The Mechanism Research Of Endothelial Progenitor Cell-derived Extracellular Vesicles In The Treatment Of Rat Glucocorticoid-induced Osteoporosis By Inhibiting The Ferroptotic Pathway On Fibroblasts

Abnormal antioxidative capabilities were observed in the pathogenesis of steroid-induced osteoporosis?SIOP?.Ferroptosis is a recently discovered type of cell death that is characterized by the overproduction of ROS in response to GPX4 and system X^c-downregulation,which is mediated by an Fe²⁺fenton reaction.However,investigations focusing on the relationship between ferroptosis and steroid-induced bone disease remain limited.In the present study,density gradient centrifugation was applied to extract primary mouse bone marrow-derived endothelial progenitor cells?EPCs?and surface markers of CD34,CD133,FLK-1 and v WF of endothelial progenitor cells were detected using flow cytometry.Purity of the extracted EPCs was identified by Dil-Ac-LDL and FITC-UEA-I fluorescence double staining.After the mouse bone marrow-derived EPCs were successfully isolated,the exosome isolation kit was combined with density gradient centrifugation to extract mouse bone marrow-derived EPCs-derived extracellular vesicles?EPC-EVs?.After the extraction was completed,Nanoparticle Tracking Analysis?NTA?method was used to detect the particle size distribution,and Western blot method was used to detect the exosome surface markers CD63,CD9,and CD81.Results of the microscopic morphology of the sample by transmission electron microscopy showed that most of the particles in the extracted sample conformed to the characteristics of exosomes and minority of sample particles are bigger EVs.On the other hand,PKH26-labeled EPCs were obtained with the incubation of fluorescent membrane dye PKH26,and PKH26-labeled EPC-EVs were further extracted from the cell supernatant.Mouse osteoblasts were co-cultured with Dil-labeled EPC-EVs and the ability of mouse osteoblasts to take up EPC-EVs was investigated by fluorescence microscopy.The results showed that mouse osteoblasts could take up EPC-EVs well.High-dose dexamethasone was used to establish a mouse SIOP model,and extracellular vesicles extracted from bone marrow-derived endothelial progenitor cells?EPC-EVs?alleviated the pathological changes in SIOP via microtomography?micro-CT?,with elevations in bone volume?BV?,bone surface?BS?,trabecular thickness?Tb.Th?,and trabecular connectivity density?Conn-D?and decreases in trabecular separation?Tb.sp?and the structure model index?SMI?.Histopathological analysis,such as haematoxylin and eosin?HE?and Masson staining,showed that EPC-EVs treatment increased the volume and density of the trabecular bone and bone marrow.RNA sequencing?RNA-seq?and bioinformatics analysis revealed subcellular biological alterations upon steroid and EPC-EVs treatment.Compared with the control,high-dose dexamethasone downregulated GPX4 and system X^C-,and the KEGG-based gene set enrichment analysis suggested that the ferroptotic pathway was activated.In contrast,combination treatment with EPC-EVs partly reversed the KEGG-mapped changes in the ferroptotic pathway at both the gene and m RNA expression levels.In addition,alterations in ferroptotic marker expression,such as SLC3A2,SLC7A11,and GPX4,were further confirmed by RNA-seq.EPC-EVs were able to reverse dexamethasone treatment-induced alterations in cysteine and several oxidative injury markers,such as malondialdehyde?MDA?,glutathione?GSH?,and glutathione disulphide?GSSG??as detected by ELISA?.In conclusion,EPC-EVs prevented mouse glucocorticoid-induced osteoporosis by suppressing the ferroptotic pathway in osteoblasts,which may provide a basis for novel therapies for SIOP in humans.