| Objective:Studies have shown that inflammatory responses play an important role in ischemic stroke and have potential neuroprotective effects by inhibiting excessive inflammatory responses.The earliest participants in the inflammatory response in the brain are microglia,which are involved in the overall development of tissue damage and repair.However,activated microglia have similar phenotypes and functions to macrophages recruited from the peripheral blood to the brain,and the two are morphologically indistinguishable.In this study,the temporal and spatial changes of microglia and macrophages after cerebral ischemic injury were investigated from the single cell and 100 cell levels,and the differences in phenotypes and functions of the two groups of cells and their specific molecular mechanisms were compared at the transcriptome and proteome levels.The gene expression profile of microglia in different stages of ischemic injury was analyzed by transcriptome sequencing,and its phenotypic and functional differences were explored.We tried to intervene the activation of microglia in the early stage of ischemic injury to reduce the inflammatory response caused by injury,so as to reduce the brain damage caused by excessive inflammatory response.Methods:1.The mouse model of cerebral artery ischemia/reperfusion injury was established,and the cerebral infarction volume of ischemic injury on day 1,3 and 7 was detected by nissl staining.Quantitative Real-time PCR(q RT-PCR)was used to detect the expression levels of M1 and m2-type inflammatory cytokines in the ischemic hemisphere at different time points after ischemic injury.Microglia and macrophages in the ischemic hemisphere at different stages of ischemic injury were analyzed by flow analysis,then separated by sorting technology.The expression levels of 42 genes in 100 microglia and macrophages at different time points were detected by single cell PCR.The expression levels of 42 genes in single microglia and macrophages at 3 d after ischemic injury were detected by single cell PCR.The expression of Ym1 and i NOS in CCR2 and CX3CR1 positive cells was detected by immunofluorescence staining.2.Microglia and macrophages from the ischemic hemisphere 3 days after ischemic injury were sorted.Gene expression profile of 100,000 cells was detected by transcriptome sequencing,and proteome profile of 400,000 cells were analyzed by quantitative proteomics.Volcano plot showed differentially expressed genes and proteins,and bioinformatics analysis was performed to analyze the biological processes and molecular functions involved in differentially expressed genes and differentially expressed proteins.Sensome,secretome,and transcriptional regulatome were analyzed to investigate the molecular mechanisms of microglia and macrophages in phenotype,function,and transcriptional regulation.Combining transcriptome and proteome,the differentially expressed genes and proteins of the two groups of cells were analyzed,and the potential new markers of the two groups of cells were searched.The expressions of CD74,Anxa1,Anxa2 and F13 a in two groups of cells were detected by flow analysis and immunofluorescence staining.3.Immunofluorescence staining was used to detect the morphology and distribution of Iba1 positive cells in brain from normal and 12 h,3 d after ischemic injury.Microglia at different stages of ischemic injury were sorted and their gene expression profiles were detected by transcriptome sequencing.GSEA analysis was used to investigate the signal pathways involved in LG12 h and 3 d microglia genes detected.The volcano plot showed differentially expressed genes of microglia at 12 h and 3 d after ischemic injury compared with normal microglia.Biological processes involved in the differential expression of genes in microglia at 12 h and 3 d were analyzed by GO analysis.The gene expression patterns of microglia at 12 h and 3 d after ischemic injury were analyzed to explore the phenotype and functional differences of microglia at different stages.The expression levels of different genes in microglia at different stages were detected by RT-q PCR.Ingenuity Pathway Analysis(IPA)was performed to analyze the classical signal pathway that involve differentially expressed genes.An inflammatory model of BV2 cells stimulated by LPS was established in vitro,and the effect of Niclosamide inhibition on the expression level of BV2 cytokines and Pcna was detected by RT-q PCR and Western blot,respectively.In vivo experiments were set up to establish a mouse model of cerebral ischemia injury.Niclosamide was injected in the early stage of injury to inhibit the phosphorylation of Stat3.RT-q PCR was used to detect the level of inflammation in the ischemic hemisphere of the brain after cerebral ischemia injury.Western blot was used to detect the expression