Mechanism Of Intervertebral Disc Degeneration Research Articles

Glucose deprivation-induced disulfidptosis in human nucleus pulposus cells: a novel pathological mechanism of intervertebral disc degeneration

BackgroundLimited supply of certain nutrients and deregulation of nucleus pulposus (NP) plays a key role in the pathogenesis of intervertebral disc degeneration (IVDD). However, whether nutrient deprivation-induced cell death, particularly disulfidptosis, contributes to the depletion of NP cells and the development of IVDD, is unknown.MethodsRNA-seq, single-cell RNA-seq, and Genome-wide DNA methylation datasets of nucleus pulposus tissue were collected for bioinformatic analysis. Predictive models of disulfidptosis related genes in IVDD were constructed by machine learning and their differential expression was analyzed. In addition, we performed cell subsets identification analysis, cell-cell communications analysis, and functional enrichment analysis of key genes in the core subset based on single-cell RNA-seq data of NP tissues isolated from one normal sample and one IVDD sample. Finally, glucose deprivation-induced disulfidptosis in human NP cells (HNPCs) was verified by various cell death inhibitors and disulfidptosis-related molecular markers.ResultsWe found the disulfidptosis signal was significantly activated in the IVDD group. Using single-cell RNA-seq analysis, we focused on the chondrocytes and found that disulfidptosis-related genes significantly highly expressed in the IVDD C4 chondrocyte subset, which was identified as a new disulfidptosis-associated cell subset. Correlation analysis revealed the negative correlation between SLC7A11 (driving gene of disulfidptosis) and the glucose transporter GLUTs (SLC2A1-4) family genes (suppressing genes of disulfidptosis) in the IVDD group. We also found obvious cell death in HNPC upon glucose starvation, while employment of various cell death inhibitors could not inhibit glucose starvation-induced death in HNPCs. Moreover, the accumulation of disulfide bonds in cytoskeletal proteins was indicated by slowed migration in non-reducible protein blotting experiments. 2-DG, a key disulfidptosis inhibitor, significantly rescued cell death caused by glucose starvation through lowering the NADP+/NADPH ratio.ConclusionsWe validated the occurrence of disulfidptosis in HPNCs and identified a novel disulfidptosis-associated cell subset, followed by experimental verification of disulfidptosis in a glucose-limited context to mimic a fall in nutrient supply during the development disc degeneration. These findings provided new insights into the pathological mechanisms of IVDD and encourage us to explore potential therapeutic targets involved in the regulation of disulfidptosis for the prevention of intervertebral disc degeneration.Graphical

Study on molecular mechanism of intervertebral disc degeneration by single cell hdWGCNA combined with transcriptome sequencing

BackgroundIntervertebral disc degeneration (IVDD) is one of the important causes of lower back pain, seriously affecting people's health and quality of life. This research employs single-cell analysis to identify the specific cellular subtypes and key regulatory genes associated with IVDD. MethodsWe analyzed the single-cell data and screened cells that closely associated with the development of IVDD. The differential expression of feature genes between IVDD and control groups was analyzed. Additionally, drugs and regulatory transcription factors that interact with feature genes were predicted and clinically validated by reverse transcription quantitative real-time PCR (RT-qPCR), immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA). ResultsOur study identified the Chond2 cell subtype associated with IVDD and selected four feature genes influencing the development of IVDD, namely IGFBP3, ACAN, VAPA and TMEM45A, through the high-dimensional weighted gene co-expression network analysis (hdWGCNA) analysis, least absolute shrinkage and selection operator (LASSO), and random forest (RF). Besides, compared to the MDD group, IGFBP3 and TMEM45A were significantly upregulated in the SDD group, while ACAN and VAPA showed no significant difference between the two groups. ELISA testing revealed a positive correlation between IGFBP3 concentration and the grading of IVDD. Furthermore, Celecoxib may be used to treat IVDD by inhibiting IGFBP3. ConclusionOur study identified the Chond2 cell subtype associated with IVDD and selected four feature genes influencing the development of IVDD, namely IGFBP3, ACAN, VAPA and TMEM45A. Our findings establish a robust theoretical foundation for the clinical diagnosis and treatment of IVDD patients.

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

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. 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. 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. 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.

Investigating the characteristics of mild intervertebral disc degeneration at various age stages using single-cell genomics.

