Identication of acute spinal cord injury and autophagy-related potential key genes (cid:0) pathways, and targeting drugs through bioinformatics analysis

Background: Acute spinal cord injury (ASCI) is considered a form of severe central nervous system damage. At present, research in the elds of spinal surgery and neurology has highlighted the complex mechanisms underlying ASCI, among which autophagy is considered to play a crucial role. Objectives: We aimed to identify the genes and molecular pathways associated with ASCI and autophagy using computational tools and publicly available data, and to identify drugs targeting the relevant genes associated with ASCI and autophagy. Materials and Methods: We used text mining to detect the ASCI and autophagy-associated genes, and the intersection of the two gene sets was selected for gene ontology analysis using the DAVID program. We then constructed protein–protein interaction networks, followed by a functional enrichment analysis, from which we obtained two signicant gene modules. Finally, the nal list of genes was queried against the Drug Gene Interaction database to nd drug candidates targeting the relevant ASCI and autophagy genes. Results: Our analysis identied 156 genes common to both the “ASCI” and “Autophagy” text mining concepts. Gene enrichment analysis yielded two signicant gene modules (20 genes), which represent six signicant signal pathways and could be targeted by 28 Food and Drug Administration (FDA)-approved drug molecules, and identied the drug–gene interactions. Conclusion: In conclusion, we presented a method to explore the potential key genes, molecular pathways and candidate drugs associated with ASCI and autophagy. As a result, in this method, we identied a total of 20 potential genes, six signicant pathways and 28 candidate drugs, which could provides a basis for new trials and the development of novel targeted therapies as potential treatments for ASCI. and MAPK14: The proteins encoded by MAPK3 and MAPK14 are the members of the mitogen-activated protein (MAP) kinase family. This kinase plays a signicant role in stress-related transcription and cell cycle regulation, as well as in genotoxic stress responses. In terms of candidate drugs targeting genes, sulfonic acid is a specic inhibitor of MAPK3 and arsenic trioxide is its specic inducer.


Introduction
Acute spinal cord injury (ASCI) is considered a form of severe central nervous system damage, which often leads to a partial or complete motor, sensory, and autonomous function loss below the injured segment, and results in are exia. Common causes of ASCI include high-altitude falls, tra c accidents, heavy pound injury, and sports injuries [1]. Since the second half of the 20th century, with the rapid development of China's economy, the annual incidence of spinal cord injuries (SCIs) in Beijing, the capital of China, has increased every year, reaching 60.6 /1 million, and the global annual incidence rate has reached 15-40 /1 million [2,3]. However, an effective treatment for ASCI remains to be found. At present, the research in the elds of spinal surgery and neurology has highlighted the complex mechanisms underlying ASCI [4,5], with autophagy being considered as one of the important processes involved [6].
Autophagy is an essential lysosome-dependent cellular catabolic pathway that degrades cytoplasmic proteins, protein aggregates, and organelles. Although under certain conditions pathologically increased autophagy has been implicated in cell death, it is considered cytoprotective under most circumstances.
Basal levels of autophagy are important for homeostasis in all types of cells, and are especially crucial in terminally differentiated cells, such as neurons and oligodendrocytes [7].
Many gene expression pro ling studies have focused on ASCI and autophagy in the last decade, and hundreds of candidate genes have been identi ed. These genes have different functions and are involved in a variety of processes [8,9]. This study was designed to identify the potential key genes and molecular pathways associated with ASCI and autophagy via bioinformatics methods, and to explore drugs targeting the relevant genes associated with ASCI and autophagy. First, we made a preliminary list of related genes by mining the literature. Subsequently, we performed functional and signaling pathway analyses using the online bioinformatics resource DAVID. Next, we constructed PPI networks of the common genes and identi ed two signi cant gene modules. Finally, based on the drug-gene interaction analysis of the nal genes, we identi ed candidate drugs. Using this approach, we identi ed some potentially important ASCI and autophagy-related genes, signi cant pathways and candidate drugs, which could provides a basis for new trials and the development of novel targeted therapies as potential treatments for ASCI.

Text mining
The web-based service GenCLip3 was used to perform text mining (http://ci.smu.edu.cn/genclip3/analysis.php). When a query is performed, GenCLip3 extracts all the gene names found in the available literature related to the search concepts [10]. We performed two queries: one for the concept termed acute spinal cord injury (ASCI), and one for the concept termed autophagy. We then extracted all the unique gene hits from each result. The intersection of these two gene sets was used in the subsequent analyses.

