Identification of common pathway and hub genes in the degeneration of both annulus fibrosus and nucleus pulposus in intervertebral disc

Abstract

Purpose

This study aimed to identify the common pathways and hub genes related to oxidative stress (OS) and autophagy of both annulus fibrosus (AF) and nucleus pulposus (NP) in intervertebral disc degeneration (IDD) based on the data obtained from the Gene Expression Omnibus (GEO) database.

Methods

The Gene expression data for human intervertebral discs was obtained from the GEO database, including the AF and NP of both non-degenerated disc and degenerated disc. The differentially expressed genes (DEGs) were identified using the limma package in R language. DEGs related to OS and autophagy were obtained using Gene Ontology (GO) database. Analyses of the GO, signaling pathways, protein-protein interaction (PPI) networks, and hub genes were performed using AnnotationDbi package, DAVID, GSEA, STRING database, and Cytoscape software, respectively. Finally, the online tool of NetworkAnalyst and the Drug Signatures database (DSigDB) were used to screen for transcriptional factors and potential drugs of the hub genes.

Results

There were 908 genes associated with OS and autophagy found. A total of 52 DEGs were identified, included five upregulated and 47 downregulated genes. These DEGs were mainly involved in mTOR signaling pathway and the NOD-like receptor signaling pathway. The top 10 hub genes were CAT, GAPDH, PRDX1, PRDX4, TLR4, GPX7, GPX8, MSRA, RPTOR, GABARAPL1. Besides, FOXC1, PPARG, RUNX2, JUN, and YY1 were identified as the key regulatory factors of hub genes. L-cysteine, oleanolic acid, and berberine were potential therapeutic agents for the treatment of IDD.

Conclusions

Common hub genes, signaling pathways, transcription factors, and potential drugs associated with OS and autophagy were identified, which provides significant basis for further mechanism research and drug screening of IDD.

Keywords

Oxidative stress gene expression omnibus autophagy intervertebral disc degeneration hub genes

Introduction

Intervertebral disc degeneration (IDD) is a common musculoskeletal disorder,¹ which not only threatens the quality of a patient’s life but also increases the economic burden on society on a global scale.² The prevalence of IDD continues to increase as the population ages.³ Previous researches have confirmed that IDD is a consequence of genetic, aging, traumatic, and obesity, and it is characterized by annulus fibrosus (AF) tears, matrix degradation and water loss in the nucleus pulposus (NP), and calcification of cartilage endplate.^4–6 However, the mechanisms involved in IDD progression remain unknown. In spite of the surgical treatments are quite effective in pain relief for severe IDD, there is still a lack of interventions to delay or even reverse the progression of degeneration in the early and middle stages of the disease. Therefore, further research related to the mechanisms of IDD is necessary, which may contribute to the development of novel therapeutic approaches.

Oxidative stress (OS) and autophagy play significant roles in the development of IDD. OS due to the imbalance between reactive oxygen species (ROS) generation and ROS elimination in the degenerative disc can not only lead to a decline in normal cell numbers of NP,⁷ but also result in damage to the extracellular matrix.⁸ The gradual accumulation of ROS can also damage cellular components (CCs) such as DNA, lipids, and proteins.⁹ In addition, OS can also affect cell repair by regulating autophagy through multiple pathways. Autophagy activated by various stimuli is a protective mechanism which can suppress senescence and apoptosis of NP cells.^10,11 However, most of the current research is focused on the degeneration of NP. Studies on the common mechanism of the NP and AF in IDD are still lacking.

Gene chip, which could detect the gene expression profiling of specific tissues and cells, has been extensively utilized to investigate the mechanisms of diseases at the gene level. Therefore, this study aims to explore the common pathways and hub genes related to OS and autophagy of both AF and NP in IDD by bioinformatics analysis. Moreover, the key transcription factors were also predicted to provide a reference for further research of IDD.

Materials and methods

Data collection and preprocessing

The gene expression data set for human intervertebral discs, GSE147383, was downloaded from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo). GSE147383 was based on the GPL570 platform (Affymetrix Human Genome U133 Plus 2.0 Array) and consisted of four NP samples (normal/degenerated = 2/2) and four AF samples (normal/degenerated = 2/2).

Differentially expressed genes (DEGs) were identified by using the Limma package in R. Absolute log2 fold change (logFC) > 1 and p value <0.05 were set as the cutoff criteria. The ggplot package was used to draw the heat map and volcano plot. The genes related to OS and autophagy in homo sapiens were obtained from the Gene Ontology (GO) database (http://geneontology.org/). The intersection of the genes searched from the GO database and the genes from the GSE147383 was considered as OS and autophagy related genes (OSA-genes) of both NP and AF in IDD.

