Identification of cellular senescence-related genes and immune cell infiltration characteristics in intervertebral disc degeneration.

Background: Intervertebral disc degeneration (IDD) progression involves multiple factors, including loss of nucleus pulposus cells and extracellular matrix as the basic pathological mechanism of degeneration, and is closely related to cellular senescence and immune cell infiltration. The aim of study was to identify critical cellular senescence-related genes and immune cell infiltration characteristics in IDD. Methods: Four datasets, including GSE70362, GSE112216, GSE114169, and GSE150408, were downloaded from the Gene Expression Omnibus database. The senescence-related genes were acquired from the CellAge Database and intersected with differentially expressed genes (DEGs) between IDD and control samples for senescence-related DEGs (SRDEGs). Protein-protein interaction (PPI) network analysis was performed to obtain ten hub SRDEGs. A consensus cluster analysis based on these hub genes was performed to divide the patients into clusters. The functional enrichment, and immune infiltration statuses of the clusters were compared. Weighted gene co-expression network analysis was used to identified key gene modules. The overlapping genes from key modules, DEGs of clusters and hub SRDEGs were intersected to obtain potential biomarkers. To verify the expression of potential biomarkers, quantitative polymerase chain reaction (qPCR) and immunohistochemistry were performed by using human intervertebral disc tissues. Results: In the GSE70362 dataset, a total of 364 DEGs were identified, of which 150 were upregulated and 214 were downregulated, and 35 genes were selected as SRDEGs. PPI analysis revealed ten hub SRDEGs and consensus cluster analysis divided the patients into two clusters. Compared to Cluster 2, Cluster 1 was highly enriched in extracellular matrix organization and various metabolic process. The level of Follicular T helper cells in the Cluster 1 was significantly higher than that in the Cluster 2. IGFBP3 and NQO1 were identified as potential biomarkers. The remaining 3 datasets, and the result of qPCR and immunohistochemistry showed that the expression levels of NQO1 and IGFBP3 in the degenerated group were higher than those in the control or treatment groups. Conclusion: Senescence-related genes play a key role in the development and occurrence of IDD. IGFBP3 and NQO1 are strongly correlated with immune infiltration in the IDD and could become novel therapeutic targets that prevent the progression of IDD.

Research Progress on the Role of Cartilage Endplate in Intervertebral Disc Degeneration.

Low back pain significantly impacts individuals' quality of life, with intervertebral disc degeneration (IDD) being a primary contributor to this condition. Currently, IDD treatment primarily focuses on symptom management and does not achieve a definitive cure. The cartilage endplate (CEP), a crucial nutrient-supplying tissue of the intervertebral disc, plays a pivotal role in disc degeneration. This review examines the mechanisms underlying CEP degeneration, summarizing recent advancements in understanding the structure and function of CEP, the involvement of various signaling pathways, and the roles of cartilage endplate stem cells (CESCs) and exosomes (Exos) in this process. The aim of this review is to provide a comprehensive reference for future research on CEP. Despite progress in understanding the role of CEP in IDD, the mechanisms underlying CEP degeneration remain incompletely elucidated. Future research poses significant challenges, necessitating further investigations to elucidate the complexities of CEP.

The Expression of Toll-like Receptors in Cartilage Endplate Cells: A Role of Toll-like Receptor 2 in Pro-Inflammatory and Pro-Catabolic Gene Expression.

