| Background: Spinal cord injury(SCI)is a devastating condition that usually results in irreversible loss of sensory and voluntary motor function below the site of injury.The pathophysiologic process of spinal cord injury includes primary mechanical injury and a secondary series of cascading injury process events that cause progressive degeneration and necrosis of the spinal cord.These secondary events include ischemia,hypoxia,vascular injury,post-ischemic reperfusion,free radical formation,sodium-and calciummediated cellular injury,excitotoxicity of glutamate and disturbances in ion homeostasis,oxidative cellular damage,intense inflammatory response,edema,demyelination,Wallerian degeneration,axonal atrophy,and neuronal cell death.Secondary damage amplifies primary damage and promotes cystic degeneration,matrix remodeling,and glial scar evolution and formation thereby preventing neurological recovery.The biological process of secondary injury after spinal cord injury is known to involve different cells and molecules in the immune,vascular and nervous systems.For example,Plexin-B2,encoded by Plxnb2,was originally thought to be an axon guidance molecule that is upregulated in injury-activated microglia and macrophages and is required for motor-sensory recovery after spinal cord injury.SIRT6,a member of the sirtuin family,plays an important role in controlling DNA repair,telomere maintenance,and inflammatory responses.Upregulation of SIRT6 can alleviate inflammation and oxidative stress and prevent apoptosis in spinal cord injury.Interferon gene stimulating factor(STING)activates innate immune responses regulated by type I interferon and plays a key role in the regulation of inflammation.Knockdown of STING resulted in phosphorylation of NF-?B and map kinase(MAPK),which attenuated the inflammatory response and promoted functional recovery after spinal cord injury in mice.Analysis of genetic changes contributes to the understanding of the molecular mechanisms of injury caused by cascading,waterfall-like,complex networks of the molecular regulation of gene expression and downstream signaling pathways following spinal cord injury.The main clinical treatment for secondary injury after acute spinal cord injury is through high-dose hormone methylprednisolone shock therapy,which causes multiple side effects making it more controversial.Although the latest treatment methods are many,such as blood pressure control,therapeutic hypothermia,cerebrospinal fluid drainage,drug therapy: riluzole,minocycline,GM-1 ganglioside,cytokine therapy: fibroblast growth factor,granulocyte colony-stimulating factor,hepatocyte growth factor,myelinassociated pathway protein antibody and inhibitor therapy,cell therapy: neural stem/precursor cells,mesenchymal stem cells The effect of cell therapy: neural stem/progenitor cells,MSCs,olfactory sphincter cells,and biomaterials transplantation therapy,etc.,is still not very satisfactory.Bioinformatics uses computer science to collect,process,and analyze big data obtained by gene chips and sequencing technologies.It allows us to perform large-scale screening of genetic changes in disease development at the microscopic level.It has become an effective method for studying the mechanisms of refractory diseases.Research is increasingly using gene microarray technology to identify aberrantly expressed genes that are closely linked to disease pathogenesis.These genes regulate inflammatory factors,chemokines,various protein molecules and cellular signaling pathways through translation and transcription,deriving distinct protein phenotypes that cause alterations in normal biological functions and ultimately lead to disease onset,development,and evolution.In recent years,microarray technology has been widely used to study a variety of biological mechanisms,and more and more research using microarray technology,to study the pathophysiology of the disease,especially in oncology,genetics,immunology,and inflammatory diseases have numerous studies,but relatively little bioinformatics research on trauma,such as spinal cord injury,while due to the complexity of life science systems,the phenotype of organisms and genes are not simply One to one correspondence,thousands of protein interactions are involved in molecular signal transduction,regulation,and action in a network that affects the downstream biological functions.In the physiological and pathological processes of spinal cord injury,these genes to molecular signaling pathways to protein-protein interactions,ultimately causing functional changes,often involve the participation and mutual regulation of these numerous genes or protein molecules.With the rapid development of bioinformatics technologies that integrate computer science,advanced mathematics,statistics,bioengineering,and other scientific expertise,such as big data analysis,natural language processing,machine learning,neural networks,deep learning,artificial intelligence,data mining,nanomaterials,and cellular simulation,etc.,the study of spinal cord injury provides multidisciplinary,multi-technical,multi-faceted,and multi-faceted support.support.Through