| BackgroundEndogenous neural stem cells(NSCs)are critical for the remyelination of axons following spinal cord injury(SCI).Cell-cell communication plays a key role in the regulation of the differentiation of NSCs.Astrocytes act as immune cells that encounter early inflammation,forming a glial barrier to prevent the spread of destructive inflammation following SCI.In addition,the cytokines released from astrocytes participate in the regulation of the differentiation of NSCs.However,the effect of factors released by inflammation-stimulated astrocytes on the differentiation of NSCs after SCI is not clear.Clarifying the effect of these cytokines on the communication of NSCs-NSCs provides a new idea for the treatment of spinal cord injury.Objective1.To explore the effect of inflammatory stimulated astrocyte secretion conditioned medium(LPS-As-CM)on the differentiation of NSCs and the regeneration of axonal myelin sheath after SCI in rats and its mechanism;2.To explore the effect of LPS-As-NSCs-EVs on the differentiation of NSCs and the regeneration of axonal myelin sheath after SCI in rats and its mechanism.Methods1.The effect of LPS-As-CM on differentiation of NSCs and regeneration of axonal myelin sheath after SCI in rats:(1)Cell experiment:primary culture of astrocytes and NSCs,extraction of CM from astrocytes and LPS-stimulated astrocytes,and treatment of NSCs respectively.The differentiation of NSCs was observed by WB analysis and immunofluorescence staining;The content difference of BMPs protein in CM from different sources was detected by ELISA to find the mechanism of differentiation difference of NSCs.(2)Animal experiment:SD rats with SCI were treated with CM from different sources.The regeneration of axonal myelin sheath after SCI was observed by WB analysis and immunofluorescence staining;The nerve function of SCI rats was evaluated by BBB score and inclined plate test.2.The effect of LPS-As-NSCs-EVs on the differentiation of NSCs and the regeneration of axonal myelin sheath after SCI in rats:(1)Cell experiment:NSCs from different sources were treated with CM respectively,and NSCs-EVs,As-NSCs-EVs and LPS-As-NSCs-EVs were extracted,and NSCs from different sources were treated with NSCs-EVs respectively.The differentiation of NSCs was observed by WB analysis and immunofluorescence staining;RT-PCR was used to detect the difference in the expression of miRNA related to axonal myelin regeneration in NSCs from different sources,and to find out the mechanism leading to the difference in differentiation of NSCs.(2)Animal experiment:SD rats with SCI were treated with NSCs-EVs from different sources.The regeneration of axonal myelin sheath after SCI was observed by WB analysis and immunofluorescence staining;The nerve function of SCI rats was evaluated by BBB score and inclined plate test.(3)Study on the mechanism of action of miRNA-22-3p in LPS-As-NSCs-EVs:① Bioinformatics analysis:search for possible target genes of miRNA-22-3p through TargetScan,GO and Pathway analysis;The correlation between miRNA-22-3p and target gene was determined by RT-PCR and double luciferase gene report analysis.②The correlation and mechanism between LPS-As-NSCs-EVs and miRNA-22-3p function were verified in cell and animal experiments by WB analysis,immunofluorescence staining and RT-PCR technology.Results1.The effect of LPS-As-CM on the differentiation of NSCs and the regeneration of axonal myelin sheath after SCI in rats:(1)Differentiation results:In the cell experiment,compared with the control group,the NSCs treated with As-CM and LPS-As-CM differentiated more into astrocytes and inhibited their differentiation into oligodendrocytes;Moreover,LPS-As-CM has a stronger effect on promoting the differentiation of NSCs into astrocytes.In animal experiments,SCI rats treated with LPS-As-CM group obtained the lowest oligodendrocyte and the highest astrocyte around the injured cavity;The SCI rats treated with LPS-As-CM group obtained the worst neurological function score.(2)Validation results of BMPs in LPS-As-CM:Through ELISA,we found that the content of BMP2 in LPS-As-CM was significantly higher than that in As-CM;And the increase was more obvious than other BMPs.By adding BMP inhibitor Noggin and BMP2 in the presence of LPS-As-CM to treat NSCs and SCI rats separately,it was verified that the differentiation of neural stem cells induced by LPS-As-CM and the inhibition of neural function recovery after spinal cord injury depended on BMP2.2.The effect of LPS-As-NSCs-EVs on the differentiation of NSCs and the regeneration of axonal myelin sheath after SCI in rats:(1)Differentiation results:In the cell experiment,compared with the control group,the NSCs treated by NSCs-EVs group,As-NSCs-EVs group and LPS-As-NSCs-EVs group differentiated more into oligodendrocytes and inhibited their differentiation into astrocytes;Moreover,LPS-As-NSCs-EVs have a stronger effect on promoting the differentiation of NSCs into oligodendrocytes.In animal experiments,SCI rats treated with LPS-As-NSCs-EVs group obtained the highest oligodendrocytes and the lowest astrocytes around the injured cavity;The SCI rats treated with LPS-As-NSCs-EVs group obtained the best neurological function score.(2)Screening results of miRNAs in different NSCs-EVs:RT-PCR technology was used to detect the expression of axonal myelin regeneration related miRNAs in NSCs-EVs from different sources.It was found that the content of miRNA-22-3p in LPS-As-NSCs-EVs increased significantly,and the increase was the largest compared with other miRNAs.