INTRODUCTION: The vertebral cartilage endplate (CEP), crucial for intervertebral disc health, is prone to degeneration linked to chronic low back pain, disc degeneration, and Modic changes (MC). While it is known that disc cells express toll-like receptors (TLRs) that recognize pathogen- and damage-associated molecular patterns (PAMPs and DAMPs), it is unclear if CEP cells (CEPCs) share this trait. The CEP has a higher cell density than the disc, making CEPCs an important contributor. This study aimed to identify TLRs on CEPCs and their role in pro-inflammatory and catabolic gene expression. METHODS: Gene expression of TLR1-10 was measured in human CEPs and expanded CEPCs using quantitative polymerase chain reaction. Additionally, surface TLR expression was measured in CEPs grouped into non-MC and MC. CEPCs were stimulated with tumor necrosis factor alpha, interleukin 1 beta, small-molecule TLR agonists, or the 30 kDa N-terminal fibronectin fragment. TLR2 signaling was inhibited with TL2-C29, and TLR2 protein expression was measured with flow cytometry. RESULTS: Ex vivo analysis found all 10 TLRs expressed, while cultured CEPCs lost TLR8 and TLR9 expression. TLR2 expression was significantly increased in MC1 CEPCs, and its expression increased significantly after pro-inflammatory stimulation. Stimulation of the TLR2/6 heterodimer upregulated TLR2 protein expression. The TLR2/1 and TLR2/6 ligands upregulated pro-inflammatory genes and matrix metalloproteases (MMP1, MMP3, and MMP13), and TLR2 inhibition inhibited their upregulation. Endplate resorptive capacity of TLR2 activation was confirmed in a CEP explant model. CONCLUSIONS: The expression of TLR1-10 in CEPCs suggests that the CEP is susceptible to PAMP and DAMP stimulation. Enhanced TLR2 expression in MC1, and generally in CEPCs under inflammatory conditions, has pro-inflammatory and pro-catabolic effects, suggesting a potential role in disc degeneration and MC.

Single-cell RNA sequencing reveals the differentiation and regulation of endplate cells in human intervertebral disc degeneration.

Low back pain (LBP) is largely attributed to intervertebral disc degeneration (IVDD), of which the endplate changes are an important component. However, the alterations in cell fate and properties within the endplates during degeneration remain unknown. Here, we firstly performed the single-cell RNA-sequencing analysis (scRNA-seq) of the cells focusing on degenerative human endplates. By unsupervised clustering of the 8,534 single-cell based on the gene expression, we identified nine distinct cell types. We employed Gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis, and the single-cell regulatory network inference and clustering (SCENIC) to determine the enriched pathways and transcriptional activities across seven chondrocyte subpopulations. Furthermore, two cell fates of chondrocyte differentiation were found by trajectory analysis, one was enriched in inflammation-related genes, and the other was related to extracellular matrix (ECM). Additionally, the intercellular interactions of macrophages (MA) and chondrocytes, T cells/natural killer cells (T/NK) and chondrocytes were examined by ligand-receptor pairs analysis, showing the important regulative function of FN1 from MA and CD74 from T/NK during endplate degeneration. Overall, our findings provide novel perspectives on the endplate degeneration at the single-cell level and a whole-transcriptome size.

Effect of electroacupuncture on expression of autophagy-related proteins in nucleus pulposus of cervical spondylosis rabbits. / ç”µé’ˆå¯¹é¢ˆæ¤Žç— å®¶å ”é«“æ ¸ç»†èƒžè‡ªå™¬ç›¸å ³è›‹ç™½è¡¨è¾¾çš„å½±å“.

OBJECTIVES: To observe the effects of electroacupuncture (EA) on the morphological changes of intervertebral disc tissues, apoptosis of nucleus pulposus cells, and the protein expression of Unc-51 like autophagy-activated kinase 1 (ULK1), homologous series of yeast Atg6 (Beclin1), and light chain protease complication 3 type (LC3) in nucleus pulposus tissue of cervical spondylosis rabbits, so as to explore the role of cellular autophagy in EA treatment of cervical spondylosis. METHODS: A total of 24 New Zealand white rabbits were randomly divided into blank, model and EA groups, with 8 rabbits in each group. In the EA group, both sides of the cervical (C)3-C6 "Jiaji" (EX-B2) were stimulated by EA (2 Hz/100 Hz, 1 mA) for 25 min, once daily for 5 days in a course, with a 2-day interval between courses, totaling 4 treatment courses. X-ray was used to assess cervical spine radiographic changes and evaluate radiographic scores；transmission electron microscopy was used to observe ultrastructural changes in nucleus pulposus cells；HE staining was used to observe morphological changes of intervertebral disc tissues and conduct pathological scoring；TUNEL staining was used to observe apoptosis rate of nucleus pulposus cells；Western blot was performed to detect protein expression levels of ULK1, Beclin1, and LC3 in nucleus pulposus tissue. RESULTS: Compared with the blank group, rabbits in the model group showed significantly higher cervical spine radiographic scores (P<0.01), higher pathological scores of intervertebral disc tissues (P<0.05), increased apoptosis rate of nucleus pulposus cells (P<0.01), and decreased expression levels of ULK1, Beclin1, and LC3Ⅱ proteins in nucleus pulposus tissue (P<0.05). Compared with the model group, the EA group showed significantly lower pathological scores of intervertebral discs (P<0.05), lower apoptosis rate of nucleus pulposus cells (P<0.01), and higher protein expression levels of ULK1, Beclin1, and LC3Ⅱ in nucleus pulposus tissue (P<0.01). Rabbits in the blank control group exhibited generally normal organelle structures in nucleus pulposus tissues with few autophagic vacuoles, indicative of early stages of autophagy；while those in the model group showed disrupted organelle structures with cytoplasmic condensation and those in the EA group exhibited autophagosomes with double-membrane structures in nucleus pulposus tissues. CONCLUSIONS: EA promotes the expression of ULK1, Beclin1, and LC3Ⅱ proteins in nucleus pulposus tissues, reduces apoptosis of nucleus pulposus cells, and improves intervertebral disc degeneration.