Introduction: Intervertebral disc degeneration often occurs in the elderly population, but in recent years, there has been an increasing incidence of disc degeneration in younger individuals, primarily with mild degeneration. Methods: In order to explore the underlying mechanisms of disc degeneration in both young and aging individuals, we collected four types of nucleus pulposus (NP) single-cell sequencing samples for analysis based on Pfirrmann grading: normal-young (NY) (Grade I), normal-old (NO) (Grade I), mild degenerative-young (MY) (Grade II-III), and mild degenerative-old (MO) (Grade II-III). Results: We found that most NP cells in NO and MY samples exhibited oxidative stress, which may be important pathogenic factors in NO and MY groups. On the other hand, NP cells in MO group exhibited endoplasmic reticulum stress. In terms of inflammation, myeloid cells were mainly present in the degenerative group, with the MY group showing a stronger immune response compared to the MO group. Interestingly, dendritic cells in the myeloid lineage played a critical role in the process of mild degeneration. Discussion: Our study investigated the molecular mechanisms of intervertebral disc degeneration from an age perspective, providing insights for improving treatment strategies for patients with disc degeneration at different age groups.

M1 macrophage-derived exosomes promote intervertebral disc degeneration by enhancing nucleus pulposus cell senescence through LCN2/NF-κB signaling axis

Intervertebral disc degeneration (IVDD) is the primary factor contributing to low back pain (LBP). Unlike elderly patients, many young IVDD patients usually have a history of trauma or long-term abnormal stress, which may lead to local inflammatory reaction causing by immune cells, and ultimately accelerates degeneration. Research has shown the significance of M1-type macrophages in IVDD; nevertheless, the precise mechanism and the route by which it influences the function of nucleus pulposus cell (NPC) remain unknown. Utilizing a rat acupuncture IVDD model and an NPC degeneration model induced by lipopolysaccharide (LPS), we investigated the function of M1 macrophage-derived exosomes (M1-Exos) in IVDD both in vivo and in vitro in this study. We found that M1-Exos enhanced LPS-induced NPC senescence, increased the number of SA-β-gal-positive cells, blocked the cell cycle, and promoted the activation of P21 and P53. M1-Exos derived from supernatant pretreated with the exosome inhibitor GW4869 reversed this result in vivo and in vitro. RNA-seq showed that Lipocalin2 (LCN2) was enriched in M1-Exos and targeted the NF-κB pathway. The quantity of SA-β-gal-positive cells was significantly reduced with the inhibition of LCN2, and the expression of P21 and P53 in NPCs was decreased. The same results were obtained in the acupuncture-induced IVDD model. In addition, inhibition of LCN2 promotes the expression of type II collagen (Col-2) and inhibits the expression of matrix metalloproteinase 13 (MMP13), thereby restoring the equilibrium of metabolism inside the extracellular matrix (ECM) in vitro and in vivo. In addition, the NF-κB pathway is crucial for regulating M1-Exo-mediated NPC senescence. After the addition of M1-Exos to LPS-treated NPCs, p-p65 activity was significantly activated, while si-LCN2 treatment significantly inhibited p-p65 activity. Therefore, this paper demonstrates that M1 macrophage-derived exosomes have the ability to deliver LCN2, which activates the NF-κB signaling pathway, and exacerbates IVDD by accelerating NPC senescence. This may shed new light on the mechanism of IVDD and bring a fresh approach to IVDD therapy.Graphical

Effect of PI3K/AKT/HIF-1α signaling pathway on oxidative stress and apoptosis in nucleus pulposus cells.

