Multi-omics And Bioinformatics-based Analysis Of The Inflammatory Response After Spinal Cord Injury

ObjectiveThe aim of this study was to investigate the key molecular signatures involved in the pathophysiological changes that occur during the course of spinal cord injury(SCI)through proteomics and bioinformatics.Then,transcriptomic changes in macrophages and microglia after SCI were analyzed to reveal the molecular mechanisms underlying the inflammatory response in the mid-to-late stage of SCI.Finally,the role of APOE in the inflammatory response was clarified by comparing the recovery of motor function and histological features in APOE-/-mice and wild-type mice after SCI.MethodsDifferentially expressed proteins at 3,7 and 14 days after SCI in rats versus controls were analyzed using 4-D label-free quantitative proteomics and the differential proteins were compared with differentially expressed genes from two public datasets(GSE115067 and GSE45006).This was followed by a time-course analysis of the protein profiles to find clusters with consistent expression patterns during SCI progression.Finally,the expression and cellular localization of the target proteins were verified using immunoblotting(WB)and immunofluorescence staining.In addition,we performed data mining on the single-cell transcriptome dataset(GSE159638)to find differential genes and pathways in macrophages and microglia at 10 and 21 days after injury.We also searched for genes whose expression was upregulated in macrophages and microglia at 10 and 21 days after injury by a time-course analysis and their functional characteristics.It was also verified by the SCI model of mice,WB,immunofluorescence staining and electron microscopy.Finally,the role of APOE in the inflammatory response after SCI was assessed by comparing the behavioral,immunofluorescence staining and electron microscopy changes of APOE-/mice and wild-type mice after SCI.ResultsThe proteins that were differentially expressed at 3,7 and 14 days after SCI in rats were mainly enriched in the complement and coagulation cascades,cholesterol metabolism and lysosomal pathways,of which 75 genes were also differentially expressed in the public dataset,such as ANXA1,C1QC,CTSZ,GM2A,GPNMB and PYCARD.Protein clusters that were upregulated at all 3 time points after SCI were associated with immune response,lipid regulation,lysosome pathway,and myeloid cells,and five of these proteins were validated by WB and immunofluorescence staining.In the single-cell dataset,macrophages were mainly involved in leukocyte migration and chemotaxis in the early stage of injury,and in the middle and late stage of injury,they were mainly involved in lymphocyte mediated immunity and antigen processing and presentation.Microglia were involved in response to tumor necrosis factor,DNA replication,and reactive oxygen species metabolic process in the early stage of injury,and mainly involved in lysosome organization,lipid transport and storage,and remyelination in the mid-to-late stage of injury.APOE,a hub gene for macrophages and microglia in the mid-to-late stage of injury,was significantly upregulated,and was also involved in biological processes such as endocytosis,lipid localization,oxidative stress and inflammatory response.Both macrophages and microglia phagocytosed myelin debris after SCI,and highly expressed APOE gene,accompanied by a significant increase in intracellular lipid droplet accumulation and lysosomes.Compared with wild-type mice,APOE-/-mice recovered motor function significantly worse after SCI,accompanied by increased inflammatory infiltration,while astrocytes phagocytosed myelin and promoted inflammatory cell infiltration.ConclusionThe complement and coagulation cascades,cholesterol metabolism and lysosome pathways are the main pathways involved in the progression of SCI in rats,and proteins that are consistently upregulated in the acute and subacute phases are associated with myeloid cells,immune response,lipid regulation and lysosomal function.APOE expression is significantly elevated in both macrophages and microglia in the mid-to-late stage of injury and is associated with multiple genes and pathways.APOE may affect motor function recovery by modulating the inflammatory response.