Network pharmacology and experimental validation to reveal the pharmacological mechanisms of Astragaloside â £ in treating intervertebral disc degeneration.

This study aims to identify potential targets and regulatory mechanisms of Astragaloside Ⅳ (AS-Ⅳ) in treating intervertebral disc degeneration (IDD) through network pharmacology analysis with experimental validation. Lumbar spine instability (LSI) mouse models were first established and treated with AS-Ⅳ. Micro-CT, safranin O-fast green staining, IDD score, RT-PCR and immunohistochemistry staining were employed to demonstrate the effect of AS-Ⅳ. Network pharmacology was used to predict the signaling pathways and potential targets of AS-Ⅳ in treating IDD. RT-PCR and immunohistochemistry staining were used to elucidate and validate the mechanism of AS-Ⅳ in vivo. Animal experiments showed that AS-Ⅳ maintained disc height and volume, improved matrix metabolism in LSI mice, and restored Col2α1, ADAMTS-5, Aggrecan, and MMP-13 expression in degenerated discs. Network pharmacology analysis identified 32 cross-targets between AS-Ⅳ and IDD, and PPI network analysis filtered out 11 core genes, including ALB, MAPK1, MAPK14 (p38 MAPK), EGFR, TGFBR1, MAPK8, MMP3, ANXA5, ESR1, CASP3, and IGF1. Enrichment analysis revealed that 7 of the 11 core target genes enriched in the MAPK signaling pathway, and AS-Ⅳ exhibited stable binding to them according to molecular docking results. Experimental validation indicated that AS-Ⅳ reversed mRNA levels of 7 core targets in degenerated disc tissues in LSI mice. Immunohistochemistry staining further revealed that AS-Ⅳ treatment mainly depressed IDD-elevated protein levels of EGFR, p38 MAPK and CASP3 in the annulus fibrosus. This study elucidates that AS-Ⅳ alleviates lumbar spine instability-induced IDD in mice, suggesting the mechanism may involve inhibition of the EGFR/MAPK signaling pathway.

Assessment of Tie2-Rejuvenated Nucleus Pulposus Cell Transplants from Young and Old Patient Sources Demonstrates That Age Still Matters.

Cell transplantation is being actively explored as a regenerative therapy for discogenic back pain. This study explored the regenerative potential of Tie2+ nucleus pulposus progenitor cells (NPPCs) from intervertebral disc (IVD) tissues derived from young (<25 years of age) and old (>60 years of age) patient donors. We employed an optimized culture method to maintain Tie2 expression in NP cells from both donor categories. Our study revealed similar Tie2 positivity rates regardless of donor types following cell culture. Nevertheless, clear differences were also found, such as the emergence of significantly higher (3.6-fold) GD2 positivity and reduced (2.7-fold) proliferation potential for older donors compared to young sources. Our results suggest that, despite obtaining a high fraction of Tie2+ NP cells, cells from older donors were already committed to a more mature phenotype. These disparities translated into functional differences, influencing colony formation, extracellular matrix production, and in vivo regenerative potential. This study underscores the importance of considering age-related factors in NPPC-based therapies for disc degeneration. Further investigation into the genetic and epigenetic alterations of Tie2+ NP cells from older donors is crucial for refining regenerative strategies. These findings shed light on Tie2+ NPPCs as a promising cell source for IVD regeneration while emphasizing the need for comprehensive understanding and scalability considerations in culture methods for broader clinical applicability.