The aim of the study was to explore the expression of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT)/hypoxia-induced factor-1 alpha (HIF-1α) signaling pathway and the apoptosis of nucleus pulposus cells (NPCs) under different oxygen concentrations to clarify the biological characteristics of NPCs and the molecular mechanism of intervertebral disc degeneration (IVDD). Normal and degenerated human NPCs were collected. Leibovitz's medium with 100 μmol/L CoCl2 was used to establish a hypoxic culture environment, and 100 μmol/L H2O2 was used to establish an oxidative stress culture environment. Third-generation NPCs were divided into 6 groups: normal NPCs + hypoxia, normal NPCs + normoxia, normal NPCs + oxidative stress, degenerated NPCs + hypoxia, degenerated NPCs + normoxia, and degenerated NPCs + oxidative stress. Normal NPCs + hypoxia was used as the experimental control group. Cell viability and proliferation were detected by using the Cell Counting Kit-8 (CCK-8) method. Cell apoptosis rate was assessed by flow cytometry, and expression levels of PI3K, AKT, and HIF-1α were determined by Real-Time-Polymerase Chain Reaction (RT-PCR) and Western blotting. The cell proliferation rate of both normal and degenerated NPCs decreased with increasing oxygen concentration. Conversely, the apoptosis rate increased as the oxygen concentration increased (p<0.05). Compared with the control group, whether the cells degenerated had a very significant impact on the apoptosis rate (p<0.001), and oxygen concentration also had a highly significant impact on both the cell proliferation rate and apoptosis rate (both p<0.001). The interaction between cell degeneration and oxygen concentration significantly affected both cell proliferation and apoptosis rates (p<0.05). Considering the expression levels of PI3K, AKT, and HIF-1α, normal NPCs had the highest levels under low oxygen concentrations, followed by oxidative stress and normoxia. In degenerated NPCs, the expression levels also decrease as the oxygen concentration increases. The PI3K/Akt/HIF-1α signaling pathway plays a significant role in inhibiting oxidative stress, antagonizing NPC apoptosis, and consequently delaying IVDD.

Degenerated nucleus pulposus cells derived exosome carrying miR-27a-3p aggravates intervertebral disc degeneration by inducing M1 polarization of macrophages

BackgroundIntervertebral disc degeneration (IVDD) is a major contributor to spinal disorders. Previous studies have indicated that the infiltration of immunocytes, specifically macrophages, plays a crucial role in the advancement of IVDD. Exosomes (exo) are believed to play a significant role in intercellular communication. This study aims to investigate the role of exosomes derived from degenerated nucleus pulposus (dNPc) in the process of macrophages M1 polarization.MethodsNucleus pulposus (NP) tissue and nucleus pulposus cells (NPc) were collected from patients with intervertebral disc degeneration (IVDD) and idiopathic scoliosis. Immunohistochemistry analysis was performed to determine the number of M1 macrophages in NP tissue. Subsequently, exosomes derived from degenerated NP cells (dNPc-exo) and non-degenerated NP cells (nNPc-exo) were collected and co-cultured with M0 macrophages, which were induced from THP-1 cells. The M1 phenotype was assessed using western blot, flow cytometry, immunofluorescence staining, and qRT-PCR. RNA-sequencing analysis was conducted to examine the expression levels of microRNAs in the dNPc-exo and nNPc-exo groups, and qRT-PCR was performed to investigate the effect pf different microRNA to induce macrophage polarization. Furthermore, western blot and qRT-PCR were employed to demonstrate the regulatory effect of microRNAs carried by dNPc-exo on downstream target signaling pathways in macrophages. Finally, an animal model of IVDD was utilized to investigate the impact of dNPc-exo on inducing M1 polarization of macrophages and its role in the IVDD process.ResultsIn this study, we observed an increase in the number of M1 macrophages as the intervertebral disc (IVD) degraded. Additionally, we discovered that dNPc releases exosomes (dNPc-exo) could promote the polarization of macrophages towards the M1 phenotype. Notably, through RNA-sequencing analysis of dNPc-exo and nNPc-exo groups, we identified miR-27a-3p as a highly expressed miRNA in the dNPc-exo group, which significantly influences the induction of M1 polarization of macrophages. And then, we discovered that dNPc-exo has the ability to transport miR-27a-3p and target the PPARγ/NFκB/PI3K/AKT signaling pathway, thereby influencing the M1 polarization of macrophages. We conducted experiments using rat model of IVDD and observed that the exosomes carrying miR-27a-3p actually induced the M1 polarization of macrophages and exacerbated the degradation of IVD.ConclusionIn conclusion, our findings highlight the significant role of dNPc-exo in IVDD process and provide a basis for further investigation into the mechanism of IVDD and the potential of exosome-based therapy.Graphic abstract

Biomaterials delivery strategies to repair degenerated intervertebral discs by regulating the inflammatory microenvironment.

