Intervertebral disc degeneration plays an essential role in the development of DS and DH. Oxidative stress and cell senescence play important roles in IDD through participating in regulation of matrix metabolism, proinflammatory phenotype, autophagy and senescence in intervertebral disc cells.(21) In this study, DH-related, DS-related, oxidative stress-related and cell senescence-related genes were analyzed respectively. After analysis, 428 OS-genes were obtained, including 33 OS-DEGs. 267 CS-genes were obtained, including 18 CS-DEGs.
For OS-genes, GO results showed that most of these genes acted in the cytoplasm and plasma membrane. The identified proteins were mainly involved in regulation of apoptotic process, response to oxidative stress and response to hydrogen peroxide. Hydrogen peroxide is a by-product of oxygen metabolism in organisms and plays an integral part in oxidative stress.(22) This indicates that hydrogen peroxide is produced in IDD and has different effect on DH and DS. GSEA and KEGG analysis results showed that the target proteins were mainly involved in IL-6 Jak STAT3 signaling pathway, calcium signaling pathway, GnRH signaling pathway, and Phospholipase D signaling pathway. IL-6 JAK STAT3 signaling pathway regulates coregulate B cell differentiation, plasma cytogenesis, and the acute phase reaction and is involved in the pathogenesis of IVD degeneration.(23, 24) Meanwhile, this pathway can induced skeletal muscle atrophy which may reduce spine muscle support and may be one cause of spondylolisthesis.(25) Calcium is an essential and complex signaling pathway among bone and muscle degenerative diseases. Calcium signaling pathway can regulate the intracellular calcium concentration. Some experiments have shown that intervertebral mechanical factors appear to regulate responses of the intervertebral disc cells through mechanisms involving intracellular Ca2+ transients and cytoskeletal remodeling.(26)
For CS-genes, GO results showed that most of these genes acted in the focal adhesion. The identified proteins were mainly involved in protein phosphorylation, signal transduction, intracellular signal transduction, MAPK cascade and cellular response to DNA damage stimulus. Protein phosphorylation combined with different signal transduction played a significant role in cell senescence.(27-29) This indicates that cell senescence is different between DS and DH. Meanwhile, oxidative stress can enhances cell senescence through MAPK pathway.(30) This suggests Oxidative Stress and Cell Senescence might interact with each other between DS and DH. GSEA and KEGG analysis results showed that the target proteins were mainly involved in heme metabolism, estrogen response and MAPK signaling pathway. Heme can induce cytotoxicity and ferroptosis within tissues and accelerate the progressive degeneration of herniated nucleus pulposus.(31, 32) As for estrogen response, estrogen may affect IVD inflammation through directly affecting the level of pro-inflammatory cytokines and modulation of SP.(33) Estrogen deficiency were also related to IDD induced by spinal instability and disc degeneration related to osteoporosis.(34) Meanwhile, the expression of estrogen receptor correlated with the severity of facet arthritis. which might be a causative factor for degenerative spondylolisthesis.(35, 36) The activation of the MAPK/ERK pathway promotes the degradation of the IVD extracellular matrix, cell aging, apoptosis, and inflammatory responses which is closely related to intervertebral degenerative diseases.(37)
The top 10 hub genes related to OS were EGFR, MMP2, MPO, COL1A1, ARG1, SPHK1, MMP14, CAT, PDGFRB. All these genes were significantly upregulated except for CAT, MPO and ARG1 which were significantly downregulated. To verify the expression of these genes between DH and DS, relevant literatures were searched and summarized in Table 1. These genes need to be further verified by experiments, especially in the disc between DH and DS.
Table 1. The Expression of Related Genes about OS by Literature Search.
