Effects Of Spib Regulating ApoE/Sorl1 Mediated Microglia/neuron Communication In Neuropathic Pain

BackgroundNeuropathic pain(NP)is one of the most commonly cured and difficult and stubborn chronic diseases.Pain perception helps organisms respond appropriately to harmful stimuli in order to protect tissues from further damage.But if the pain persists for a long time,chronic pain can develop.The spinal cord is the pain signal from the peripheral nervous system to the middle station of the central nervous system.As the first-level damage experience transfer center,the spinal cord is responsible for integrating the damage sensing information of the peripheral nerve to the back corner of the spinal cord through the fiber,and then passed it to the brain through the projection neuron to cause pain.In this study,we have combined analysis of the scRNA-seq and single-cell assay for transposase-accessible chromatin sequencing(scATAC-seq)by using spared nerve injury(SNI)model.The transcription factor Spib regulating ApoE/Sorll mediated the role of microglia/neuron communication in the process of Neuropathic pain.Methods1.Download the scRNA-seq dataset GSE134003 with spinal cord samples from SNI mouse models using the GEO database.The cell population was identified by RStudio software,and microglia were extracted to further subdivide the subpopulation.Subsequently,cellular communication analysis was performed on individual cell populations.ApoE-Sorll,a ligand receptor pair with large variation and high activity,was selected from the ligand-receptor pairs of microglia-neurons for follow-up study.2.scATAC sequencing of spinal cord tissue from mouse SNI models,combined with scRNA data,we defined 5 cell populations.Specific motif analysis of single-cell ATAC sequencing data by RStudio software showed that the microglial ligand ApoE was the target gene of the microglial heterosexual transcription factor Spib,and downstream functional enrichment analysis was performed on the receptor Sorl1-related protein.3.Mouse SNI model was established to detect the localization of Spib,ApoE and microglia and Sorl1 and neurons by immunofluorescence4.In LPS stimulation BV2 microglia,the expression of Spib was knocked down,and ApoE,Sorl1 and inflammatory factor indexes were detected by real-time real-time PCR(qRT-PCR).Result1.Bioinformatics analysis was performed on the single-cell transcriptome data GSE 134003 of mouse SNI model spinal cord samples in GEO database,and differential genes were screened,and the expression of ApoE and Sorll in the spinal cord of SNI mice was significantly increased,and cell communication analysis screened ApoE-Sorll with high activity in the ligand receptor pair of microglia-neuron.2.In LPS-induced BV2 microglial activation model,ApoE and high expression,knockdown Spib inhibits the expression of ApoE,and knockdown Spib reduces the expression of related inflammatory indexes.3.Mouse SNI model was established,the behavioral changes of mice were observed,the mechanical pain threshold changes of mice were detected,spinal cord tissues were extracted for single-cell ATAC sequencing,motif,footprint analysis and target gene prediction were carried out to screen out that ApoE was the target gene of Spib.4.Mouse SNI model was established,and the localization and expression changes of Spib,ApoE and Sorll were observed by immunofluorescence in spinal cord tissue.ConclusionThe expression of Spib,ApoE and Sorll in the spinal cord tissues of SNI mice increased,knockdown Spib reduced the release of inflammatory factors,and microglial Spib regulated ApoE-Sorll mediated microglia/neuronal cell communication,thereby promoting the development of neuropathic pain...