GO and KEGG analysis

GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed using the clusterProfiler package. Gene annotation was carried out using the ‘AnnotationDbi’ and ‘org.Hs.eg.db’ Bioconductor R packages. The visualization was done using the dplyr package and the ggplot2 package.

Protein-protein interaction network analysis

Protein-Protein Interaction (PPI) networks of DEGs was constructed using the online database resource Search Tool for the Retrieval of Interacting Genes (STRING) (https://string-db.org). A combined score >0.4 was considered statistically significant. The PPI network of the results was drawn using Cytoscape 3.9.1 software. The top 10 hub genes were then determined using the cytoHubba plugin and the maximal clique centrality (MCC) approach.

Prediction of transcription factor and potential drugs

NetworkAnalyst web tool was used to determine the transcriptional factors (TFs) of the hub genes and build the gene-TF regulatory network. Potential drugs/compounds targeting the hub genes were identified using the Drug Signatures database (DSigDB) via Enrichr for follow-up experiments. DSigDB is a new gene set resource that relates drugs/compounds and their target genes and consists of 17,389 unique compounds covering 19,531 genes.¹² The top 10 drugs are presented according to p value, with p < 0.05 considered to be statistically significant. In addition, the verification of related pharmacological effects is made by literature review.

Results

Screening of candidate genes

A total of 20,460 genes were detected in the GSE147383 dataset, and 1021 DEGs were identified using the Limma package. Figure 1 displays the volcanic map of GSE147383 as well as the heat map of all genes. The GO database was searched for 1010 genes associated with OS and autophagy, of which 908 genes were detected in the GSE147383 dataset. Fifty-two DEGs, including five upregulated and 47 downregulated genes related to OS and autophagy, were obtained from the intersection between the DEGs and OSA-genes (Figure 2).

Figure 1.

(a) Volcano plot of differentially expressed genes (DEGs). The red nodes represent upregulated DEGs with p value <0.05 and |logFC|>1. (b) Heat map and clustering analysis of all genes.

Figure 2.

The Venn diagram. DEGs: Differentially expressed genes screened from GSE147383. OSA-genes: Oxidative stress and autophagy related genes downloaded from gene ontology database.

GO and KEGG analysis

The results of GO analysis, including biological process (BP), CC, and molecular function (MF), are shown in Figure 3. The BP of DEGs related to OS and autophagy (OSA-DEGs) was mainly enriched in response to OS, regulation of autophagy, cellular response to chemical stress, and macroautophagy. In the CC category, ‘phagocytic vesicle membrane’, ‘vacuolar membrane’, ‘phagocytic vesicle’, ‘autophagosome’, and ‘endocytic vesicle membrane’ were the main enriched terms. At the level of MF aspect, antioxidant activity, peroxidase activity, reductase activity, catalase activity, transmembrane receptor protein kinase activity were significantly enriched. KEGG pathway annotation showed that DEGs mainly participate in pathways of neurodegeneration, amyotrophic lateral sclerosis, shigellosis, NOD-like receptor signaling pathway, and mTOR signaling pathway (Figure 4).

Figure 3.

GO enrichment map with DEGs related to oxidative stress and autophagy. The top five enriched terms in biological process, cellular component, molecular function were included.

Figure 4.

Top 10 KEGG pathway enrichment map of DEGs related to oxidative stress and autophagy.

PPI network and potential transcription factors

The PPI network map of OSA–DEGs is shown in Figure 5. There are 54 edges and 52 nodes in the network diagram. The top 10 hub genes were CAT, GAPDH, PRDX1, PRDX4, TLR4, GPX8, GPX7, MSRA, RPTOR, and GABARAPL1. The TFs-genes network diagram is shown in Figure 6. A total of five transcription factors, including PPARG (degree: five; betweeness: 124.64), FOXC1 (degree: seven; betweeness: 293.84), RUNX2 (degree: four; betweeness: 29.39), YY1 (degree: four; betweeness: 42.03), JUN (degree: four; betweeness: 74.43), were found.

Figure 5.

PPI network diagram of DEGs related to oxidative stress and autophagy. The red color represents the higher degree of nodes. The network diagram contains 52 nodes and 54 edges. The top 10 genes are selected as CAT, GAPDH, PRDX1, PRDX4, TLR4, GPX7, GPX8, MSRA, RPTOR, and GABARAPL1.

Figure 6.

Transcriptional factors-genes network diagram. The red circle represents the hub genes, and the blue quadrilateral represents the transcription factor. The top five transcription factors are FOXC1, PPARG, RUNX2, JUN and YY1.

