Microglia MS4A6C Involves In Inflammatory Response After Spinal Cord Injury And Mechanisms

Spinal cord injury initiates neurological motor and other physiological dysfunctions.At present,there is no feasible and effective treatment.Therefore,exploring how to attenuate the continuous deterioration of pathology after SCI has become a much-needed and significant medical problem in the field of neurological diseases worldwide.Microglia are the innate immune cells of the central nervous system,which play an important role in the maintenance of environmental homeostasis in the nervous system,anti-infective immunity and nerve system injury repair.However,the specific role and molecular mechanism of microglia in inflammatory response and tissue repair after SCI need to be clarified.MS4A6C（murine homolog of human MS4A6A）is a member of the four-time transmembrane domain membrane molecule MS4A gene family.Studies have shown that MS4A6C is mainly expressed on the membrane surface of immune cells such as microglia.In the mouse model of experimental autoimmune encephalomyelitis,single cell RNA sequencing and molecular biological verification experiments showed that the expression of MS4A6C in microglia was significantly up-regulated,suggesting that it may play an important role in microglia-mediated nervous system inflammation,but the spatio-temporal characteristics of MS4A6C expression in microglia membrane under the condition of spinal cord injury are not clear.What is the specific role and molecular mechanism of microglial membrane MS4A6C in inflammatory response and tissue repair after spinal cord injury?In-depth study of these important scientific issues will help to clarify the biological role of microglia in SCI,and is expected to find specific intervention targets that can reduce spinal cord injury and promote repair,which has important biological significance and clinical application value.Objectives:1.To investigate the temporal variation and spatial localization of microglia membrane MS4A6C expression under the pathological condition of spinal cord injury.2.To determine the effect and mechanism of MS4A6C knockdown on microglia inflammatory response induced by lipopolysaccharide.3.Transcriptome sequencing to explore the molecular changes and mechanism of knockdown of MS4A6C on lipopolysaccharide induced inflammatory response.Methods:1.Analysis of serum titers after immunization with MS4A6C peptide antigen and verification of antibodies by mouse spinal cord tissue.The 8-week-old male C57BL/6J mice were operated on spinal cord injury.The mice were sacrificed on day 3,7 and 14 after operation,and the expression of MS4A6C was detected by Q-PCR and Western blotting.Immunofluorescence was used to detect local IBA-1⁺-MS4A6C⁺expression in microglia after mouse spinal cord injury.2.The expression of other members of the MS4A family and MS4A6C at 6h,12h and 24h is determined by Q-PCR after lipopolysaccharide stimulation in BV2 microglia.Conditions explored with MS4A6C sh RNA lentivirus infected BV2 microglia;Incubation of BV2 microglial concentration with LPS.Q-PCR was detected inflammatory cytokine of TNF-α、IL-1βand IL-6 expression after MS4A6C knockdown3.Transcriptome sequencing was performed for microglia LV3-NC+LPS group and LV3-MS4A6C-660+LPS group.Overview of sequencing data quality control.Regional distribution of reference genome alignment.Analysis of genes expression levels.Statistics of down-regulation frequency of differentially expressed genes.Volcano plot analysis of differentially expressed genes expression levels.Cluster analysis of differentially expressed genes and GO function of differentially expressed genes with KEGG pathway enrichment analysis.Differentially expressed genes GDF15,Ube2L3,Zfand3,Med6 and GBP2 were detected by Q-PCR assay.Results:1.8-week-old C57BL/6J male mice,a spinal cord injury model was successfully established,and the expression levels of inflammatory factors were significantly increased in the local area of the mouse damaged spinal cord.Mice were immunized five times with MS4A6C polypeptide antigen,blood was collected,and the serum titers were measured by ELISA method.Serum titer≥32K,which the best results were verified by using mouse serum antibodies of M01 and M05.MS4A6C m RNA levels in spinal cord injured mice showed an increasing trend and were significantly higher than sham mice;Similarly,MS4A6C protein levels were elevated 3d after SCI.Immunofluorescence assay revealed significant local of mouse injured spinal cord IBA-1⁺-MS4A6C⁺microglial infiltration.The number of microglia was increased in the local area of the mouse damaged spinal cord.2.The expression of MS4A family members were all altered in BV2 microglial cells after LPS stimulation.MS4A6C m RNA expression increased with the time of LPS stimulation,showing a tendency of elevation followed decrease in MS4A family members,with 7-fold elevation compared with the sham at 12h and the highest elevation in MS4A family members.After 50 MOI MS4A6C sh RNA lentivirus infection with BV2 microglia for 48h and LPS 50ng/m L stimulation for 12h,MS4A6C gene knockdown was about 60%-70%,which reached a significant level compared with the control.Knockdown of MS4A6C in BV2 microglia reduced inflammation and inflammatory cytokine of TNF-α、IL-1βand IL-6 expression levels were decreased.3.A total of 8 libraries were constructed by transcriptome sequencing,including 4 LV3-NC+LPS libraries and 4 LV3-MS4A6C-660+LPS chain specific libraries.Strand specific library libraries yielded 398814810 high-quality sequencing data.After quality control analysis,Q20%、Q30%and GC content also conformed to the requirements for subsequent analysis.After alignment,most of the sequencing data aligned mainly to the exon region.Comparing the LV3-NC+LPS group with the LV3-MS4A6C-660+LPS group revealed that140 genes were upregulated and 29 genes were downregulated.Go enrichment analysis showed that the differentially expressed genes involved cytoplasm、cell membrane、extracellular space、extracellular region、protein binding、metal ion binding and calcium ion binding etc.KEGG pathway enrichment analysis showed that the differentially expressed genes involved in extracellular matrix receptor interaction、focal adhesion、infection and other pathway enrichment.Conclusion1.The expression of MS4A6C m RNA and protein are up-regulated in the area of spinal cord injury,and the infiltration of MS4A6C⁺+IBA-1⁺microglia in the local area of spinal cord injury increased significantly.2.Knockdown of MS4A6C in BV2 microglia reduced inflammatory response and inflammatory cytokine of TNF-α、IL-1βand IL-6 expression levels are decreased.3.Transcriptome sequencing indicates that MS4A6C regulates the inflammatory response of BV2 microglia after LPS stimulation,and the knockdown of MS4A6C induces changed extracellular matrix interaction、differential expression of tissue repair related genes and enrichment of pathways.