Biological information analysis has been widely applied to help scientists identify new therapeutic targets by exploring gene changes in the process of disease development (26, 27). In the present study, we focused on inflammation as the primary etiological factor impacting disc degeneration, and IL-1β was selected as a representative inflammatory cytokine (28). Therefore, we obtained gene expression profiling data from GEO microarray datasets containing 4 samples of degenerative intervertebral disc cells stimulated with 102 pM IL-1β and 4 controls. The key genes that were affected during disc degeneration caused by inflammatory cytokine exposure were analyzed using bioinformatics analysis, and 260 DEGs were obtained (161 upregulated and 99 downregulated). Subsequently, GO annotation and KEGG pathway enrichment analyses of the DEGs revealed their participation in several cellular processes (e.g., the regulation of chemotaxis and taxis) and signaling cascades related to IVDD development (e. g., the interactions of cytokine-cytokine receptor interaction and rheumatoid arthritis). A PPI network based on these genes was constructed to obtain the top 10 hub genes: CXCL8, CXCL1, CCL20, CXCL2, CXCL5, CXCL3, CXCL6, C3, PF4, and GPER1. Subsequent analysis confirmed their involvement in important IVDD-related pathways. Our results suggest that these hub genes might be potential biological targets in IVD diagnosis and for the development of therapeutic drugs.
In terms of GO enrichment analysis, we found that most of DEGs were mainly involved in the regulation of chemotaxis and taxis, cytokine activity, cytokine receptor binding, and chemokine receptor binding. Regarding the KEGG pathway, we similarly found that most of DEGs were primarily enriched in the cytokine-cytokine receptor interaction, TNF signaling pathway, chemokine signaling pathway. In addition, CXCL5 and CXCL8 are the most significantly upregulated genes in our analysis. Inflammation has been correlated with IDD but its role remains controversial. Previous studies have shown that pathological inflammation of IVD and peridiscal space is characterized by increased levels of pro-inflammatory cytokines and chemokines (29-31). Cytokines and chemokines have three modes of action: (1) recruiting inflammatory cells and activating phagocytosis, (2) stimulating the production of other inflammatory mediators and MMPs, and (3) enhancing matrix degradation (32-37). And these effects can activate a cascade of detrimental self-promoting events that exacerbate the degeneration of IVD. Furthermore, experimental studies have reported the contribution of pro-inflammatory cytokines in degenerated painful IVD (38, 39).
In addition, our study also showed that the DEGs were mostly enriched in the extracellular region and extracellular matrix. A normal adult IVD is composed of a vascular tissue, which contain a large amount of extracellular matrix (ECM), and the disc cells are responsible for maintaining the integrity of the ECM (40). Changes in the metabolic balance of IVD cells affect the quality and quantity of the ECM and its functional properties, and these changes can, therefore, be related to disc degeneration (41). The underlying molecular mechanisms of IVDD have been previously investigated, and ECM degradation and inflammation have been confirmed to play a critical role in accelerating IVDD progression. Le et al. reported that IL-1β induced the expression of MMPs (16). And the study of Vo et al. found that upregulation of MMP and ADAMTS expression and enzymatic activity is implicated in disc ECM destruction, leading to the development of IDD (42). Our bioinformatics analysis also showed that MMP-3 significantly increased in the degenerative intervertebral disc cells stimulated with 102 pM IL-1β than controls.
Furthermore, we constructed a PPI network the top 10 hub genes - CXCL8, CXCL1, CCL20, CXCL2, CXCL5, CXCL3, CXCL6, C3, PF4, and GPER1 - were identified. It is known that leukocytes, such as neutrophils, macrophages, and lymphocytes, mainly participate in the inflammatory defense response of the body. Chemokines produced by mammalian cells during inflammation are a class of chemotactic and inducible small molecule peptides that are ubiquitous and play an important role in acute and chronic inflammation (43). Based on key cysteine residues involved in disulfide bonds, chemokines are classified as C, CC, CXC, and CX3C (44). Among the top 10 hub genes, CXCL8, CXCL1, CXCL2, CXCL5, CXCL3, CXCL6, and PF4 belong to the CXC family of chemokines, CCL20 belongs to the CC family, and C3 belongs to the C family.