(3)Research results of miRNA-22-3p mechanism in LPS-As-NSCs-EVs:Targetscan and GO,Pathway analysis,RT-PCR and double luciferase gene report analysis confirmed that miRNA-22-3p plays a biological role in SCI through targeted regulation of KDM3A.Verify that miRNA-22-3p passes miRNA-22-3p/KDM3A/TGF by WB analysis,immunofluorescence staining and RT-PCR-β Axis regulates the differentiation of neural stem cells and the recovery of neural function after spinal cord injury.Conclusion1.The effect of LPS-As-CM on the differentiation of NSCs and the regeneration of axonal myelin sheath after SCI in rats:LPS stimulates astrocytes to secrete more BMP2 protein in their CM,thus promoting the differentiation of NSCs into astrocytes,and inhibiting the recovery of neural function in rats after SCI;2.The effect of LPS-As-NSCs-EVs on differentiation of NSCs and regeneration of axonal myelin sheath after SCI in rats:BMP2 protein in LPS-As-CM enriched miRNA-22-3p in NSCs-EVs,and miRNA-22-3p enriched miRNA-22-3p/KDM3A/TGF-β Axis promotes the differentiation of NSCs into oligodendrocytes,and promotes the recovery of neural function in rats after SCI.BackgroundEndogenous neural stem cells are essential for the myelin regeneration of axons after spinal cord injury.Intercellular communication plays a key role in regulating the differentiation of neural stem cells.Neurons are the most basic structural and functional units of the nervous system.After spinal cord injury,neurons are stimulated by inflammation,resulting in a large number of deaths in the injury center.In the early stage of spinal cord injury research,many scholars focused on how to promote the regeneration of neurons after injury.However,the regeneration ability of neurons is extremely low,and in the secondary injury stage of spinal cord injury,inflammation continues to stimulate residual neurons,which still participate in the intercellular communication in the injury environment.The effect of EVs released by neurons stimulated by inflammation on the differentiation of NSCs after SCI is not clear.Clarifying the effect of these EVs on the communication of NSCs and NSCs provides a new idea for the treatment of spinal cord injury.ObjectiveTo explore the effect of EVs secreted by inflammatory neurons on the differentiation of NSCs and the regeneration of axonal myelin sheath after SCI in rats and its mechanism.Methods1.Cell experiment:(1)The primary cultured neurons and NSCs,the EVs of neurons with different treatments were extracted by ultracentrifugation,and the electron microscope was used to confirm the existence of neuron-EVs;The diameter of neuron-EVs was measured by nanoparticle tracking analyzer;WB analysis of markers of neuron-EVs.(2)PKH-26 labeled EVs for cell and tissue tracing to determine whether NSCs can phagocytize neuron-EVs.(3)NSCs were treated with EVs from different sources,and the differentiation of NSCs was observed by immunofluorescence staining.(4)RT-PCR was used to detect the difference of miRNA-21-5p in neuron-EVs from different sources,and to find the mechanism of differentiation difference of NSCs.(5)MiRNA-21-5p mimics/inhibitor were used to up-regulate/down-regulate the expression of miRNA-21-5p in NSCs,and the effect of miRNA-21-5p on the differentiation of NSCs was evaluated by immunofluorescence staining and WB.2.Animal experiment:(1)SD rats with SCI were treated with neuron-EVs from different sources,and the regeneration of axonal myelin sheath after SCI was observed by immunofluorescence staining;The nerve function of SCI rats was evaluated by BBB score and inclined plate test.(2)MiRNA-21-5p agomir/antagomir is used to up-regulate/down-regulate the expression of miRNA-21-5p in vivo;SCI rats were treated with miRNA-21-5p agomir and LPS-neuron-EVs+miRNA-21-5p antagomir respectively.The regeneration of axonal myelin sheath after SCI was evaluated by immunofluorescence staining;The nerve function of SCI rats was evaluated by BBB score and inclined plate test.3.Research on the mechanism of action of miRNA-21-5p in neuron-EVs:(1)Bioinformatics analysis:search for possible target genes of miRNA-21-5p through TargetScan;The correlation between miRNA-21-5p and target gene was determined by RT-PCR and double luciferase gene report analysis.(2)Through WB analysis,immunofluorescence staining and RT-PCR technology,the correlation between neuron-EVs and miRNA-21-5p function and the mechanism of miRNA-21-5p action were verified in cell and animal experiments.ResultsNeuron-EVs can be engulfed by NSCs in vitro and in vivo,and can promote the differentiation of NSCs into oligodendrocytes and the myelin regeneration of axons in SCI rats;However,neuron-EVs after inflammatory stimulation lost their biological effects.It was confirmed by RT-PCR that inflammatory stimulation could up-regulate miRNA-21-5p in neuron-EVs.The correlation between miRNA-21-5p and SMAD7 target gene was determined by TargetScan,RT-PCR and double luciferase gene report analysis.It was verified by WB analysis,immunofluorescence staining and RT-PCR that miRNA-21-5p in neuron-EVs could target SMAD 7 and up-regulate TGF-β/SMAD2 signal transduction pathway leads to the excessive formation of astrocyte scar boundary and the failure of myelin regeneration in rats with spinal cord injury;In addition,these effects can be eliminated by adding miRNA-21 inhibitor/antagomir.ConclusionInflammatory stimuli are able to upregulate the expression of miRNA-21-5p within neuron EVs,inhibiting the beneficial biological effects of remyelination after SCI by activating TGF-β/SMAD2 signaling pathways through targeting SMAD7. |
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