Transcriptome analysis of the diseased intervertebral disc tissue in patients with spinal tuberculosis.

OBJECTIVE: To investigate the differential expression genes (DEGs) in spinal tuberculosis using transcriptomics, with the aim of identifying novel therapeutic targets and prognostic indicators for the clinical management of spinal tuberculosis. METHODS: Patients who visited the Department of Orthopedics at the Second Hospital, Lanzhou University from January 2021 to May 2023 were enrolled. Based on the inclusion and exclusion criteria, there were 5 patients in the test group and 5 patients in the control group. Total RNA was extracted and paired-end sequencing was conducted on the sequencing platform. After processing the sequencing data with clean reads and annotating the reference genome, FPKM normalization and differential expression analysis were performed. The DEGs and long non-coding RNAs (LncRNAs) were analyzed for Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment. The cis-regulation of differentially expressed mRNAs (DE mRNAs) by LncRNAs was predicted and analyzed to establish a co-expression network. RESULTS: This study identified 2366 DEGs, with 974 genes significantly upregulated and 1392 genes significantly downregulated. The upregulated genes are associated with cytokine-cytokine receptor interactions, tuberculosis, and TNF-α signaling pathways, primarily enriched in biological processes such as immunity and inflammation. The downregulated genes are related to muscle development, contraction, fungal defense response, and collagen metabolism processes. Analysis of LncRNAs from bone tuberculosis RNA-seq data detected a total of 3652 LncRNAs, with 356 significantly upregulated and 184 significantly downregulated. Further analysis identified 311 significantly different LncRNAs that could cis-regulate 777 target genes, enriched in pathways such as muscle contraction, inflammatory response, and immune response, closely related to bone tuberculosis. There are 51 genes enriched in the immune response pathway regulated by cis-acting LncRNAs. LncRNAs that regulate immune response-related genes, such as upregulated RP11-451G4.2, RP11-701P16.5, AC079767.4, AC017002.1, LINC01094, CTA-384D8.35, and AC092484.1, as well as downregulated RP11-2C24.7, may serve as potential prognostic and therapeutic targets. CONCLUSION: The DE mRNAs and LncRNAs in spinal tuberculosis are both associated with immune regulatory pathways. These pathways promote or inhibit the tuberculosis infection and development at the mechanistic level and play an important role in the process of tuberculosis transferring to bone tissue.

Dysregulated lipid metabolism and intervertebral disc degeneration: the important role of ox-LDL/LOX-1 in endplate chondrocyte senescence and calcification.

BACKGROUND: Lipid metabolism disorders are associated with degeneration of multiple tissues and organs, but the mechanism of crosstalk between lipid metabolism disorder and intervertebral disc degeneration (IDD) has not been fully elucidated. In this study we aim to investigate the regulatory mechanism of abnormal signal of lipid metabolism disorder on intervertebral disc endplate chondrocyte (EPC) senescence and calcification. METHODS: Human intervertebral disc cartilage endplate tissue, cell model and rat hyperlipemia model were performed in this study. Histology and immunohistochemistry were used to human EPC tissue detection. TMT-labelled quantitative proteomics was used to detect differential proteins, and MRI, micro-CT, safranin green staining and immunofluorescence were performed to observe the morphology and degeneration of rat tail intervertebral discs. Flow cytometry, senescence-associated ß-galactosidase staining, alizarin red staining, alkaline phosphatase staining, DCFH-DA fluorescent probe, and western blot were performed to detect the expression of EPC cell senescence, senescence-associated secretory phenotype, calcification-related proteins and the activation of cell senescence-related signaling pathways. RESULTS: Our study found that the highly expressed oxidized low-density lipoprotein (ox-LDL) and Lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) in human degenerative EPC was associated with hyperlipidemia (HLP). TMT-labelled quantitative proteomics revealed enriched pathways such as cell cycle regulation, endochondral bone morphogenesis and inflammation. The rat model revealed that HLP could induce ox-LDL, LOX-1, senescence and calcification markers high expression in EPC. Moreover, we demonstrated that ox-LDL-induced EPCs senescence and calcification were dependent on the LOX-1 receptor, and the ROS/P38-MAPK/NF-κB signaling pathway was implicated in the regulation of senescence induced by ox-LDL/LOX-1 in cell model. CONCLUSIONS: So our study revealed that ox-LDL/LOX-1-induced EPCs senescence and calcification through ROS/P38-MAPK/NF-κB signaling pathway, providing information on understanding the link between lipid metabolism disorders and IDD.