Gene Ontology (GO) Enrichment And Pathway Analysis
Gene ontology [11] is a structured vocabulary of terms describing gene products according to their biological process (BP), molecular function (MF), and cellular component (CC). The Kyoto Encyclopaedia of Genes and Genomes (KEGG) [12] provides data resources of known biological metabolic pathways.
We used DAVID [13], a web-accessible program that integrates functional genomic annotations with intuitive graphical summaries, to view the GO and KEGG enrichment of the common genes. A p value of < 0.05 was considered statistically signi cant.

PPI networks and module analysis
The Search Tool for the Retrieval of Interacting Genes (STRING, Version 11.0) [14] database was used to retrieve the common genes, encoded proteins and PPI network information. This database contains over 24.6 million proteins and 2 billion interactions observed in 5,090 organisms. We uploaded the common genes to the STRING database and set the interaction score to > 0.900 (highest con dence) as the signi cance threshold. Following this, PPI networks were constructed using the Cytoscape software [15].

Page 4/20
The Molecular Complex Detection (MCODE) built in Cytoscape is an automated method that was used to analyse highly interconnected modules as molecular complexes or clusters. The analysis parameters were set to default. The functional enrichment analysis was executed for the common genes, from which two signi cant gene modules were identi ed with p < 0.05 set as the signi cance threshold.

Drug-gene Interaction
We used the Drug Gene Interaction Database (DGIdb, http://www.dgidb.org) to explore drug-gene interactions in the nal list of genes, which were used as the potential targets in a search for existing drugs [16]. Our criteria for drug selection required FDA-approval and the presence of de ned drug-gene interactions. These candidate drugs targeting the genes/pathways relevant to ASCI and autophagy may represent potential treatment strategies.

Text mining
Based on the data mining strategy, 4178 genes identi ed were related to autophagy, 210 genes were related to ASCI, and 156 genes were common to both lists ( Fig. 1 and Table 1).

GO enrichment and pathway analysis
To further explore the potential targets of these common genes in ASCI and autophagy, we performed GO and pathway analyses on these common genes with the criterion of having a p value of < 0.05 (Fig. 2). Figure 3 shows the top six signi cant terms for each of the following: the BP, CC, MF, and KEGG pathways of the common genes, respectively.
We also show the annotation of the common genes. As shown in Table 2, in the BP group, the common genes were mainly enriched for genes involved in the regulation of programmed cell death and regulation of the apoptotic process. In the CC group, the common genes were mainly enriched for genes associated with the extracellular space, axon, neuron projection, cell body, neuron part, and membrane raft. In the MF group, the common genes were primarily enriched for genes associated with cytokine receptor binding, receptor binding, growth factor activity, enzyme binding, cytokine activity, and identical protein binding. In the KEGG pathway group, the enrichment was observed for genes in the tumour necrosis factor (TNF) signaling pathway, cancer pathways, as well as hepatitis B, Chagas disease, neurotrophin, and hypoxiainducible factor-1 (HIF-1) signaling pathways.

PPI network and module analysis
All the common genes were uploaded to the STRING website and were analysed using the Cytoscape software. A total of 146 nodes with 451 edges with scores > 0.900 (highest con dence) were selected to construct the PPI networks (Fig. 3). Two signi cant modules were selected using the MCODE plug-in. Module 1 consisted of 12 nodes/genes and 32 edges (Fig. 4), which were mainly associated with response to the peptide (BP), cytosol (CC), phosphatase binding (MF), and the mammalian target of rapamycin (mTOR) signaling pathway (KEGG) ( Table 3). Module 2 consisted of 8 nodes/genes and 18 edges (Fig. 5), which were mainly associated with the apoptotic process (BP), extracellular space (CC), protease binding (MF), and Toll-like receptor signaling pathway (KEGG) ( Table 4).