of p-Stat3,IL6 and Pcna.Results:1.At the tissue level,the expression level of M1-type inflammatory cytokines was higher in the early stage and M2-type in the later stage of cerebral ischemia injury,reflecting the process of tissue damage,inflammation and repair.The gene expression patterns of microglia and macrophages were different,the former showed the characteristics of transient activation in the early stage of ischemic injury,while the latter was in the state of continuous activation after ischemic injury.The two types of cells have different phenotypes,among which microglia were the main source of early M1-type inflammatory cytokines,while macrophages entering later stage were the main source of M2-type inflammatory cytokines.The transformation of M1-M2 was the result of the transformation of two cell types,reflected the main response cell types at different stages of injury.2.Combining flow cell sorting,transcriptomics and proteomics,a systematic biological analysis method was established.The differences in phenotypes and functions between activated microglia and macrophages infiltrating into the brain induced by cerebral ischemia injury were described from the perspectives of cell level,gene level and protein level.The two groups of cells have a unique set of genes in sensing external stimuli,secreted proteins and transcriptional regulation.During the peak period of tissue inflammation in cerebral ischemia injury,macrophages dominated the pathways related to inflammatory response,and the most typical biological process of microglia in this stage was cell cycle.In addition,proteins specifically expressed by two groups of cells were found.Anxa1 and CD74 can be used as potential markers to bind CD45 and be used to distinguish microglia and macrophages in the flow analysis,while Anxa2 can be used to mark activated microglia and macrophages and distinguish them from microglia in resting state.3.This study used flow sorting and transcriptomics sequencing system to analyze and compare the gene expression profiles of microglia at 12 h and 3 d after cerebral ischemia.Bioinformatics analysis showed that the gene expression profile of microglia 12 h after ischemia was mainly related to inflammatory response,while the 3d expression profile was mainly related to cell proliferation and cell cycle regulation.Classical pathway analysis showed that genes highly expressed in microglia at 12 h were involved in multiple inflammatory pathways,including Stat3 pathway,acute phase response signal,il-6 signal,TREM1 signal,neural inflammatory signal pathway,among which Stat3 pathway was the most significant one.The genes with high expression in 3d microglia are mainly involved in protein synthesis.Interfering with the Stat3 signaling pathway inhibited the activated microglia and their inflammatory factors induced by LPS stimulation.Interfering with the Stat3 signaling pathway during early ischemia,down-regulated the expression of inflammatory cytokines after injury.Conclusion:1.Flow cytometry sorting and single-cell PCR were used to investigate the dynamic changes of microglia and macrophages after cerebral ischemia injury at the cellular level.Microglia were briefly activated in the early stage of ischemic injury and were the main source of early M1-type inflammatory cytokines.The macrophages showed continuous activation,which was the main source of M2-type inflammatory cytokines.The transformation of tissue level M1 and M2 phenotypes was the result of cell type conversion.2.Combined with flow cytometry sorting,second-generation sequencing and quantitative proteomics,a set of systematic biological analysis method was established.The role of activated microglia and infiltrating macrophages in feel stimulation,protein secretion effect and transcriptional regulation were elucidated from the level of cells,genes and proteins.At the peak of inflammation after ischemic injury,macrophages played a leading role in inflammatory response,and microglia were involved in cell cycle and other processes.Potential markers to distinguish microglia from macrophages were identified.Anxa1 and CD74 could be used for flow cytometry.Anxa2,on the other hand,could be used to distinguish between resting and activated microglia cells and macrophages in immunofluorescence staining experiments.3.Based on transcriptome sequencing,the phenotype,functional changes and molecular mechanism of microglia at different stages of cerebral ischemic injury were systematically analyzed.A key signaling pathway for microglia activation in the early stage of cerebral ischemia injury was discovered.After the early intervention of the Stat3 pathway,the inflammatory response after brain injury was inhibited,thereby reducing the secondary tissue damage caused by excessive inflammatory response. |
PDF Full Text Request