The role of melatonergic system in intervertebral disc degeneration and its association with low back pain: a clinical study.

Objective: The mechanisms of intervertebral disc degeneration (IVDD) in low back pain (LBP) patients are multiples. In this study, we attempt to investigate whether melatonergic system plays a potential role in IVDD patients with LBP by analyzing their clinical specimens. The fucus will be given to the correlation between the melatonin receptor expression and intervertebral disc tissue apoptosis. Methods: In this clinical study, 107 lumbar intervertebral disc nucleus pulposus (NP) specimens from patients with LBP were collected with patients' consents. The disc height (DH) discrepancy ratio, range of motion and sagittal parameters of the pathological plane were measured and Pfirrmann grade was used to classified the grades of IVDD level. Discs at grades 1-3 were served as normal control and grades 4-5 were considered as IVDD. The expression levels of melatonin receptor 1A (MT1) and 1B (MT2) were measured by immunohistochemistry. The apoptosis of NP was assessed using TUNEL staining. Their potential associations among MT1/2, DH, apoptosis, sagittal parameters with IVDD and LBP were evaluated with statistical analysis. Results: The incidence of IVDD was positively associated with age and negatively related to VAS scores for LBP (p < 0.001). Patients with higher degree of IVDD also have higher DH discrepancy ratio (p < 0.001), higher prevalence of lumbar instability (p = 0.003) and higher cell apoptosis compared to the control. Nevertheless, no statistically significant correlation was identified between Pfirrmann grade and lumbar sagittal parameters. MT1 and MT2 both were highly expressed in the NP tissues. Importantly, MT1 expression but not MT2 was significantly increased in the intervertebral disc tissue of patients with IVDD and its level correlated well with cell apoptosis level and the severity of IVDD as well as lower VAS scores for LBP. Conclusion: The highly elevated MT1 expression was found in NP tissues of patients with IVDD and LBP compared to the control. This phenomenon probably reflects the compensating response of the body to the pathological alteration of the IVDD and LBP. Therefore, these findings provide the novel information to use selective agonists of MT1 to target IVDD and LBP clinically.

Enzymatic Digestion of the Intervertebral Disc Alters Intradiscal Injection and Leakage Mechanics.

Intradiscal injection is required to deliver therapeutic agents to the intervertebral disc (IVD) nucleus pulposus (NP). However, injectate leakage following needle retraction may result in decreased treatment efficacy and adverse side effects. While enzymatic digestion is a common research approach for simulating degeneration in healthy animal IVDs, contributions to the leakage phenomenon are unknown. In this study, bovine caudal discs were treated with injection into the NP of either a tris buffer control, collagenase (to primarily target collagen), or trypsin (to primarily target proteoglycans) and then injected with fluorescent saline using a through-puncture defect protocol. Pressure-volume records during injection were used to determine volume and pressure at leakage. Discs were then frozen, transected, and photographed to visualize injectate dispersion. Collagenase treatment resulted in a large increase in injectate dispersion, along with a decrease in injection pressure relative to control. Trypsin treatment resulted in a moderate increase in dispersion, with no associated effect on pressure. This study concludes that care should be taken when employing enzymatic digestion to simulate IVD degeneration, as NP tissue disruption may affect both retention and dispersion of subsequent therapeutic injections.