Intervertebral disc degeneration (IVDD) is one of the leading causes of lower back pain. Although IVDD cannot directly cause death, it can cause pain, psychological burdens, and economic burdens to patients. Current conservative treatments for IVDD can relieve pain but cannot reverse the disease. Patients who cannot tolerate pain usually resort to a strategy of surgical resection of the degenerated disc. However, the surgical removal of IVDD can affect the stability of adjacent discs. Furthermore, the probability of the reherniation of the intervertebral disc (IVD) after surgery is as high as 21.2%. Strategies based on tissue engineering to deliver stem cells for the regeneration of nucleus purposes (NP) and annulus fibrosus (AF) have been extensively studied. The developed biomaterials not only locally withstand the pressure of the IVD but also lay the foundation for the survival of stem cells. However, the structure of IVDs does not provide sufficient nutrients for delivered stem cells. The role of immune mechanisms in IVDD has recently become clear. In IVDD, the IVD that was originally in immune privilege prevents the attack of immune cells (mainly effector T cells and macrophages) and aggravates the disease. Immune regulatory and inflammatory factors released by effector T cells, macrophages, and the IVD further aggravate IVDD. Reversing IVDD by regulating the inflammatory microenvironment is a potential approach for the treatment of the disease. However, the biological factors modulating the inflammatory microenvironment easily degrade in vivo. It makes it possible for different biomaterials to modulate the inflammatory microenvironment to repair IVDD. In this review, we have discussed the structures of IVDs and the immune mechanisms underlying IVDD. We have described the immune mechanisms elicited by different biological factors, including tumor necrosis factors, interleukins, transforming growth factors, hypoxia-inducible factors, and reactive oxygen species in IVDs. Finally, we have discussed the biomaterials used to modulate the inflammatory microenvironment to repair IVDD and their development.

Sulforaphane Delays Intervertebral Disc Degeneration by Alleviating Endoplasmic Reticulum Stress in Nucleus Pulposus Cells via Activating Nrf-2/HO-1.

Intervertebral disc degeneration (IVDD) is one of the main causes of low back pain, which brings heavy burdens to individuals and society. The mechanism of IVDD is complex and diverse. One of the important reasons is that the abnormal accumulation of reactive oxygen species (ROS) in nucleus pulposus cells (NPCs) leads to endoplasmic reticulum stress (ERS), which causes increased apoptosis of NPCs. Nuclear factor E2-related factor 2 (Nrf-2) and its downstream antioxidant proteins are key molecular switches for sensing oxidative stress and regulating antioxidant responses in cells. Sulforaphane (SFN), a natural compound derived from Brassicaceae plants, is a Nrf-2 agonist that displays potent antioxidant potential in vitro and in vivo. Here, we used advanced glycation end products (AGEs) to construct an in vitro degeneration model of NPCs, and we found that AGEs elevated ROS level in NPCs and caused severe ERS and apoptosis. While SFN can promote the entry of Nrf-2 into the nucleus and increase the expression level of heme oxygenase 1 (HO-1) in vitro, thus clearing the accumulated ROS in cells and alleviating ERS and cell apoptosis. Moreover, the protection of SFN on NPCs was greatly attenuated after HO-1 was inhibited. We also used AGEs to construct a rat IVDD model. Consistent with the in vitro experiments, SFN could attenuate ERS in NPCs in vivo and delay disc degeneration in rats. This study found that SFN can be used as a new and promising agent for the treatment of IVDD.

A20 ameliorates disc degeneration by suppressing mTOR/BNIP3 axis-mediated mitophagy.

The pathological mechanism of intervertebral disc degeneration (IDD) is an unanswered question that we are committed to exploring. A20 is an anti-inflammatory protein of nucleus pulposus (NP) cells and plays a protective role in intervertebral disc degeneration. This study aims to investigate the molecular mechanism by which A20 attenuates disc degeneration. The proteins of interest were measured by immunoblotting, immunofluorescence, ELISA assay, and immunohistochemical technique to conduct related experiments. Immunofluorescence assays and mitochondrial membrane potential (JC-1) were used to assess mitophagy and mitochondrial fitness, respectively. Here, we demonstrated that A20 promoted mitophagy, attenuated pyroptosis, and inhibited the degradation of the extracellular matrix, consequently significantly ameliorating disc degeneration. Mechanistically, A20 reduces pyroptosis and further suppresses cellular mTOR activity. On the one hand, A20-induced mTOR inhibition triggers BNIP3-mediated mitophagy to ensure mitochondrial fitness under LPS stimulation, as a result of mitigating mitochondrial dysfunction induced by LPS. On the other hand, A20-induced mTOR inhibition reduces the loss of mitochondrial membrane potential and the generation of Mitochondrial ROS. The study revealed that A20 promotes BNIP3-mediated mitophagy by suppressing mTOR pathway activation against LPS-induced pyroptosis.