CXCL8 is a chemotactic factor that attracts neutrophils, basophils, and T-cells. It is involved in neutrophil activation and modulates both acute and chronic inflammation. Ahn et al. found that CXCL8 mRNA expression was associated with the development of radicular pain by back extension and suggested that CXCL8 participates in the pathomechanism of nerve root inflammation in lumbar disc herniations (45). Pederson et al. found that patients with lumbar radicular pain due to disc herniation have increased serum levels of CXCL8 (46). Wang et al. also found higher CXCL8 concentrations in the serum of lumbar disc herniation (LDH) patients (47). A recent study reported that CXCL8 was elevated in the cerebrospinal fluid (CSF) of chronic low back pain (LBP) patients with IVDD, compared to pain-free subjects with or without IVDD, and they supported that the IL-8 signaling pathway is a viable therapy for chronic LBP (48). Palada et al. considered that neuroinflammation mediated by elevated CXCL8 concentrations in the CSF and CXCL8 mediated periphery-to-CNS (central nervous system) inflammatory cross-talk contributes to pain in LDH patients (49). Burke et al. reported that the increased level of CXCL8 within the nucleus pulposus may be related to neovascularization in patients with discogenic pain (50). Importantly, the protein expression of CXCL8 is significantly increased, concordant with the histological degenerative tissue changes in human NP (51). Similarly, Zhang et al. found that annulus fibrosus samples from LBP patients had an elevated IL-8 expression compared to controls with scoliosis (52). These experimental results are consistent with the results of our analysis. In the present study, the expression of CXCL8 in IL-1β-exposed samples was increased by 7.88-fold compared to control samples, indicating that increased levels of IL-1β affected the expression of the low-back-pain-related factors in intervertebral disc cells, which might become a new target for treating related diseases.
Chemokine CXCL5, which was increased by 9.83-fold in the present study, also called epithelial neutrophil-activating peptide 78 (ENA-78), belongs to the CXC family of chemokines that carry a glutamate-leucine-arginine (ELR) motif and binds to the G-protein CXCR2 to recruit neutrophils and promote angiogenesis (53, 54). CXCL5 has been shown to facilitate nociceptive input transmission in the pathogenesis of inflammatory pain (55). Dawes et al. reported that the chemokine CXCL5 is a peripheral mediator of ultraviolet B (UVB)-induced inflammatory pain, likely in humans and rats (56). Xu et al. reported that the upregulation of spinal CXCL5 and CXCR2 is involved in neuropathic pain after nerve injury by regulating GSK-3β activity in a rat model of chronic constriction injury (CCI) of the sciatic nerves (57). In addition, CXCL5 overexpression has been observed in several malignancies, such as pancreatic ductal adenocarcinoma, demonstrating its role in tumor carcinogenesis (58). This finding showed that the role of CXCL5 in intervertebral discs merits further research.
Chemokine CXCL6, also known as granulocyte chemotactic protein 2 (GCP2), recruits inflammatory cells to the site of inflammation by binding to receptors CXCR1 and CXCR2. Sandell et al. reported that the expression of CXCL6 was significantly increased in IL-1β-treated human chondrocytes (59). CXCL6 expression in IL-1β-exposed samples was increased by 3.2-fold, which was consistent with the results of a previous study. CXCL6 has also been detected in conditioned medium of induced degenerative discs in organ culture and may contribute to the chemotactic response of induced-degenerative discs (60). Grad et al. reported that significantly elevated levels of systemic blood plasma concentrations of chemokine CXCL6 were observed in individuals with moderate/severe disc degeneration, in comparison with a control group with no signs of disc degeneration, according to the MRI results (61). However, long-term medically treated patients with persistent chronic back pain expressed lower levels of the chemokine CXCL6 compared to pain-free healthy controls (62).
