Role And Mechanism Of Hypoxic Mesenchymal Stem Cell-derived Small Extracellular Vesicles In Regulating Astrocytes Polarization To Alleviate Spinal Cord Injury

Objective:Spinal cord injury（SCI）repair is one of the most challenging clinical problems.After SCI,different phenotypes of reactive astrocytes regulate the central nervous system microenvironment,affecting the repair of SCI.Our group has found mesenchymal stem cell derived small extracellular vesicle（MSC-s EV）can regulate astrocytes phenotypes to alleviate SCI rats.Oxygen partial pressure has been reported to be one of the important factors affecting MSC growth and cell function;hypoxic culture can promote MSC secretion of s EV and improve its efficacy in tissue damage repair.However,the role and mechanism of hypoxia-cultured MSC-s EV（Hyp-MSC-s EV）in SCI repair is not completely understood.Thus,This study aims to compare the regulatory effects of convention-cultured MSC-s EV（Con-MSC-s EV）and Hyp-MSC-s EV on astrocytes phenotype and the therapeutic effects of SCI models,to find new options for SCI treatment and to elucidate their molecular mechanisms.Methods:（1）MSC were cultured separately in normoxia（18%O₂）and hypoxia（1%O₂）incubators.CCK-8,Western blot,and real-time quantitative reverse transcription polymerase chain reaction（q RT-PCR）were used to evaluate MSC proliferation,migration,and stemness.Con-MSC-s EV and Hyp-MSC-s EV were isolated by using ultrafiltration and then characterized.（2）SD（Sprague Dawley）rat spinal cord injury models were established,and Con-MSC-s EV and Hyp-MSC-s EV were intravenously injected at the same concentration for treatment.BBB（basso-beattie-bresnahan）score,tissue immunofluorescence,q RT-PCR,and Western blot were used to compare the hind limb motor function,spinal cord tissue repair,neuronal axon regeneration,and astrocytes phenotype polarization of rat models after Con-MSC-s EV and Hyp-MSC-s EV treatments.（3）Primary astrocytes were stimulated with lipopolysaccharide（LPS）in vitro and treated with Con-MSC-s EV and Hyp-MSC-s EV respectively.The markers of astrocytes phenotypes and inflammation were detected by cell immunofluorescence,q RT-PCR,and Western blot.（4）m RNA transcriptome sequencing was used to analysis the signal pathways in astrocytes that were significantly altered after treatments with Con-MSC-s EV and Hyp-MSC-s EV.Overexpression plasmids were used to transfect key protein to verify the role of NOD-like receptor（NLR）pathway in astrocytes.（5）The active proteins in Hyp-MSC-s EV were screened by s EV proteomics.The specific mechanism was verified by Western blot,co-immunoprecipitation（Co-IP）,immunofluorescence staining and cycloheximide（CHX）assay.Results:（1）Hypoxic culture promoted MSC proliferation and migration and maintained stemness characteristics.（2）Compared to Con-MSC-s EV,Hyp-MSC-s EV more effectively promoted spinal cord tissue repair and improved hind limb motor function in SCI rats.（3）In vitro and vivo,Hyp-MSC-s EV could effectively inhibit the A1 astrocytes phenotype and promote their polarization towards the A2 phenotype,thereby alleviating the inflammatory response.（4）NLR signalling pathway were significantly upregulated in astrocytes under inflammation induction.Guanylate-binding protein 1（GBP1）could mediate astrocytes toward A1 phenotype,thus activating their neurotoxicity,while Hyp-MSC-s EV could inhibit the activation of GBP1.（5）Protein spectrum analysis revealed that Erbb2 interacting protein（ERBIN）was rich in Hyp-MSC-s EV.Co-IP and CHX experiments confirmed that ERBIN could bind to NOD2 and mediate its degradation,thereby inhibiting GBP1 transcription and reversing the astrocytes A1 phenotype.Conclusions:Hyp-MSC-s EV can mediate the degradation of NOD2 via delivering the active protein ERBIN,which in turn suppress GBP1 and reverse the A1 phenotype of astrocytes,thus alleviating SCI.This study elucidates the mechanism by which Hyp-MSC-s EV regulate the polarization of astrocytes to alleviate SCI,providing a new target and potential strategy for the treatment of SCI.