|
Express
|
Conclusion
|
EGFR
|
Upregulation
|
EGFR involved in pain hypersensitivity and other sensory abnormalities after disc herniation.(38)
|
MMP2
|
Upregulation
|
MMP-2 increased in IDD and might be related to the elastin degradation and fibrosis of the ligamentum flavum.(39)
|
MPO
|
Downregulation
|
ROS mediated oxidative injury to NP cells resulting in premature cell death. MPO as oxidative stress response proteins were found in IDD.(40)
|
COL1A1
|
Upregulation
|
Polymorphisms of COL1A1 gene have been identified as contributors for disc degeneration.(41)
|
ARG1
|
Downregulation
|
PlGF expressed in MSCs increase the expression of M2/anti-inflammatory genes like ARG1 and may improve spinal fusion through macrophage recruitment and polarization. (42)
|
SPHK1
|
Upregulation
|
SPHK1 as the key gene of lncRNA could be potential biomarkers for the diagnosis of IDD(43)
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MMP14
|
Upregulation
|
MMP14 promotes loss of type II collagen and contributes to the development of IDD.(44)
|
CAT
|
Downregulation
|
CAT improved the pathological condition of intervertebral disc tissues with increased GAG and COL2 expression, as well as reduced inflammation.(45)
|
PDGFRB
|
Upregulation
|
Muscle fibers from IVD herniations had focal regions of degeneration, and that the most common cell type observed within the degenerating regions were PDGFRβ-positive (FAP) cells. (46)
|
AQP1
|
Upregulation
|
AQP1 expression leads to decreased water content in the IVDs, which results in degeneration.(47)
|
IDD = intervertebral degenerative disease; NP = nucleus pulposus; IVD = intervertebral disc
The EGFR gene provides instructions for making a receptor protein called the epidermal growth factor receptor, signaling pathways within the cells are triggered through this receptor that promote cell growth and proliferation. Disc herniation will induce secrete epiregulin (EREG) receptors EGFR up-regulation and induce sensory abnormalities and pain hypersensitivity.(38)Meanwhile, EGFR in LF samples showed a significant increase on the dorsal side which may be a reason for altered spinal stress that induces spondylolisthesis.(48) MMP2 gene provides instructions for making an enzyme called matrix metallopeptidase 2. The activity of matrix metallopeptidase 2 appears to be important for repair of damaged tissues, and inflammation. Matrix metallopeptidase 2 also plays a role in bone remodeling. As polymorphisms in the promoter region of the MMP-2 gene increased, the increase expression of MMP2 can destroy the protein of disc and accelerates the degeneration of the intervertebral disc.(49) Previous report has shown that active MMP2 were significantly higher in the spinal stenosis samples than that in the disc herniation samples, and this situation might be related to the elastin degradation and fibrosis of the ligamentum flavum which may decrease the stability of the spine.(39) MPO activates acute inflammation causing neuropathic pain in patient and accelerating disc degeneration. The down-regulation of MOP gene expression in DS may be due to the alleviation of acute inflammation, which needs further study. (40, 50) COL1 is a main element of extracellular matrix and a necessary component to provide tensile strength to the intervertebral disc. Polymorphisms of COL1A1 gene have been identified as likely contributors for disc degeneration.(41) PIGF improves spinal fusion by promoting the expression of M2 anti-inflammatory factor (ARG1, CD163 and so on) to recruit and polarize MSCs through macrophages. Conversely, the reduction of ARG1 may be a factor leading to the destabilization of spondylolisthesis.(42) SPHK1 leads to increased expression of S1P and can be used as a biomarker in response to changes caused by cytokine and mechanical stress changes.(51) The expression of MMP14 promoted loss of type II collagen.(52) Researchers have hypothesized that overexpression of MMP-14 leads to overall disc degradation mainly through the activation of MMP2.(53) The CAT gene provides instructions for making pieces catalase which breaks down hydrogen peroxide (H2O2) molecules. H2O2 induced oxidative stress, inflammation, apoptosis and enhanced the formation of markers of DNA damage. (54, 55) Another research shown that Modification of hADSCs by catalase improved the pathological condition of intervertebral disc tissues with increased GAG and COL2 expression, as well as reduced inflammation.(45) As a result, the up-regulation of CAT gene might be an adaptative response to against oxidative stress in DH. PDGFRβ-positive cells play an important role in muscle degenerating. When the coordination of inflammatory cues is disrupted, these cells may take on fibroblastic or adipogenic phenotypes which can reduce the protective and supportive role of the muscle.(46) Increased AQP1 expression is associated with greater disc hydration. AQP1 may have a role in the dehydration associated with disc degeneration.(47)
The top 10 hub genes related to CS were MAPK12, BLK, CAV1, CDK18, PIM1, CXCL1, NOTCH3, NUAK1, MAP3K6, MAP4K1. Excepted for BLK, PIM1, CXCL1 and MAP4K1 which were significantly downregulated, other genes were significantly upregulated. To verify the expression of these genes between DH and DS, relevant literatures were also searched and summarized in Table 2.
Table 2. The Expression of Related Genes about CS by Literature Search.
