Effects And Mechanisms Of UTX Regulating Macrophage To Phagocytize Myelin Debris On Neural Functional Recovery After Spinal Cord Injury

Objective:UTX（Ubiquitously Transcribed tetratricopeptide repeat,X chromosome）,as a histone demethylation modification enzyme,is mainly responsible for removing the dimethylation/trimethylation of H3K27 sites and can directly sense oxygen to control chromatin reprogramming and cell fate.However,it is not clear whether UTX is also involved in regulating the fate and function of macrophage in the ischemic and hypoxic damaged spinal cord tissue.Therefore,the study aimed to explore the effects and mechanisms of UTX regulating macrophage to phagocytize myelin debris on neural functional recovery after spinal cord injury in mice,analyze the pathophysiological process of UTX regulating macrophage myelin debris phagocytosis and affecting spinal cord injury repair,and provide basic experimental theoretical basis for seeking potential therapeutic targets of spinal cord injury.Methods:1.The brain tissues of C57BL/6 mice aged 8-10 weeks were collected by gradient sucrose supercentrifugation.The cortical primary neurons of newborn C57BL/6 mice were extracted and treated with myelin debris at different concentration.The axon length of cortical neurons was detected by immunofluorescence to verify the biological activity（neurotoxicity）of myelin debris.The spinal cord injury model of8-10 weeks C57BL/6 mice was established by modified Allen’s method.The temporal and spatial pattern of macrophages phagocytosis myelin debris after spinal cord injury in mice was characterized by immunofluorescence.Western Blot and q RT-PCR were used to detect UTX expression at different days post injury.Data sets related to acute spinal cord injury in mice were selected after searching the GEO public database of high-throughput sequencing data sets.Single-cell sequencing data were analyzed by using R language software,and the temporal and spatial pattern of UTX in macrophages in the injuried site were characterized by immunofluorescence.2.Lys MCre mice were mated with Utx^flox/flox mice to construct conditional knockout macrophage Utx transgenic mice(Utx^-/-).The mouse tails were cut and the genotype was identified by agarose gel electrophoresis(Utx^-/-were experimental group mice,and Utx^+/+were littermate control group mice).Hair colour,body weight,sensation,movement,and nerve electrophysiology measurements of both hind limbs were compared between experimental group(Utx^-/-)and littermate control group(Utx^+/+).Established a mouse spinal cord injury model.Spinal cord tissue was obtained at the 7th day after injury,and the expression level of UTX in macrophages was detected by immunofluorescence to verify the knockout efficiency of UTX.Bone marrow derived macrophages（BMDMs）of experimental group(Utx^-/-)and control group(Utx^+/+)were respectively isolated and cultured.Immunofluorescence,Western Blot and q RT-PCR were used to verify the knockout efficiency of UTX within macrophages in vitro.3.In vitro,bone marrow derived macrophages were isolated and cultured from mice in experimental group(Utx^-/-)and mice in control group(Utx^+/+).After intervention with myelin debris at a concentration of1mg/m L for different periods of time,immunofluorescence was used to detect the macrophages phagocytosis myelin debris.The mouse spinal cord injury model was established and spinal cord tissue was obtained from experimental mice(Utx^-/-)and control mice(Utx^+/+)at 7th and 14th day after spinal cord injury.Immunofluorescence was used to detect macrophages phagocytosis myelin debris.BMS scores and hind limb cortical motor evoked potential（c EMP）of mice in experimental group(Utx^-/-)and in littermate control group(Utx^+/+)were evaluated to reflect the recovery of sensory and motor functions after spinal cord injury at different days after spinal cord injury comparatively.Combined with Allen’s percussion method and spinal cord transection method,the mouse spinal cord injury-transection model was established.Based on the mouse spinal cord injury model and injury-transection model,spinal cord specimens were collected at the 28th day after surgery.Axonal regeneration after spinal cord injury and after spinal cord injury-transection of mice in experimental group(Utx^-/-)and littermate control group(Utx^+/+)was determined by immunofluorescence.Based on the spinal cord injury model of mice in experimental group(Utx^-/-)and control group(Utx^+/+),spinal cord specimens were collected at 7th and14th day after injury.Oil red O staining was used to detect lipid deposition in the spinal cord injury area and Shanghai Synchrotron radiation Fourier transform infrared（SR-FTIR）microspectroscopy was used to characterize the material composition and distribution of spinal cord tissues.Based on bone marrow derived macrophages of experimental mice(Utx^-/-)and control mice(Utx^+/+),after 24h intervention with myelin debris at a concentration of 1mg/m L,Oil red O staining was performed to detect lipid deposition in macrophages and Shanghai Synchrotron radiation Fourier transform infrared（SR-FTIR）microspectroscopy was used to analyze the difference of substance composition within macrophages at single cell level.4.Spinal cord injury models of mice in the experimental group(Utx^-/-)and control group(Utx^+/+)were established by modified Allen’s attack method.Macrophages in the spinal cord injury area of mice in the experimental group(Utx^-/-)and control group(Utx^+/+)were