Pip5k1γ promotes anabolism of nucleus pulposus cells and intervertebral disc homeostasis by activating CaMKII-Ampk pathway in aged mice.

Degenerative disc disease (DDD) represents a significant global health challenge, yet its underlying molecular mechanisms remain elusive. This study aimed to investigate the role of type 1 phosphatidylinositol 4-phosphate 5-kinase (Pip5k1) in intervertebral disc (IVD) homeostasis and disease. All three Pip5k1 isoforms, namely Pip5k1α, Pip5k1ß, and Pip5k1γ, were detectable in mouse and human IVD tissues, with Pip5k1γ displaying a highest expression in nucleus pulposus (NP) cells. The expression of Pip5k1γ was significantly down-regulated in the NP cells of aged mice and patients with severe DDD. To determine whether Pip5k1γ expression is required for disc homeostasis, we generated a Pip5k1γfl/fl; AggrecanCreERT2 mouse model for the conditional knockout of the Pip5k1γ gene in aggrecan-expressing IVD cells. Our findings revealed that the conditional deletion of Pip5k1γ did not affect the disc structure or cellular composition in 5-month-old adult mice. However, in aged (15-month-old) mice, this deletion led to several severe degenerative disc defects, including decreased NP cellularity, spontaneous fibrosis and cleft formation, and a loss of the boundary between NP and annulus fibrosus. At the molecular level, the absence of Pip5k1γ reduced the anabolism of NP cells without markedly affecting their catabolic or anti-catabolic activities. Moreover, the loss of Pip5k1γ significantly dampened the activation of the protective Ampk pathway in NP cells, thereby accelerating NP cell senescence. Notably, Pip5k1γ deficiency blunted the effectiveness of metformin, a potent Ampk activator, in activating the Ampk pathway and mitigating lumbar spine instability (LSI)-induced disc lesions in mice. Overall, our study unveils a novel role for Pip5k1γ in promoting anabolism and maintaining disc homeostasis, suggesting it as a potential therapeutic target for DDD.

Nuclear factor κB overactivation in the intervertebral disc leads to macrophage recruitment and severe disc degeneration.

Persistent inflammation has been associated with severe disc degeneration (DD). This study investigated the effect of prolonged nuclear factor κB (NF-κB) activation in DD. Using an inducible mouse model, we genetically targeted cells expressing aggrecan, a primary component of the disc extra cellular matrix, for activation of the canonical NF-κB pathway. Prolonged NF-κB activation led to severe structural degeneration accompanied by increases in gene expression of inflammatory molecules (Il1b, Cox2, Il6, and Nos2), chemokines (Mcp1 and Mif), and catabolic enzymes (Mmp3, Mmp9, and Adamts4). Increased recruitment of proinflammatory (F4/80+,CD38+) and inflammatory resolving (F4/80+,CD206+) macrophages was observed within caudal discs. We found that the secretome of inflamed caudal disc cells increased macrophage migration and inflammatory activation. Lumbar discs did not exhibit phenotypic changes, suggestive of regional spinal differences in response to inflammatory genetic overactivation. Results suggest prolonged NF-κB activation can induce severe DD through increases in inflammatory cytokines, chemotactic proteins, catabolic enzymes, and the recruitment and activation of macrophage cell populations.

Progenitor-like cells contributing to cellular heterogeneity in the nucleus pulposus are lost in intervertebral disc degeneration.

The nucleus pulposus (NP) in the intervertebral disc (IVD) arises from embryonic notochord. Loss of notochordal-like cells in humans correlates with onset of IVD degeneration, suggesting that they are critical for healthy NP homeostasis and function. Comparative transcriptomic analyses identified expression of progenitor-associated genes (GREM1, KRT18, and TAGLN) in the young mouse and non-degenerated human NP, with TAGLN expression reducing with aging. Lineage tracing using Tagln-CreERt2 mice identified peripherally located proliferative NP (PeriNP) cells in developing and postnatal NP that provide a continuous supply of cells to the entire NP. PeriNP cells were diminished in aged mice and absent in puncture-induced degenerated discs. Single-cell transcriptomes of postnatal Tagln-CreERt2 IVD cells indicate enrichment for TGF-ß signaling in Tagln descendant NP sub-populations. Notochord-specific removal of TGF-ß/BMP mediator Smad4 results in loss of Tagln+ cells and abnormal NP morphologies. We propose Tagln+ PeriNP cells are potential progenitors crucial for NP homeostasis.