Identification of candidate drugs

The top 10 potential drugs, including streptozotocin, cumene hydroperoxide, nadide, L-cysteine, hydrogen peroxide, oleanolic acid, dioxidanide, 1,2-naphthoquinone, serotonin, and berberine, were identified (Table 1). CAT, GAPDH, PRDX4, PRDX1, and TLR4 are targeted by these drugs. Nadide has coenzyme activity in redox reactions. L-cysteine, oleanolic acid, and berberine have antioxidant activity, while other drugs have pro-oxidant activity.

Table 1.

Candidate drugs (top 10) identified from gene–drug interaction enrichment analysis.

Drug	p-value	Combined score	Odds ratio	Target genes	Related pharmacological effect
Streptozotocin	6.41 × 10⁻¹⁰	11,739.73	554.61	PRDX4; PRDX1; CAT; TLR4	Pro-oxidant activity
Cumene hydroperoxide	5.66 × 10⁻⁵	2324.81	237.73	PRDX1; GAPDH	Pro-oxidant activity
Nadide	5.66 × 10⁻⁵	2324.81	237.73	CAT; GAPDH	Coenzyme activity in redox reactions
L-cysteine	9.05 × 10⁻⁵	431.86	46.39	PRDX1; GAPDH; TLR4	Antioxidant activity
Hydrogen peroxide	9.49 × 10⁻⁵	422.66	45.63	PRDX4; PRDX1; TLR4	Pro-oxidant activity
Oleanolic acid	9.71 × 10⁻⁵	1646.74	178.23	PRDX1; CAT	Antioxidant, anti-inflammatory, and anti-apoptotic activities
Dioxidanide	1.48 × 10⁻⁴	1256.48	142.54	CAT; GAPDH	Pro-oxidant activity
1,2-Naphthoquinone	1.65 × 10⁻⁴	1174.13	134.82	CAT; GAPDH	Pro-oxidant activity
Serotonin	2.20 × 10⁻⁴	976.58	115.97	CAT; GAPDH	Pro-oxidant activity
Berberine	6.79 × 10⁻⁴	471.62	64.65	GAPDH; TLR4	Antioxidant and anti-inflammatory activities

Discussion

The development of IDD is significantly influenced by NP and AF degeneration. The elucidation of the common mechanism of the two may provide a better therapeutic target for the early intervention of IDD.

In this study, DEGs associated with OS and autophagy in both NP and AF were identified. The majority of these genes act in the cytoplasm and encode different proteins that are involved in the cellular response to OS and autophagy, according to GO findings. This suggests that a general imbalance of OS and autophagy dysfunction in the degenerated disc. KEGG analysis results showed that the pathways involving the products of DEGs mainly were neurodegeneration, amyotrophic lateral sclerosis, NOD-like receptor signaling pathway, and mTOR signaling pathway. The majority of the genes involved in these pathways experienced downregulation. In degenerated disks however, nerve endings have been detected deeper inside the AF^13,14 and occasionally even in the NP.¹⁵ The pathways of neurodegeneration and amyotrophic lateral sclerosis may be involved in intradiscal nerve fiber degeneration and the development of discogenic back pain.

The NOD-like receptors regulate cell death by modulating damage-associated molecular patterns and metabolic sensors, as well as inflammation via signal transduction. Recent research has shown that LINC00917 can regulate the proliferation, inflammation, and pyroptosis of NP cells via targeting miR-149-5p/NOD-like receptor protein one axis.¹⁶

The mTOR signaling pathways are involved in a number of BPs via various cellular mechanisms. Induction of pro-autophagy, anti-apoptotic, anti-matrix catabolism, and anti-aging effects were observed in intervertebral disc cell experiments. Yurube T et al.¹⁷ observed the selective RNA interference-mediated and pharmacological inhibition of mTOR complex one was protective against inflammation-induced disc cellular apoptosis, senescence, and extracellular matrix catabolism, through the induction of autophagy. In addition, resveratrol in combination with 17β -estradiol increases levels of activated phosphorylated mTOR and phosphorylated glycogen synthase kinase-3β leading to the down-regulation of caspase-3, inhibition of IL-1β induced NP cell apoptosis and recovery of cell viability.¹⁸