|
Express
|
Conclusion
|
MAPK12
|
Upregulation
|
The activation of MAPK pathway promotes the degradation of the IVD extracellular matrix, cell aging, apoptosis, and inflammatory responses.[15]
|
BLK
|
Downregulation
|
Clones derived from populations of progenitor cells expressing tyrosine protein kinase receptor TIE-2 had superior ability to proliferate, produce ECM and self-renew.(56)
|
CAV1
|
Upregulation
|
Cav1 associated with senescent phenotype plays an important role in IVD, cellular mechanotransduction and inflammatory responses.(57, 58)
|
CDK18
|
Upregulation
|
None
|
PIM1
|
Downregulation
|
PIM1 encodes a serine/threonine protein kinase that regulates cell proliferation, survival, differentiation and apoptosis.(59)
|
CXCL1
|
Downregulation
|
During senescence, NF-κB activation increases the expression of CXCL1 and other CXCR2. CXCL1 alone can also cause senescence, which has important implications in malignant tumors.[40]
|
NOTCH3
|
Upregulation
|
Notch3 was elevated in human cells during senescence activated by various senescence-inducing stimuli.(60)
|
NUAK1
|
Upregulation
|
The levels of NUAK1 are upregulated during senescence whereas its ectopic expression triggers a premature senescence.[42]
|
MAP3K6
|
Upregulation
|
MAP3K6 is closely related to the MAPK signaling pathway which is related to cell senescence. [10, 44]
|
MAP4K1
|
Downregulation
|
MAP4K1 was identified as one of the 12 senescence-regulating kinases encoding gene.(61)
|
IVD = intervertebral disc; ECM = extracellular matrix
MAPK12 is one of[1] the four p38 MAPKs which act in the cascades of cellular responses evoked by extracellular stimuli such as pro-inflammatory cytokines or physical stress leading to direct activation of transcription factors.(62) The activation of MAPK pathway promotes the degradation of the IVD extracellular matrix, cell aging, apoptosis, and inflammatory responses.(37) BLK gene encodes a nonreceptor tyrosine-kinase of the src family which is typically involved in cell proliferation and differentiation. Research has shown that clones derived from populations of progenitor cells expressing tyrosine protein kinase receptor TIE-2 had superior ability to form spheroid colonies, proliferate, produce ECM, in vitro and in vivo multipotent differentiation capacity, and ability to self-renew(56). Therefore, the reduction of tyrosine protein kinase may be related to the reduction of the repair ability of disc. The downregulation of BLK might prove that the degeneration of the IVD is more serious after spondylolisthesis. CAV1, associated with the senescent phenotype expressed in IVD tissue and excessive mechanical stimulation, promotes Cav1-mediated signaling and appears to promote inflammation in the annulus fibrosus cells.(57, 58) There are currently no relevant studies related to CDK18. PIM1 encodes a serine/threonine protein kinase that regulates cell proliferation, survival, differentiation and apoptosis. PIM1 is also a direct target of IL-6/STAT3 signaling and mediates cytokine-induced cellular senescence.(59, 63) During senescence, NF-κB activation increases the expression of CXCL1 and CXCR2. CXCL1 alone can also cause senescence, which has important implications in malignant tumors.(64) The downregulation of CXCL1 might indicate the senescence of DS is different with tumor. Notch3, a member of Notch family transmembrane receptors, is elevated in human cells during senescence activated by various senescence-inducing stimuli. This upregulation of Notch3 is required for the induction of p21 expression in senescent cells.(60) Downregulation of the AMPK-related protein kinase 5 (ARK5 or NUAK1) results in extension of the cellular replicative lifespan. The levels of NUAK1 are upregulated during senescence whereas its ectopic expression triggers a premature senescence.(65) The regulation of MAP3K6 is closely related to the MAPK signaling pathway, which is related to both oxidative stress and cell senescence.(30, 66) MAP4K1 was identified as one of the 12 senescence-regulating kinases encoding gene.(61) MAP4K1, which may up-regulate phosphorylation of JUN or MYC, plays roles in stress responses and growth regulation.(67)
The TFs-genes network of OS-gene showed that NFIC and FOXC1 played important roles between DH and DS. Abnormally elevated levels of NFIC can interact with the miR-200b promoter, leading to the activation of the transcription of miR-200b. MiR-200b represses autophagy and triggers apoptosis by directly targeting the autophagy-related gene Ambra1.(68) Meanwhile, NFIC impairs osteoblast differentiation and bone formation, and increases bone marrow adipocytes. (69) FOXC1 can regulate the expression of FOXO1A and bind to a conserved element in the FOXO1A promoter. Reduced FOXC1 expression increases cell death in response to oxidative stress.(70, 71)
The TFs-genes network of CS-gene showed that GATA2, FOXC1 and SREBF1 play important roles between DH and DS. GATA2 gene encodes a member of the GATA family of zinc-finger transcription factors. Researches have indicated GATA2 and SOD-1 gene expressions were decreased in senescent cells lower than young cells. (72) FOXC1 is linked to adipo-osteoprogenitor cell differentiation.(73) In addition to the above-mentioned role in oxidative stress, FOXC1 can also repair aged endothelial progenitor cells which have senescent potential.(74) SREBP-1 gene, which involved in the metabolism of endosterols and lipids, could enhance lipogenesis involved in senescence via SREBP-1 induction.(75) Silencing of Nrf2 leads to premature senescence. Activators of Nrf2 increase the expression of genes linked to SREBF1.(76-78) Meanwhile, KDM1A-mediated attenuation of SREBF1 transcriptional activities as the underlying mechanism for the suppression of de novo lipogenesis by oxidative stress.(79)