Effect of compressive and tensile forces on glucose concentration and cell viability within the intervertebral disc: A finite element study.

Understanding the role of mechanical force on tissue nutrient transport is essential, as sustained force may affect nutrient levels within the disc and initiate disc degeneration. This study aims to evaluate the time-dependent effects of different compressive force amplitudes as well as tensile force on glucose concentration and cell viability within the disc. Based on the mechano-electrochemical mixture theory, a multiphasic finite element model of the lumbar intervertebral disc was developed. The minimum glucose concentration and minimum cell density in both normal and degenerated discs were predicted for different compressive force amplitudes, tensile force, and corresponding creep time. Under high compressive force, the minimum glucose concentration exhibited an increasing and then decreasing trend with creep time in the normal disc, whereas that of the degenerated disc increased, then decreased, and finally increased again. At steady state, a higher compressive force was accompanied by a lower glucose concentration distribution. In the degenerated disc, the minimum cell density was negatively correlated with creep time, with a greater range of affected tissue under a higher compressive force. For tensile force, the minimum glucose concentration of the degenerated disc raised over time. This study highlighted the importance of creep time, force magnitude, and force type in affecting nutrient concentration and cell viability. Sustained weight-bearing activities could deteriorate the nutrient environment of the degenerated disc, while tensile force might have a nonnegligible role in effectively improving nutrient levels within the degenerated disc.

MGST1 May Regulate the Ferroptosis of the Annulus Fibrosus of Intervertebral Disc: Bioinformatic Integrated Analysis and Validation.

BACKGROUND: The objective of this research was to identify differentially expressed genes (DEGs) related to ferroptosis in the annulus fibrosus (AF) during intervertebral disc degeneration (IDD). METHODS: We analyzed gene data from degenerated and normal AF obtained from the GSE70362 and GSE147383 datasets. An analysis to determine the functional significance of the DEGs was conducted, followed by the creation of a network illustrating the interactions between proteins. We further analyzed the immune infiltration of the DEGs and determined the hub DEGs using LASSO regression analysis. Finally, we identified the hub ferroptosis-related DEGs (FRDEGs) and verified their expression levels using Real-time quantitative polymerase chain reaction (RT-qPCR), Western blot, Immunohistochemical Staining (IHC), and Immunofluorescence (IF). RESULTS: By analyzing the GSE70362 and GSE147383 datasets, we identified 118 DEGs. In degenerative AF groups, we observed a significant increase in immune infiltration of resting memory CD4+ T cells. LASSO regression analysis revealed 9 hub DEGs. The construction of a Receiver Operating Characteristic (ROC) curve yielded an Area Under the Curve (AUC) value of 0.762. Furthermore, we found that MGST1 is a hub gene related to ferroptosis. Our examination of immune infiltration indicated that MGST1 primarily influences macrophage M0 in different immune cell expression groups. Finally, our observations revealed a marked upregulation of MGST1 expression in the degenerated annulus fibrosus tissue. CONCLUSION: Our findings indicate an upsurge in MGST1 levels within degenerative AF, potentially playing a crucial role in the exacerbation of IDD. These findings provide a foundation for further exploration of the pathological mechanisms underlying IDD and offer potential drug targets for intervention.

Identification of key eRNAs for intervertebral disc degeneration by integrated multinomial bioinformatics analysis.

