The Study Of Subacute Spinal Cord Injury Microenvironment By Proteomics Analysis

【Objective】Spinal cord injury(SCI)is a disease associated with high disability and mortality rates.The subacute phase of SCI may play a major role in the process of secondary injury.Changes in the expression levels of proteins have been shown to play key roles in many central nervous system(CNS)diseases.Nevertheless,the roles of proteins in the subacute phase of SCI are not clear.In this study,the expression level of differentially expressed proteins(DEPs)after SCI in rats was detected by isobaric tagging for relative and absolute protein quantification(iTRAQ)technique,and the functional annotation of DEPs was carried out to find the potential targets of subacute phase,so as to provide theoretical basis for repairing SCI.【Methods】1.Experimental grouping: 72 adult female Wistar rats were randomly divided into 8 groups(9 rats in each group).Group A,sham group.Group B,0 hour post-SCI.Group C,4 hour post-SCI.Group D,8 hour post-SCI.Group E,1 day post-SCI.Group F,3 days post-SCI.Group G,1 week post-SCI.Group H,2 weeks post-SCI.2.Establishment of SCI animal model: the use of New York University(NYU)Impactor Model II was used to establish T10 SCI model of Wistar rats with the weight of 10 grams and height of 25 mm.The control group only performed laminectomy at the same level.The experimental group and the control group rats were received cardiac perfusion at 0 hour,4 hours,8 hours,1 day,3 days,1 week and 2 weeks after SCI respectively,and 1 cm tissue of spinal cord around the injured area was removed.3.Proteomics analysis: iTRAQ technique was performed to detect the expression level of differentially expressed proteins between SCI group and control group.Then we analyzed the enrichment of Gene Ontology(GO)function,Kyoto Encyclopedia of Genes and Genomes(KEGG)signal pathway,and the Search Tool for the Retrieval of Interacting Genes(STRING)protein-protein interaction(PPI)network,and the top 10 high-degree core nodes were identified.【Results】1.Differentially expressed proteins after SCI:Compared with the control group,in group B,the number of DEPs was 68,of which 51 were up-regulated and 17 were down-regulated;in group C,the number of DEPs was 76,of which 72 were up-regulated and 4 were down-regulated;in group D,the number of DEPs was 88,of which 84 were up-regulated and 4 were down-regulated;in group E,the number of DEPs was 64,of which 61 were up-regulated and 3 were down-regulated;in group F,the number of DEPs was 176,of which 114 were up-regulated and 62 were down-regulated;in group G,the number of DEPs was 295,of which 204 were up-regulated and 91 were down-regulated;in group H,a total of 162 DEPs were identified,among which 101(62.35%)were up-regulated and 61(37.65%)were down-regulated.2.Bioinformatics analysis of differentially expressed proteins 2 weeks post-SCI: This study focused on the bioinformatics analysis of differentially expressed proteins 2 weeks post-SCI.According to the GO enrichment results,transporter activity,monovalent inorganic cation transmembrane transporter activity,lysosome,neurotransmitter transport and response to steroid hormone were the enriched GO terms.The KEGG analyses results showed that lysosome,glycosaminoglycan degradation,phagosome,cGMP-PKG signaling pathway and thyroid hormone synthesis were enriched pathways.The top 10 high-degree hub nodes included serum albumin(Alb),calmodulin(Calm1),vimentin(Vim),apolipoprotein E(ApoE),synaptophysin(Syp),protein disulfide-isomerase(P4hb),the CD68 molecule(CD68),elongation factor 1-alpha 2(Eef1a2),Ras-related protein Rab-3A(Rab3a)and galectin(Lgals3)according to the STRING network.【Conclusion】1.In this study,differential protein expression profiles of subacute phase of SCI were successfully established.We found that Alb,Calm1,Vim,Apoe,Syp,P4 hb,Cd68,Eef1a2,Rab3 a and Lgals3 may be key proteins that related to the repair of SCI.2.In this study,lysosome,glycosaminoglycan degradation,phagosome,cGMP-PKG signaling pathway and thyroid hormone synthesis were found to be the key signal pathway in subacute phase of SCI.3.The mechanism of differential protein in subacute phase of SCI was further explored,which may provide a new idea for repairing SCI.