Discussion
We classi ed and summarised the nal list of 20 genes, their targeted drugs, and the signaling pathways involved.
Genes, gene-targeted drugs, and gene-mediated signaling pathways associated with neuroin ammation in ASCI.
NFKB1 and RelA NFKB1 and RelA are important members of the NFKB family. NF-κB is a rapidly acting primary transcription factor found in all cell types. It is involved in the cellular responses to stimuli, such as cytokines and stress, and plays a key role in regulating the immunological response to infections. In terms of candidate drugs targeting genes, thalidomide is a speci c antagonist of NFKB1.
IL1B The protein encoded by IL1B is a member of the interleukin 1 cytokine family. This cytokine is an important mediator of the in ammatory response, and is involved in a variety of cellular activities, including cell proliferation, differentiation, and apoptosis. In terms of candidate drugs targeting genes, canakinumab, gallium and nitrate rilonacept are its inhibitor.
NLRP3 Jiang et al. demonstrate that pharmacologic suppression of NLRP3 in ammasome activation controls neuroin ammation, attenuates mitochondrial dysfunction, alleviates the severity of spinal cord damage, and improves neurological recovery after SCI [17].
TLR4 The protein encoded by TLR4 is a member of the Toll-like receptor (TLR) family which plays a fundamental role in pathogen recognition and activation of innate immunity. They recognize pathogenassociated molecular patterns that are expressed on infectious agents, and mediate the production of cytokines necessary for the development of effective immunity.
IL6 IL6 encodes a cytokine that functions in in ammation and the maturation of B cells. The protein is primarily produced at sites of acute and chronic in ammation, where it is secreted into the serum and induces a transcriptional in ammatory response through interleukin 6 receptor, alpha. In terms of candidate drugs targeting genes, siltuximab is a speci c inhibitors of this gene.

STAT3
The protein encoded by STAT3 is a member of the STAT protein family. In response to cytokines and growth factors including IFNs, EGF, IL5, IL6, HGF, LIF and BMP2, STAT family members are phosphorylated by the receptor associated kinases, and then form homo-or heterodimers that translocate to the cell nucleus where they act as transcription activators. This protein mediates the expression of a variety of genes in response to cell stimuli, and thus plays a crucial role in many cellular processes such as cell growth and apoptosis. In terms of candidate drugs targeting genes, acitretin is a speci c inhibitor of this gene.
The prognosis of spinal cord injury (SCI) is closely related to secondary injury, which is dominated by neuroin ammation. Considerable studies have shown that autophagy can be activated through the NLR and TLR/NF-κB signaling pathways after SCI. However, activation of autophagy can inhibit the activity of these pathways, thus playing a role in reducing the in ammatory response and protecting the injured spinal cord [18,19,20,21,22].
Genes, gene-targeted drugs, and gene-mediated signaling pathways associated with complement and coagulation cascades after ASCI Spinal cord ischemia, due to thrombosis after SCI, is one of the important mechanisms of secondary SCI. The complement and coagulation cascades involve two major genes, SERPINC1 and PROC. SERPINC1: The protein encoded by this gene, antithrombin III (ATIII), is a plasma protease inhibitor and a member of the serpin superfamily. In terms of candidate drugs targeting genes, activatorheparin calcium is a speci c activator of it.
PROC This gene encodes a vitamin K-dependent plasma glycoprotein. The encoded protein is cleaved to its activated form by the thrombin-thrombomodulin complex. This activated form contains a serine protease domain and functions in degradation of the activated forms of coagulation factors V and VIII.
Albumin (ALB) reduces oedema of the injured area after ASCI ALB gene encodes the most abundant protein in human blood. This protein functions in the regulation of the blood plasma colloid osmotic pressure and acts as a carrier protein for a wide range of endogenous molecules, including hormones, fatty acids, and metabolites, as well as exogenous drugs.
Following acute SCI, 5% ALB is often used in the clinic to increase the plasma colloid osmotic pressure, to reduce the oedema of the injured area. ALB is also routinely used in neurosurgical procedures and often combined with mannitol 20% (MAN). Palmaers et al., demonstrated that clinically relevant dilutions of MAN+ALB showed signi cant inhibition of blood coagulation and platelet function [23].
Three signi cant signaling pathways, which include ve key genes, reduce apoptosis and neuroin ammation in ASCI by enhancing autophagy.
The AMPK/mTOR signaling pathway Zhou et al. showed that SPT ameliorates the AMPK/mTOR signaling-induced autophagy and thereby improves functional recovery in SCI-induced rats [24]. Furthermore, Meng et al. and Wang et al. showed that resveratrol promoted functional recovery and inhibited neuroin ammation through the activation of autophagy, mediated by the AMPK/mTOR pathway following SCI [25,26]. In general, the activation of this pathway enhances autophagy to protect the injured spinal cord.
The phosphatidylinositol-3-kinase (PI3K)/protein kinase B(Akt)/mTOR signaling pathway Wang et al. showed that autophagy protects spinal cord neurons against PI3K/Akt/mTOR-mediated apoptosis after mechanical injury [27]. Furthermore, Li et al. showed that melatonin (MT) can improve the recovery of locomotor function by enhancing autophagy as well as reducing apoptosis after SCI in rats, probably via the inhibition of the PI3K/Akt/mTOR signaling pathway [28]. In conclusion, the inhibition of this pathway can enhance autophagy to protect the injured spinal cord.