BACKGROUND: Intervertebral disc degeneration (IVDD) is a common degenerative condition leading to abnormal stress distribution under load, causing intervertebral stenosis, facet joint degeneration, and foraminal stenosis. Very little is known about the molecular mechanism of eRNAs in IVDD. METHODS: Gene expression profiles of 38 annulus disc samples composed of 27 less degenerated discs (LDs) and 11 more degenerated discs (MDs) were retrieved from the GEO database. Then, differentially expressed enhancer RNAs (DEeRNAs), differentially expressed target genes (DETGs), and differentially expressed transcription factors (DETFs), hallmark of cancer signalling pathways according to GSVA; the types and quantity of immune cells according to CIBERSORT; and immune gene sets according to ssGSEA were analysed to construct an IVDD-related eRNA network. Then, multidimensional validation was performed to explore the interactions among DEeRNAs, DETFs and DEGs in space. RESULTS: A total of 53 components, 14 DETGs, 15 DEeRNAs, 3 DETFs, 5 immune cells, 9 hallmarks, and 7 immune gene sets, were selected to construct the regulatory network. After validation by online multidimensional databases, 21 interactive DEeRNA-DEG-DETF axes related to IVDD exacerbation were identified, among which the C1S-CTNNB1-CHD4 axis was the most significant. CONCLUSION: Based upon the results of our study, we theorize that the C1S-CTNNB1-CHD4 axis plays a vital role in IVDD exacerbation. Specifically, C1S recruits CTNNB1 and upregulates the expression of CHD4 in IVDD, and subsequently, CHD4 suppresses glycolysis and activates oxidative phosphorylation, thus generating insoluble collagen fibre deposits and leading to the progression of IVDD. Overall, these DEeRNAs could comprise promising therapeutic targets for IVDD due to their high tissue specificity.

Lipid Peroxidation, Reduced Glutathione, and Glutathione Peroxidase Levels in Intervertebral Discs of Patients with Lumbar Degenerative Disc Disease.

BACKGROUND Either a reduction in antioxidant levels or an accumulation of reactive oxygen species can heighten susceptibility to oxidative damage in disc cells. To date, no research has investigated the levels of lipid peroxidation products (thiobarbituric acid reactive substances [TBARs]), reduced glutathione (GSH), and glutathione peroxidase (GPx) in excised human lumbar disc tissues affected by degenerative disease. Therefore, this study aimed to evaluate lipid peroxidation products in excised disc tissues from patients with degenerative disc disease. MATERIAL AND METHODS Forty-two patients were enrolled. Patients were divided into lumbar disc degeneration (LDD) and nonlumbar disc degeneration (nonLDD) groups according to Pfirrmann classification. Intervertebral discs were obtained from all patients during the operation and were homogenized for analysis. TBARs levels were measured using fluorometry. GSH levels and GPx activity were quantified spectrophotometrically using a kinetic method. RESULTS TBARs levels in excised discs from LDD patients (5.18±4.14) were significantly higher than those from nonLDD patients (2.56±1.23, P=0.008). The levels of TBARs tended to increase with the severity of degeneration according to the Pfirrmann classification. However, these 2 groups showed no significant differences in reduced glutathione levels or glutathione peroxidase activity (P>0.05). Patients with LDD exhibited a worse health-related quality of life, reflected in lower utility and EQ-VAS scores and higher Oswestry disability index scores. CONCLUSIONS There was a notable increase in lipid peroxidation products in the excised intervertebral discs of patients with LDD. This finding suggests that oxidative stress may contribute to the development of disc degeneration.

The role of oxidative stress in intervertebral disc degeneration: Mechanisms and therapeutic implications.

Oxidative stress is one of the main driving mechanisms of intervertebral disc degeneration(IDD). Oxidative stress has been associated with inflammation in the intervertebral disc, cellular senescence, autophagy, and epigenetics of intervertebral disc cells. It and the above pathological mechanisms are closely linked through the common hub reactive oxygen species(ROS), and promote each other in the process of disc degeneration and promote the development of the disease. This reveals the important role of oxidative stress in the process of IDD, and the importance and great potential of IDD therapy targeting oxidative stress. The efficacy of traditional therapy is unstable or cannot be maintained. In recent years, due to the rise of materials science, many bioactive functional materials have been applied in the treatment of IDD, and through the combination with traditional drugs, satisfactory efficacy has been achieved. At present, the research review of antioxidant bioactive materials in the treatment of IDD is not complete. Based on the existing studies, the mechanism of oxidative stress in IDD and the common antioxidant therapy were summarized in this paper, and the strategies based on emerging bioactive materials were reviewed.