multidisciplinary progress,joint development and cooperation,we analyze a large amount of genetic and protein network data from the molecular environment in the organism,simulate biological processes and functions,and explore the laws and algorithms behind the mechanisms,dynamically monitor the changes and progress of tissue repair,reconstruction,and regeneration,and enable more effective intervention and treatment,so that people can have a more intuitive and profound understanding of the physiological,pathological and functional mechanisms behind spinal cord injury and other diseases.This will provide a more intuitive and profound understanding of the physiological,pathological,and functional mechanisms behind spinal cord injury and other diseases.The secondary cascade waterfall-like inflammatory storm response after spinal cord injury is characterized by the involvement of multiple genes,however,little is known about the potential molecular mechanisms underlying the interaction of these genes and the downstream signaling pathways,protein-molecular network interactions,etc.,in the response to injury and the recovery of neurological function.Therefore,to better explore and understand the molecular mechanisms after spinal cord injury,the identification and screening of the differential temporal expression of these genes at different time points after spinal cord injury,the molecular signaling pathways enriched by these genes,and the exploration of downstream protein molecular networks are expected to provide new insights and ideas for spinal cord injury research through bioinformatics studies at the gene level.Purpose: To gain insight into the molecular mechanisms at different time points after spinal cord injury,this study was conducted in the following three parts: Part I:Bioinformatics analysis of differential genes at different time points after spinal cord injury(overall analysis)Part II: Screening of CCL2 autophagy-related HUB genes and molecular signaling pathways after spinal cord injury based on a combination of GEO database and autophagy database and validation(targeted analysis).Part III: Regulation of autophagy,inflammation,and apoptosis by CCL2 via PI3 K signaling pathway in a spinal cord medullary damage cell model(PC-12 cells).Part IV: Effects of CCL2 on functional recovery after spinal cord injury in rats via PI3 K signaling pathway.Methods: Gene expression profile data of rats(GSE45006 and GSE464)were downloaded from the GEO public database high-throughput sequencing website,from which the gene expression matrix files of each key time point after spinal cord injury were screened,and the data of each time point were annotated with gene probes,transformed,samples were merged,batch effects were corrected,data were cleaned,and normalized by computer Perl and R language.The normalized files were obtained by using the R language limma package to obtain differentially expressed significant genes,and these differentially expressed genes were subjected to gene ontology enrichment analysis(GO enrichment analysis),Kyoto Gene and Genome Encyclopedia pathway analysis(KEGG enrichment analysis),and gene set enrichment analysis(GSEA enrichment analysis).STRING online database was used to analyze and visualize the protein-protein interaction network of these differentially expressed genes,and the 30 key genes with the highest weights at each key time point were obtained by Cytoscape software and the MCC algorithm of Cytohubba plug-in.ROC curves were drawn based on Hub gene expression data and post-spinal cord injury status.We used the p ROC package in R language to plot the ROC curves for each Hub gene.The expression of these Hub genes in the normal and spinal cord injury groups was inferred from this data matrix,and the correlation of these Hub genes with clinical diagnosis after spinal cord injury was analyzed.Prediction of genes associated with regeneration was performed by GOPLOT in R language.The gene expression matrix files of autophagy genes were obtained by taking the intersection of the autophagy gene database with the aforementioned GEO database rat gene expression database,and the differential expression heat map of these autophagy genes and their expression in spinal cord injured and normal control spinal cord tissues were derived.The key autophagy-related genes CCL2 and the molecular signaling pathway PI3 K,which are closely related to the regenerative repair of spinal cord injury,were screened out from these autophagy genes,and finally,the autophagy and function of CCL2 autophagy gene and PI3 K molecular signaling pathway were experimentally validated by cellular and animal levels.Results: In this experiment,we downloaded two rat gene expression profile data from GEO open database,including thoracic spine matrix data from the normal and spinal cord injury groups at different time points after SCI,for analysis.We obtained 20 up-regulated genes and 20 down-regulated genes that were significantly differentially expressed at each time point,and performed GO analysis on these DEGS,and obtained three categories of biological processes(BP),cellular