selected by flow cytometry at 7th day after spinal cord injury.RNA-seq was used to detect the differential expression genes before and after conditional knockout of Utx in macrophages.The phagocytic gene sets in GSEA and NCBI databases were searched,and bioinformatics analysis was combined with the intersection of up-regulation and down-regulation differential genes to screen out the differential genes that promote macrophage myelin debris phagocytosis.By referring to relevant literatures and NCBI database,candidate target genes were further screened from differential exprerssion genes according to the function and expression abundance in macrophages.Bone marrow derived macrophages of experimental mice(Utx^-/-)and control mice(Utx^+/+)were isolated,and differential expression of candidate target genes（target proteins）in macrophages were detected by q RT-PCR and Western Blot.Isolated macrophages from the experimental group(Utx^-/-)mice and control mice(Utx^+/+).After down-regulating the target gene and blocking the target protein with recombinant lentiviral vector and blocking antibody respectively,1mg/m L myelin debris was used to for 24 hours intervention.Immunofluorescence was use to detected the macrophage phagocytosis myelin debris.Results:1.Myelin debris with bioactive was collected successfully.Immunofluorescence results showed that macrophages rarely phagocytosis myelin debris within 7 days and clearly phagocytosis myelin debris from 10th to 14th day in the injured area after spinal cord injury.Western Blot and q RT-PCR results showed that UTX expression was relatively high within 7 days and low from 10th to 14th day after spinal cord injury.Bioinformatics analysis indicated Utx expression in macrophages after spinal cord injury.Immunofluorescence results also showed that UTX expression was relatively high in macrophage nucleus at 7th day,but low from 10th to 14th day after spinal cord injury.2.Conditional knockout of macrophages’Utx transgenic mice(Utx^-/-)were successfully constructed.The results showed that conditional knockout of macrophage Utx did not affect mice hair colour,body weight,sensation,movement,and nerve electrophysiology measurement results of both hind limbs.The knockout efficiency of Utx was also detected both in vivo and in vitro.3.Immunofluorescence results in vitro showed that myelin debris phagocytosis in bone marrow derived macrophages of mice in the experimental group(Utx^-/-)is more than in control group(Utx^+/+).Immunofluorescence results in vivo showed that at 7th and 14th day after spinal cord injury,macrophages in experimental group(Utx^-/-)phagocytic myelin debris were more than those in control group(Utx^+/+).The BMS scores after spinal cord injury of mice in experimental group(Utx^-/-)was much better than that in control group(Utx^+/+).Nerve electrophysiology measurement results showed that the c EMP recovery of mice in the experimental group(Utx^-/-)was significantly better than that in control group(Utx^+/+).Immunofluorescence results showed that at 28th day after spinal cord injury and injury-transection,the fluorescence density of axons in the injured area of mice in the experimental group(Utx^-/-)was higher than that in control group(Utx^+/+).Oil red O staining and SR-FTIR microspectroscopy results of injuried spinal cord indicated that lipid deposition in the spinal cord injury area of mice in experimental group(Utx^-/-)was less than that in control group(Utx^+/+)at 14th day.Oil red O staining in vitro indicated that lipid deposition in macrophages of experimental mice(Utx^-/-)was less than that of control mice(Utx^+/+).SR-FTIR was able to distinguish the two groups of macrophages,which qualitatively indicated changes in lipid components in the two groups of macrophages.4.The whole gene transcription（RNA-seq）and differentially expressed genes（DEGs）of macrophages in spinal cord injury area of experimental mice(Utx^-/-)and control mice(Utx^+/+)were obtained successfully at 7th day after spinal cord injury,and differentially expressed genes that positively regulate macrophages phagocytosis were screened after convergence DEGs with phagocytic related genes.After referring to relevant literatures and NCBI database,candidate genes Vsig4,Cd5l,Mfge8 and Cd36 were further screened according to the function and expression abundance in macrophages.Western Blot and q RT-PCR results showed that the expression of Vsig4,the most differentially expressed gene,was much higher in macrophages of experimental mice(Utx^-/-)than in control mice(Utx^+/+).Immunofluorescence results showed that down-regulation of Vsig4 by recombinant lentiviral vectors and blocking of VSGI4 by blocking antibodies both attenuated macrophage phagocytosis of myelin debris.Conclusions:1.In this study,within the first two weeks of spinal cord injury in mice,the temporal and spatial patterns of macrophage phagocytosis of myelin debris shows an inverse trend to the spatial and temporal distribution of UTX expression in macrophages.2.Conditional knockout of macrophage Utx promotes macrophage phagocytosis myelin debris,alleviates lipid deposition in the spinal cord injury site,and is beneficial for axonal regeneration and neurological function recovery after spinal cord injury in mice.3.Conditional knockout of Utx can reverse Vsig4 downregulation and enhance the ability of macrophage phagocytosis myelin debris,which is beneficial for axonal regeneration and nerve function repair after spinal cord injury in mice.