The AMPK-forkhead box O-3 (FOXO3) signaling pathway
Zhang et al. demonstrated that liraglutide was therapeutically bene cial in treating spinal contusion injury and its underlying mechanism was the activation of autophagic responses through the AMPK-FOXO3 signaling pathway [29]. Thus, the activation of this pathway can enhance autophagy to protect the injured spinal cord.
AKT1 The serine-threonine protein kinase encoded by the AKT1 gene is catalytically inactive in serumstarved primary and immortalized broblasts. AKT1 and the related AKT2 are activated by the plateletderived growth factor. It was shown that the activation occurs through phosphatidylinositol 3-kinase. In the developing nervous system, AKT is a critical mediator of growth factor-induced neuronal survival. In terms of candidate drugs targeting genes, arsenic trioxide is a speci c inducer of this gene. Everolimus and Nel navir are speci c inhibitors of it.
FOXO3: FOXO3 belongs to the forkhead family of transcription factors, which are characterized by a distinct forkhead domain. This gene likely functions as a trigger for apoptosis through the expression of genes necessary for cell death.
CDKN1B: CDKN1B encodes a cyclin-dependent kinase inhibitor, which shares a limited similarity with CDK inhibitor CDKN1A/p21. The encoded protein binds to and prevents the activation of cyclin E-CDK2 or cyclin D-CDK4 complexes, and thus controls the cell cycle progression at G1. The degradation of this protein, which is triggered by its CDK dependent phosphorylation and subsequent ubiquitination by SCF complexes, is required for the cellular transition from quiescence to the proliferative state.
MAPK3 and MAPK14: The proteins encoded by MAPK3 and MAPK14 are the members of the mitogenactivated protein (MAP) kinase family. This kinase plays a signi cant role in stress-related transcription and cell cycle regulation, as well as in genotoxic stress responses. In terms of candidate drugs targeting genes, sulfonic acid is a speci c inhibitor of MAPK3 and arsenic trioxide is its speci c inducer.
There are ve signi cant genes, which can be used as important directions for future research in ASCI Beta/A4 protein precursor (APP) Although a precursor of Alzheimer amyloid, substantial evidence suggests that APP is involved in the regulation of neuronal growth and survival. Recently, Bowes et al. have demonstrated that the trophic properties of APP are completely preserved in a 17-amino acid sequence. If APP is neurotrophic, then it would be anticipated that the administration of the growth-promoting segment of the APP 17-mer peptide might attenuate the neuronal dysfunction or loss or behavioural de cits associated with neuronal injuries, such as those accompanying CNS ischemia [30].

Cystatin C (CST3)
Cystatin C, which belongs to the type II cystatin gene family, is a potent inhibitor of lysosomal proteinases. Zhang et al. showed that CysC levels are increased in patients with acute SCI, possibly as a direct result of the injury. Serum CysC is a potential biomarker of SCI [31]. The protein encoded by MAP3K7 is a member of the serine/threonine protein kinase family. This kinase mediates the signaling transduction induced by TGF beta and morphogenetic protein (BMP), and controls a variety of cell functions including transcription regulation and apoptosis.
There are two limitations in the our study: Firstly, the information on the functions or roles of the nal list of 20 genes have not been veri ed through experiments but via databases used. Thus, further molecular biological experiments are required to con rm the function of these identi ed genes. Secondly, not all existing gene interactions are known for a given drug. Therefore, it is possible that drugs which could potentially be useful were missed or ignored because their gene interactions have not yet been fully elucidated.

Conclusion
In conclusion, the mechanisms underlying secondary SCI, usually include in ammation, ischemia, oedema, and apoptosis. Autophagy could regulate these injury mechanisms through different genes and their signaling pathways, thus playing a role in protecting the damaged spinal cord. In this article, we presented a method to explore the potential key genes, molecular pathways and candidate drugs associated with ASCI and autophagy. As a result, in this method, we identi ed a total of 20 potential genes, six signi cant pathways and 28 candidate drugs, which could provides a basis for new trials and the development of novel targeted therapies as potential treatments for ASCI. However, further molecular biological experiments are required to con rm the function of these identi ed genes, molecular pathways and candidate drugs in ASCI and autophagy.  The most signi cant module 1 from the PPI network