrocytes may be associated with the upregulation of the Nrf2/GPX4 signaling pathway,thereby inhibiting ferroptotic responses and reducing chondrocyte damage.
作者
张文豪
易林
向文远
方锐
梁治权
Zhang Wenhao;Yi Lin;Xiang Wenyuan;Fang Rui;Liang Zhiquan(Fourth Clinical Medical College,Xinjiang Medical University,Xinjiang,Urumqi 830000,China;不详)
出处
《疑难病杂志》
2025年第8期991-997,共7页
Chinese Journal of Difficult and Complicated Cases
基金
国家自然科学基金资助项目(82360934)
新疆维吾尔自治区自然科学基金(2023D01C145)
新疆维吾尔自治区重大科技专项项目(2023A03007-3)
新疆维吾尔自治区青年托举人才项目(2023TSYCQNTY0050)
新疆维吾尔自治区重点研发专项(2021B03006-2)
新疆维吾尔自治区科技创新领军人才—高层次领军人才项目(2022TSYCLJ0007)。
作者简介
通信作者:方锐,E-mail:xjfrdoctor@163.com。