composition(CF),and molecular functions(MF)that these DEGS were enriched into.The main enrichments in the biological processes category were the following: regulation of neurotransmitter levels,vesicle-mediated transport in synapses,learning or memory,cognition,neurotransmitter transport,regeneration,wound healing,response to lipopolysaccharide,and regulation of membrane potential.Among them,the following are mainly enriched in the cellular composition category: neuronal synaptic terminals,transport vesicles,postsynaptic membrane,asymmetric synapses,postsynaptic density,postsynaptic specialization,neuron-to-neuron synapses,distal axons,synaptic membranes,and axon terminals.Among them,the following molecular functional categories were mainly enriched:ligand-gated ion channel activity,metal ion transmembrane transporter activity,carboxylic acid-binding,organic acid-binding,cell adhesion molecule binding,gated channel activity,ligand-gated channel activity,integrin binding,transmitter-gated ion channel activity involved in post-synaptic membrane potential regulation and neurotransmitter receptor activity involved in post-synaptic membrane potential regulation.Methods: Gene expression profile data of rats(GSE45006 and GSE464)were downloaded from the GEO public database high-throughput sequencing website,from which the gene expression matrix files of each key time point after spinal cord injury were screened,and the data of each time point were annotated with gene probes,transformed,samples were merged,batch effects were corrected,data were cleaned,and normalized by computer Perl and R language.The normalized files were obtained by using the R language limma package to obtain differentially expressed significant genes,and these differentially expressed genes were subjected to gene ontology enrichment analysis(GO enrichment analysis),Kyoto Gene and Genome Encyclopedia pathway analysis(KEGG enrichment analysis),and gene set enrichment analysis(GSEA enrichment analysis).STRING online database was used to analyze and visualize the protein-protein interaction network of these differentially expressed genes,and the 30 key genes with the highest weights at each key time point were obtained by Cytoscape software and the MCC algorithm of Cytohubba plug-in.ROC curves were drawn based on Hub gene expression data and post-spinal cord injury status.We used the p ROC package in R language to plot the ROC curves for each Hub gene.The expression of these Hub genes in the normal and spinal cord injury groups was inferred from this data matrix,and the correlation of these Hub genes with clinical diagnosis after spinal cord injury was analyzed.Prediction of genes associated with regeneration was performed by GOPLOT in R language.The gene expression matrix files of autophagy genes were obtained by taking the intersection of the autophagy gene database with the aforementioned GEO database rat gene expression database,and the differential expression heat map of these autophagy genes and their expression in spinal cord injured and normal control spinal cord tissues were derived.The key autophagy-related genes CCL2 and the molecular signaling pathway PI3 K,which are closely related to the regenerative repair of spinal cord injury,were screened out from these autophagy genes,and finally,the autophagy and function of CCL2 autophagy gene and PI3 K molecular signaling pathway were experimentally validated by cellular and animal levels.Results: In this experiment,we downloaded two rat gene expression profile data from GEO open database,including thoracic spine matrix data from the normal and spinal cord injury groups at different time points after SCI,for analysis.We obtained 20 up-regulated genes and 20 down-regulated genes that were significantly differentially expressed at each time point,and performed GO analysis on these DEGS,and obtained three categories of biological processes(BP),cellular composition(CF),and molecular functions(MF)that these DEGS were enriched into.The main enrichments in the biological processes category were the following: regulation of neurotransmitter levels,vesicle-mediated transport in synapses,learning or memory,cognition,neurotransmitter transport,regeneration,wound healing,response to lipopolysaccharide,and regulation of membrane potential.Among them,the following are mainly enriched in the cellular composition category: neuronal synaptic terminals,transport vesicles,postsynaptic membrane,asymmetric synapses,postsynaptic density,postsynaptic specialization,neuron-to-neuron synapses,distal axons,synaptic membranes,and axon terminals.Among them,the following molecular functional categories were mainly enriched: ligand-gated ion channel activity,metal ion transmembrane transporter activity,carboxylic acid-binding,organic acid-binding,cell adhesion molecule binding,gated channel activity,ligand-gated channel activity,integrin binding,transmitter-gated ion channel activity involved in post-synaptic membrane potential regulation and neurotransmitter receptor activity involved in post-synaptic membrane potential regulation.Kyoto gene and genomic encyclopedia