The new ceRNA crosstalk between mRNAs and miRNAs in intervertebral disc degeneration.

Degeneration of the intervertebral disc has been linked to lower back pain. To date, pathophysiological mechanisms of intervertebral disc degeneration (IDD) remain unclear; it is meaningful to find effective diagnostic biomarkers and new therapeutic strategies for IDD. This study aimed to reveal the molecular mechanism of IDD pathogenesis from the multidimensional transcriptomics perspective. Here, we acquired IDD bulk omics datasets (GSE67567 and GSE167199) including mRNA, microRNA expression profiles, and single-cell RNA sequencing (GSE199866) from the public Gene Expression Omnibus (GEO) database. Through principal component analysis and Venn analysis, we found different expression patterns in the IDD transcription level and identified 156 common DEGs in both bulk datasets. GO and KEGG functional analyses showed these dysregulators were mostly enriched in the collagen-containing extracellular matrix, cartilage development, chondrocyte differentiation, and immune response pathways. We also constructed a potentially dysregulated competing endogenous RNA (ceRNA) network between mRNAs and miRNAs related to IDD based on microRNA target information and co-expression analysis of RNA profiles and identified 36 ceRNA axes including ZFP36/miR-155-5p/FOS, BTG2/hsa-miR-185-5p/SOCS3, and COL9A2/hsa-miR-664a-5p/IBA57. Finally, in integrating bulk and single-cell transcriptome data analyses, a total of three marker genes, COL2A1, PAX1, and ZFP36L2, were identified. In conclusion, the key genes and the new ceRNA crosstalk we identified in intervertebral disc degeneration may provide new targets for the treatment of IDD.

An in-depth analysis of the immunomodulatory mechanisms of intervertebral disc degeneration.

Intervertebral disc degeneration (IVDD) is the pathological basis of disc herniation, spinal stenosis, and other related diseases, and the lower back pain it produces lays a heavy financial burden on individuals and society. Thus, it is essential to comprehend IVDD's pathophysiology. Numerous factors, such as inflammatory factors, oxidative stress, apoptosis, matrix metalloproteinases, are linked to IVDD pathogenesis. Despite the fact that many researches has provided explanations for the pathophysiology of IVDD, these studies are typically singular, restricted, and isolated, expound only on one or two components, and do not systematically analyze and summarize the numerous influencing elements. In addition, we discovered that the incidence of many chronic diseases in the field of orthopedics may be thoroughly and systematically defined in terms of immunological systems. In order to provide a theoretical foundation for an in-depth understanding of the pathological process of IVDD and the formulation of more effective prevention and treatment measures, this review provides a comprehensive and systematic account of the pathogenesis of IVDD from the physical to the molecular barriers of the intervertebral disc, from the nucleus pulposus tissue to the cellular to the immune-molecular level.

MiR-31 from Mesenchymal Stem Cell-Derived Extracellular Vesicles Alleviates Intervertebral Disc Degeneration by Inhibiting NFAT5 and Upregulating the Wnt/β-Catenin Pathway.