CXCL1 promotes both nociceptor and central sensitization through its primary receptor CXCR2, which may be a promising target for novel analgesic drugs in pathological pain conditions triggered by either peripheral inflammation or nerve injury (63). Under varied pathological states, activated astrocytes are considered the main source of CXCL1 (64). In addition, previous studies indicated that CXCL1 might act on CXCR2 via glial-neuronal interactions in the spinal cord in several pathological pain models (65, 66). A study by Ni et al. also demonstrated that CXCL1-CXCR2 signaling plays a critical role in glial-neuron interactions and in descending facilitation of bone cancer pain (BCP) (67). In our study, CXCL1 expression was increased 3.97-fold, indicating that CXCL1 plays an important role in LBP with IVDD.
CC chemokine ligand 20 (CCL20), which is expressed by endothelial cells in several tissues, is also known as macrophage inflammatory protein 3α (MIP-3α); it is a 70-amino-acid chemokine that binds exclusively to the chemokine receptor 6 (CCR6) and recruits CCR6-expressing cells (68, 69). Zhang et al. reported that NP cells from the extruded and herniated patient group could produce abundant amounts of CCL20 (70). Subsequently, Zhang et al. suggested a potential mechanism for the recruitment of IL-17-producing cells to degenerated intervertebral discs via a CCL20/CCR6 system in vivo, while IL-17 is involved in the auto-immune process of intervertebral disc degeneration in rat models (71).
G protein-coupled estrogen receptor 1 (GPER1) is the receptor of estrogen. There are four majors naturally occurring oestrogens identified in women: oestrone (E1), oestradiol (E2), oestriol (E3), and estetrol (E4). In addition, oestrogen receptors consist of two classic nuclear receptors, estrogen receptor (ER)-α and ER-β, and the membrane-bound G-protein-coupled receptor 30 (GPR30) (72). Over the past decade, many researchers have widely studied the relevance between oestrogen and IVDD and found that oestrogen can effectively alleviate IVDD development by inhibiting the apoptosis of IVD cells. Oestrogen can decrease IVD cell apoptosis in multiple ways, including the inhibition of inflammatory cytokines IL-1β and TNF-α, reducing catabolism because of matrix metalloproteinases inhibition, upregulating integrin α2β1 and IVD anabolism, activating the PI3K/Akt pathway, decreasing the oxidative damage, and promoting autophagy (72). Song et al. found that both cytoplasmic and nuclear staining of ERα and ERβ immunoreactivity were observed in nucleus pulposus cells, and ERα and ERβ expression significantly decreased, along with the aggravation of IVD degeneration in both males and females (73). Wei et al. also showed that GPR30 with a high affinity for estrogen is expressed in the human disc NP, can mediate E2 enhanced cell proliferation and influence disc cell survival (74). In our study, GPER1 was downregulated by 2.42-fold. Therefore, IL-1β decreases the expression of GPER1 in human disc NP, which accelerates disc degeneration.
Chemokine (C-X-C motif) ligand 2 (CXCL2), also known as macrophage inflammatory protein 2 (MIP-2), belongs to the CXC chemokine family, along with growth-regulated protein β and growth-regulated oncogene-2. CXCL2 is 90% identical regarding the amino acid sequence to the related chemokine CXCL1. CXCL2, which is produced in an injured sciatic nerve by partial ligation and is secreted by monocytes, can rapidly recruit neutrophils to sites of inflammation or injured tissues through blood flow, in response to infection or injury (43, 75, 76). CXCL3 is a small cytokine belonging to the CXC chemokine family, and is also known as the GRO3 oncogene, GRO protein gamma, and macrophage inflammatory protein 2β. PF4, also known as CXCL4, which belongs to the CXCL chemokine family, is predominantly produced by megakaryocytes and α-granules of platelets and has an important role in hemostasis/thrombosis (77). Complement C3 is a central molecule in the complement system and plays a very important role in inflammatory and immune responses. Interestingly, the complement system has been implicated in cartilage degradation, and C3 has been found to be aberrantly increased in the synovial fluids from individuals with osteoarthritis and in animal models of osteoarthritis (78, 79). However, we did not retrieve the literature to identify whether IVDD is correlated with CXCL2, CXCL3, C3, and PF4.