pathway analysis of these differentially expressed genes and gene set enrichment analysis yielded the following molecular signaling pathways: GABAergic synapses,glutamate synapses,nicotine addiction,amyotrophic lateral sclerosis(ALS),c AMP signaling pathway proteoglycans in cancer,cardiomyocytes adrenergic signaling,relaxin signaling pathway insulin secretion,platelet activation,Rap1 signaling pathway,AGE-RAGE signaling pathway in diabetic complications,neuroactive ligand-receptor interactions,pancreatic secretion,amebiasis,thyroid hormone synthesis,serotonin synapses,aldosterone synthesis and secretion,proximal tubular bicarbonate recycling,synaptic vesicle recycling,cocaine addiction,salivary secretion,chemokine signaling pathway,Rheumatoid arthritis Inflammatory mediator regulation of TRP channels ECM-receptor interactions,TNF signaling pathway,long-term potentiation,osteoclast differentiation,calcium signaling pathway,computation of endocrine and other factor regulation,human cytomegalovirus infection,MAPK signaling pathway.Relaxin signaling pathway,IL-17 signaling pathway,HIF-1 signaling pathway,Toll-like receptor signaling pathway,NF-kappa B signaling pathway,apoptosis,JAK-STAT signaling pathway,PI3K-Akt signaling pathway,PPAR signaling pathway and p53 signaling pathway,and measles pathway.To obtain PPI data,genes differentially expressed at each time point were submitted to the STRING database.Cytoscape(version 3.8.0)was used to construct the protein interaction network,which shows the relationship and function of differentially expressed genes,which was built by removing isolated nodes.The 1-day PPI network consists of 451 nodes and 1877 connections.3-day PPI network consists of 513 nodes and 1869 connections.7-day PPI network consists of 531 nodes and 2344 connections.14-day PPI network consists of 451 nodes and 1877 connections.Then the histogram of the top 30 genes at each time point was obtained using R software.The 30 Hub genes with the highest weight at each time point of spinal cord injury were filtered from the differentially expressed genes by the MCC algorithm of Cytoscape plugin Cytohubba.Regeneration-associated Hub genes were obtained by combining datasets from days 1,3,7,and 14 after spinal cord injury,using the GOplot package in R language to analyze and select elements and terms of interest for gene prediction,and then taking the intersection with Hub genes to obtain regeneration-associated Hub genes.We used the human autophagy database HADb to screen for autophagy-related genes.(http://autophagy.lu/)and intersected with the above-obtained gene matrix files to obtain the autophagy-related gene matrix files,and the autophagy-related genes with significant differential expression after spinal cord injury were screened by the R language limma package.The CCL2 gene was also screened out from the autophagy-related genes.The expression of CCL2 autophagy-related genes in the control and spinal cord injury groups at different time points after spinal cord injury was analyzed by the R language "ggpubr" package,and the expression of CCL2 was significantly different in the spinal cord injury group compared with the control group.The gene matrix was calculated by the R language p ROC package for four-time points after spinal cord injury,and we found that the area under the curve of the ROC curve of CCL2 at 4 h,12 h,1 day,and 3 days after spinal cord injury were greater than 90%,91.7%,100%,100%,and 100%,respectively.Conclusion: In summary,we performed a comprehensive bioinformatic analysis of gene expression profiles in rats at four-time points after spinal cord injury.Genes with significant differential expression were successfully screened at each time point.We performed enrichment analysis of these differentially expressed genes: Kyoto gene and genome encyclopedia pathway analysis,and gene set enrichment analysis revealed that they are associated with inflammatory response,tissue regeneration,and injury healing and that PI3K-Akt,MAPK,JAK-STAT,HIF-1,NF-?B,and Toll-like molecular signaling pathways play important roles in spinal cord injury.In addition,30 Hub genes were obtained by constructing PPI,and genes that play key roles in tissue repair,reconstruction,and regeneration after spinal cord injury were obtained by the R goplot package.We used the autophagy database HADb to obtain the CCL2 autophagy-related genes And the regulation of autophagy,inflammation,and apoptosis by CCL2 in PC-12 cells through the PI3 K signaling pathway was verified,and finally,the functional recovery after spinal cord injury by inhibiting CCL2 expression in rat spinal cord injury model was verified by animal experiments.Therefore,this study obtained CCL2 autophagy-related genes and PI3 K signaling pathway by bioinformatics,and experimentally verified the function and mechanism of CCL2 in spinal cord injury,which provides a theoretical basis to further elucidate the molecular mechanism of secondary spinal cord injury and helps the formulation and development of new therapeutic strategies for spinal cord injury. |
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