In this study, we explored the regulatory mechanism of intervertebral disc degeneration (IDD) that involves miR-31 shuttled by bone marrow mesenchymal stem cell-derived extracellular vesicles (BMSC-EVs) and its downstream signaling molecules. Nucleus pulposus cells (NPCs) were isolated and treated with TNF-α to simulate IDD in vitro. The TNF-α-exposed NPCs were then cocultured with hBMSCs or hBMSC-EVs in vitro to detect the effects of hBMSC-EVs on NPC viability, apoptosis, and ECM degradation. Binding between miR-31 and NFAT5 was determined. A mouse model of IDD was prepared by vertebral disc puncture and injected with EVs from hBMSCs with miR-31 knockdown to discern the function of miR-31 in vivo. The results demonstrated that hBMSC-EVs delivered miR-31 into NPCs. hBMSC-EVs enhanced NPC proliferation and suppressed cell apoptosis and ECM degradation, which was associated with the transfer of miR-31 into NPCs. In NPCs, miR-31 bound to the 3′UTR of NFAT5 and inhibited NFAT5 expression, leading to activation of the Wnt/β-catenin pathway and thus promoting NPC proliferation and reducing cell apoptosis and ECM degradation. In addition, miR-31 in hBMSC-EVs alleviated the IDD in mouse models. Taken together, miR-31 in hBMSC-EVs can alleviate IDD by targeting NFAT5 and activating the Wnt/β-catenin pathway.

Ferroptosis: A potential target for the intervention of intervertebral disc degeneration

Ferroptosis, an iron-dependent form of programmed cell death marked by phospholipid peroxidation, is regulated by complex cellular metabolic pathways including lipid metabolism, iron balance, redox homeostasis, and mitochondrial activity. Initial research regarding the mechanism of ferroptosis mainly focused on the solute carrier family 7 member 11/glutathione/glutathione peroxidase 4 (GPX4) signal pathway. Recently, novel mechanisms of ferroptosis, independent of GPX4, have been discovered. Numerous pathologies associated with extensive lipid peroxidation, such as drug-resistant cancers, ischemic organ injuries, and neurodegenerative diseases, are driven by ferroptosis. Ferroptosis is a new therapeutic target for the intervention of IVDD. The role of ferroptosis in the modulation of intervertebral disc degeneration (IVDD) is a significant topic of interest. This is a novel research topic, and research on the mechanisms of IVDD and ferroptosis is ongoing. Herein, we aim to review and discuss the literature to explore the mechanisms of ferroptosis, the relationship between IVDD and ferroptosis, and the regulatory networks in the cells of the nucleus pulposus, annulus fibrosus, and cartilage endplate to provide references for future basic research and clinical translation for IVDD treatment.

Identification of compositional and structural changes in the nucleus pulposus of patients with cervical disc herniation by Raman spectroscopy.

PurposeCervical disc herniation (CDH) is one of the most common spinal diseases in modern society; intervertebral disc degeneration (IVDD) has long been considered as its primary cause. However, the mechanism of intervertebral disc degeneration is still unclear. The aim of the study is to examine the components and structures of proteoglycan and collagen in cervical disc herniated nucleus pulposus (NP) using a validated and convenient Raman spectra technique and histological methods to further elucidate the mechanism of IVDD at the microscopic level.MethodsOur study used a burgeoning technique of Raman spectroscopy combined with in vitro intervertebral disc NP to characterize the above mentioned research purposes. Firstly, we collected cervical disc NP samples and imaging data by certain inclusion and exclusion criteria. Then, we graded the NP of the responsible segment according to the patient’s preoperative cervical magnetic resonance imaging (MRI) T2-weighted images by Pfirrmann grading criteria while measuring the T2 signal intensity value of NP. In addition, the structure of the NP samples was evaluated by histological staining (H&E staining and Safranin-O staining). Finally, the samples were scanned and analyzed by Raman spectroscopy.ResultsA total of 28 NP tissues from 26 patients (two of these patients were cases that involved two segments) with CDH were included in this study. According to the Raman spectroscopy scan, the relative content of proteoglycans which is characterized by the ratio of the two peaks (I 1,064/ I 1,004) in the NP showed a significantly negative correlation with Pfirrmann grade (P < 0.001), while the collagen content and the NP intensity value showed a positive correlation (P < 0.001). For the microstructural characterization of collagen, we found that it may have an essential role in the degenerative process of the intervertebral disc. Moreover, histological staining (H&E staining and Safranin-O staining) showed the general structure of the NP and the distribution of macromolecules.ConclusionThe present study demonstrated the possibility of characterizing the macromolecular substances inside the cervical disc NP tissue by Raman spectroscopy. It also confirmed that macromolecular substances such as proteoglycans and collagen have some degree of alteration in content and structure during degeneration, which has a further positive significance for the elucidation of CDH’s mechanism.