The top 10 hub genes were CAT, GAPDH, PRDX1, PRDX4, TLR4, GPX7, GPX8, MSRA, RPTOR, and GABARAPL1. All of these genes were significantly downregulated. Three of the most significant enzyme families involved in the process of eliminating ROS are catalase (CAT), peroxiredoxin (PRDX), and glutathione peroxidases (GPXs). Over-expression of CAT improved the pathological condition of intervertebral disc tissues with increased GAG and COL2 expression, as well as reduced inflammation.¹⁹ Dimethyl fumarate significantly increased expression of PRDX1/4 in NP cells, which promotes the anti OS response and reduces endoplasmic reticulum stress.²⁰ Although it has been found that the median lifespan of GPX7 knockout mice decreased to 400 days from normal 760 days,²¹ the research on the role of GPX7 in IDD is still lack. The structural and in vitro functional studies of GPX7 and GPX8 indicated that they were very similar to one another. Previous researches revealed that a series of antioxidants, including polyphenols, ROS scavengers, and nonenzymatic antioxidants, can exert therapeutic effects on degenerative disc cells and IDD.⁹ The decreased expression of GAPDH, a housekeeping gene, suggests that the number of NP and AF cells was significantly decreased. TLR4, MSRA, RPTOR, and GABARAPL1 are involved in different physiological functions in different cells, and their relationship with IDD is uncertain.

The TFs-genes networks showed that PPARG, FOXC1, RUNX2, YY1, and JUN play important roles in IDD. These TFs-genes are closely related to the regulation of OS. Fatty acid-induced mitochondrial dysfunction, OS, and apoptosis could be alleviated by upregulating PPARG coactivator one alpha in hepatocytes.²² Overexpression of FOXC1 mitigated the lung injury, OS, and inflammation in rats with chronic obstructive pulmonary disease.²³ Reduced FOXC1 expression increases cell death in cultured trabecular meshwork cells in response to OS.²⁴ Inhibition of RUNX2 using short hairpin RNA blocked vascular calcification induced by OS.²⁵ The evoked YY1 amplified the NRF2-mediated ARE transcription, which protects neuronal cells against damage by potentiating antioxidant response.²⁶ By stimulating the pentose phosphate pathway, YY1 decreased intracellular ROS levels and promoted antioxidant defense by supplying increased reducing power in the form of NADPH.²⁷ Inactivation of the Jun N-terminal kinase via increased expression of JIP-1b/lB1 might protect neuronal cells from OS.²⁸ It’s meaningful to learn more about how these genes affect the intervertebral disc.

Identified hub genes with specific physiological functions can be considered potential drug targets. Most of the identified drugs have pro-oxidative activity, except L-cysteine, oleanolic acid, and berberine. The antioxidant activity of L-cysteine, oleanolic acid, and berberine could be the basis for the treatment of IDD. Ahmed E.A et al. evaluated the antioxidant activity of L-cysteine against cisplatin-induced testicular oxidative damage in rats, and found the co-administration of cisplatin with l-cysteine significantly reduced the elevation in lipid peroxides.²⁹ Han Y et al. confirmed that the apoptosis and the calcification of the human cartilage endplate cells induced by H₂O₂ can be abolished by N-acetyl-l-cysteine.³⁰ Oleanolic acid, as a triterpenoid, has anti-oxidant, anti-inflammatory, and anti-apoptotic activities. Wang JL et al. demonstrated that oleanolic acid treatments could dose-dependently ameliorate mouse spinal cord damage through impeding p38-and JNK-regulated apoptosis and inflammation.³¹ Lee ES et al. found oleanolic acid administration increased blood superoxide dismutase levels in fatty rats and inhibited the level of ROS and endoplasmic reticulum stress in cultured mesangial cells.³² Berberine is a natural compound with various pharmacological activities. Berberine treatment in vitro inhibited the expression of pro-apoptotic proteins induced by tert-butyl hydroperoxide and increased the expression of anti-apoptotic Bcl-2, as well as inhibited the production of matrix-degrading enzymes.³³ Moreover, berberine significantly mitigated OS-decreased cell viability as well as apoptosis in human NP cells and attenuated OS-induced ER stress and autophagy in a concentration-dependent manner.¹¹ Previous research indicates the therapeutic potential for these drugs. Further studies on the regulatory mechanisms between these drugs and target genes in IDD seem valuable.

There are still some limitations to this study. Firstly, the sample size of this study is small, which may affect the reliability of the conclusions. In addition, the results of this study are all based on the analysis of database data. The necessary evidence from the in vitro cell experiment is lacking. Therefore, further experiments both in vitro and in vivo are needed. In spite of these limitations, the results of the current study may provide potential directions for future research.

Conclusion

In summary, common hub genes, signaling pathways, transcription factors, and potential drugs identified in this study may be involved in the development of IDD, which promote the understanding of OS and autophagy in degenerated NP and degenerated AF. However, the level of evidence for the results is insufficient. Further studies are needed to confirm the specific effects and therapeutic targets of these genes.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Pudong New Area Health and Health Commission Clinical Characteristic Discipline Project (PWYts2021-03); Shanghai Pudong New Area Health System Excellent Young Medical Talents Training Program (PWRq2020-41); Scientific research project fund of Shanghai Municipal Health Commission (20204Y0478).

ORCID iD

Haohan Huang

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