The Role And Mechanism Of UTX Regulating SCMECs’ Exosomes-Mediated Macrophage Polarization In Spinal Cord Injury

ObjectiveOur previous research confirmed that the knockout of the UTX gene in spinal cord microvascular endothelial cells(Tek-Cre;UTX^f/f,UTX^-/-)could promote the recovery of neurological function.Still,its mechanism of action has not been thoroughly studied.UTX/KDM6A is an epigenetic regulator with a wide range of effects.The demethylation of SCMECs acts on various cytokines,and there is a close connection and communication between SCMECs and macrophages.Therefore,this study focused on the role and mechanism of epi-factor UTX/KDM6A in regulating SCMECs’exosomes-mediated macrophage polarization in spinal cord injury and searched for potential new targets for the treatment of spinal cord injury.Materials and methods:（1）Using methods such as immunofluorescence,BMS score,BMS sub-score,and nerve electrophysiological detection to observe the effect of macrophage depletion on the neural function of UTX^-/-mice to that the role of macrophages in promoting neurological recovery in UTX^-/-mice.（2）Immunofluorescence,q RT-PCR and flow cytometry were used to observe the polarization changes of macrophage phenotype in the injured area of UTX^f/fand UTX^-/-mice after SCI.To clarify the effect of UTX regulation of SCMECs on macrophage phenotype changes.（3）The effects of UTX^f/fSCMECs and UTX^-/-SCMECs on macrophage polarization in SCMECs-macrophage"non-touch"co-culture system were observed by Western Blotting,q RT-PCR and GW4869 exosome inhibitor.And to clarify whether UTX^-/-SCMECs mediate macrophage phenotype changes via exosomes.（4）Immunofluorescence and flow cytometry were used to observe the uptake of Dil-labeled SCMECs-derived exosomes by macrophages in vitro and in vivo and determine whether macrophages uptake exosomes secreted by SCMECs.（5）Western Blotting,immunofluorescence,q RT-PCR,flow cytometry,BMS score,BMS sub-score,and neurophysiological detection were used to observe the phenotype changes of UTX^f/fand UTX^-/-SCMECs-derived exosomes intervened LPS-stimulated macrophage,and at the same time in WT SCI model,UTX^f/fand UTX^-/-SCMECs-derived exosomes were injected into the tail vein and evaluate the effects of UTX^-/-SCMECs exosomes on macrophage polarization phenotype and neurological recovery after SCI.（6）UTX^f/fand UTX^-/-SCMECs were collected,miRNA sequencing was used to establish expression profiles,and the obtained differential miRNAs were subjected to bioinformatics analysis.q RT-PCR and Western Blotting were used to identify miRNA-467b-3p as differential Key miRNAs and verify whether the function of miR-467b-3p mediate macrophage polarization.Chip-q PCR was used to clarify the relationship between UTX and miRNA-467b-3p further.（7）Target Scan and miRWalk databases searched for downstream target genes of miRNA-467b-3p.And q RT-PCR,Western Blotting and dual luciferase reporter assays were used to determine whether miRNA-467b-3p directly inhibited the expression of PTEN.（8）Western Blotting,immunofluorescence,q RT-PCR and rapamycin were used to observe the expression level of the PTEN/PI3K/mTOR signalling pathway and changes in macrophage polarization after miRNA-467b-3p targeted inhibition of PTEN to clarify whether miRNA-467b-3p activates the PTEN/PI3K/mTOR signalling pathway to mediate macrophage polarization.Results（1）In the SCI model,the knockout of UTX in SCMECs can significantly promote the recovery of neurological function.Its role in promoting neural function is weakened dramatically after macrophage depletion,indicating that macrophages played an essential role in promoting the recovery of neurological function in UTX^-/-mice.（2）In the SCI model,UTX^-/-SCMECs can mediate the polarization of macrophages to M2 type in the spinal cord injury area.（3）The primary way UTX^-/-SCMECs mediate the polarization of macrophages to M2 type is via exosomes.（4）Macrophages can take up a large number of exosomes secreted by SCMECs,both in vivo and in vitro,and most SCMECs-derived exosomes were taken up by macrophages,indicating that UTX^-/-SCMECs-derived exosomes depended on macrophages to work.（5）UTX^-/-SCMECs-derived exosomes can mediate LPS-stimulated macrophage polarization to M2-type in vitro.And in the SCI model,UTX^-/-SCMECs-derived exosomes mediate the M2-type polarization of macrophages in the injured area and promote the recovery of neurological function.（6）Compared with the miRNA expression profiles in UTXf/f SCMECs samples,there were 892 differentially expressed mature miRNA sequences in UTX^-/-SCMECs samples,of which 481 were up-regulated,and 411 were down-regulated.With P-value<0.05 and log2Fold-Change>1 as the screening conditions,92 miRNAs with significant differences,54 were up-regulated,and 38 were down-regulated.q RT-PCR,Western Blotting and immunofluorescence confirmed that miR-467b-3p as a differential key miRNA could mediate the polarization of macrophages to M2 type.Further,the Chip-q PCR experiment confirmed that UTX/H3K27me2/3 has a direct binding relationship with the promoter sequence of Sfmbt2/miR-467b-3p,thereby regulating the expression of miR-467b-3p.（7）PTEN was a downstream target gene of miRNA-467b-3p,and miRNA-467b-3p can directly inhibit the expression of PTEN.（8）After miRNA-467b-3p targeted inhibition of PTEN,PI3K/AKT/mTOR signalling pathway was activated to mediate macrophage polarization.Conclusion（1）UTX^-/-SCMECs mediate the polarization of macrophages to M2-type,thereby promoting the recovery of neurological function,which is a potential therapeutic target for spinal cord injury.（2）The primary way UTX^-/-SCMECs mediate the polarization of macrophages to M2 type is via exosomes.（3）UTX^-/-SCMECs-derived exosomes depend on macrophages to work.（4）miR-467b-3p is a crucial differential miRNA in UTX^-/-SCMECs exosomes,and the epigenetic complex UTX/H3K27me2/3 has a direct binding relationship with the miR-467b-3p/Sfmbt2 promoter,which can directly regulate miR-467b-3p expression.（5）miR-467b-3p in UTX^-/-SCMECs-derived exosomes could directly inhibit the expression of PTEN.（6）UTX/miR-467b-3p activates PTEN/PI3K/mTOR signalling pathway to promote macrophage polarization to M2-type.