Biomechanical effect of intervertebral disc degeneration on the lower lumbar spine

Lumbar intervertebral disc degeneration can induce bone hyperplasia, lumbar intervertebral disc herniation and other diseases, is one of the causes of low back pain, which seriously affects people’s quality of life. And the causes of degeneration are very complex, so it is essential to understand the underlying mechanism of intervertebral disc degeneration and its influence. In this study, biomechanical effects of L4∼L5 lumbar degeneration with different degrees of degeneration were studied based on the numerical simulations. The three-dimensional finite element model of normal L2∼S1 lumbar vertebrae was established based on CT images of average adult male and verified. Several key parameters (intervertebral disc height, nucleus pulposus size, properties of different materials, etc.) of the model were modified to construct L4∼L5 models with different degrees of degeneration (grade 1, grade 2, grade 3, and grade 4). The range of motion (ROM), the intradiscal pressure of the nucleus, and the maximum Von Mises stress were determined by applying torques in different directions to simulate the four postures of flexion, extension, lateral bending, and axial rotation under compression load (500 N) to simulate the upper body weight of the human body. In different postures, with the increase of L4∼L5 degeneration degree, the ROM of the L4∼L5 degeneration segment showed a decreasing trend (Grade 4 had decrease of 41.9% to 65.2% compared to normal at different postures), while the ROM of its adjacent normal segments showed an increasing trend (L3∼L4: Grade 4 had increase of 21%–94% compared to normal at different postures; L5∼S1: Grade 4 had increase of 32%–66% compared to normal at different postures). With the increase in the degree of degeneration, nucleus pulposus pressure in the L4∼L5 degeneration segment decreased continuously under different postural conditions (Grade 4 had decrease of 25%–134.6% compared to normal at different postures), while the nucleus pulposus pressure in adjacent normal segments (L3∼L4 and L5∼S1) showed a gradually increasing trend. The maximum Von Mises stress of the three segments increased with the increasing degree of degeneration at different postures (L4∼L5: Grade 4 increased to 1.75 ∼ 4 times compared to normal at different postures). In four different models of lumbar disc degeneration, the adjacent normal segment of the disc compensates for the movement and loading pattern of the degenerated segment. At the same time, the load pattern inside the degenerated segment also changes.

O-GlcNAc transferase regulates intervertebral disc degeneration by targeting FAM134B-mediated ER-phagy

Both O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) and endoplasmic reticulum-phagy (ER-phagy) are well-characterized conserved adaptive regulatory mechanisms that maintain cellular homeostasis and function in response to various stress conditions. Abnormalities in O-GlcNAcylation and ER-phagy have been documented in a wide variety of human pathologies. However, whether O-GlcNAcylation or ER-phagy is involved in the pathogenesis of intervertebral disc degeneration (IDD) is largely unknown. In this study, we investigated the function of O-GlcNAcylation and ER-phagy and the related underlying mechanisms in IDD. We found that the expression profiles of O-GlcNAcylation and O-GlcNAc transferase (OGT) were notably increased in degenerated NP tissues and nutrient-deprived nucleus pulposus (NP) cells. By modulating the O-GlcNAc level through genetic manipulation and specific pharmacological intervention, we revealed that increasing O-GlcNAcylation abundance substantially enhanced cell function and facilitated cell survival under nutrient deprivation (ND) conditions. Moreover, FAM134B-mediated ER-phagy activation was regulated by O-GlcNAcylation, and suppression of ER-phagy by FAM134B knockdown considerably counteracted the protective effects of amplified O-GlcNAcylation. Mechanistically, FAM134B was determined to be a potential target of OGT, and O-GlcNAcylation of FAM134B notably reduced FAM134B ubiquitination-mediated degradation. Correspondingly, the protection conferred by modulating O-GlcNAcylation homeostasis was verified in a rat IDD model. Our data demonstrated that OGT directly associates with and stabilizes FAM134B and subsequently enhances FAM134B-mediated ER-phagy to enhance the adaptive capability of cells in response to nutrient deficiency. These findings may provide a new option for O-GlcNAcylation-based therapeutics in IDD prevention.

Study on the process of intervertebral disc disease by the theory of continuum damage mechanics

BackgroundRecently, more and more people suffer from low back pain triggered by lumbar degenerative disc disease. The mechanical factor is one of the most significant causes of disc degeneration. This study aims to explore the biomechanical responses of the intervertebral disc, and investigate the process of disc injury by the theory of continuum damage mechanics. MethodsA finite element model of the L4–L5 lumbar spine was developed and validated. The model not only considered changes in permeability coefficient with strain, but also included physiological factors such as osmotic pressure. Three loading conditions were simulated: (A) static loads, (B) vibration loads, (C) injury process. FindingsThe simulation results revealed that the facet joints shared massive stresses of the intervertebral discs, and prevented excessive lumbar spine movement. However, their asymmetrical position may have led to degeneration. The von Mises stress and pore pressure of annulus fibrosus showed significantly different trends under static loads and vibration loads. The von Mises stress of nucleus pulposus was not sensitive to vibration loads, but its pore pressure was conspicuously influenced by vibration loads. The injury first appeared at the posterior centre, and then, it gradually expanded along the edge of the intervertebral disc. With an increase in the loading steps, the damage rate of the intervertebral disc increased logarithmically. InterpretationThe variation in the biomechanical performance of the intervertebral disc could be attributed to the periodic movement of internal fluids. This study might be helpful for understanding the mechanism of intervertebral disc degeneration.

New Hope for Treating Intervertebral Disc Degeneration: Microsphere-Based Delivery System.

Intervertebral disc (IVD) degeneration (IVDD) has been considered the dominant factor in low back pain (LBP), and its etiological mechanisms are complex and not yet fully elucidated. To date, the treatment of IVDD has mainly focused on relieving clinical symptoms and cannot fundamentally solve the problem. Recently, a novel microsphere-based therapeutic strategy has held promise for IVD regeneration and has yielded encouraging results with in vitro experiments and animal models. With excellent injectability, biocompatibility, and biodegradability, this microsphere carrier allows for targeted delivery and controlled release of drugs, gene regulatory sequences, and other bioactive substances and supports cell implantation and directed differentiation, aiming to improve the disease state of IVD at the source. This review discusses the possible mechanisms of IVDD and the limitations of current therapies, focusing on the application of microsphere delivery systems in IVDD, including targeted delivery of active substances and drugs, cellular therapy, and gene therapy, and attempts to provide a new understanding for the treatment of IVDD.

Identification of Core Genes and Screening of Potential Targets in Intervertebral Disc Degeneration Using Integrated Bioinformatics Analysis.

Background: Intervertebral disc degeneration (IDD), characterized by diverse pathological changes, causes low back pain (LBP). However, prophylactic and delaying treatments for IDD are limited. The aim of our study was to investigate the gene network and biomarkers of IDD and suggest potential therapeutic targets. Methods: Differentially expressed genes (DEGs) associated with IDD were identified by analyzing the mRNA, miRNA, and lncRNA expression profiles of IDD cases from the Gene Expression Omnibus (GEO). The protein–protein interaction (PPI) network, Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis as well as miRNA–lncRNA–mRNA networks were conducted. Moreover, we obtained 71 hub genes and performed a comprehensive analysis including GO, KEGG, gene set enrichment analysis (GSEA), gene set variation analysis (GSVA), Disease Ontology (DO), methylation analysis, receiver operating characteristic (ROC) curve analysis, immune infiltration analysis, and potential drug identification. We finally used qRT-PCR to verify 13 significant DEGs in normal and degenerative nucleus pulposus cells (NPCs). Results: We identified 305 DEGs closely related to IDD. The GO and KEGG analyses indicated that changes in IDD are significantly associated with enrichment of the inflammatory and immune response. GSEA analysis suggested that cell activation involved in the inflammatory immune response amide biosynthetic process was the key for the development of IDD. The GSVA suggested that DNA repair, oxidative phosphorylation, peroxisome, IL-6-JAK-STAT3 signaling, and apoptosis were crucial in the development of IDD. Among the 71 hub genes, the methylation levels of 11 genes were increased in IDD. A total of twenty genes showed a high functional similarity and diagnostic value in IDD. The result of the immune cell infiltration analysis indicated that seven genes were closely related to active natural killer cells. The most relevant targeted hub genes for potential drug or molecular compounds were MET and PIK3CD. Also, qRT-PCR results showed that ARHGAP27, C15orf39, DEPDC1, DHRSX, MGAM, SLC11A1, SMC4, and LINC00887 were significantly downregulated in degenerative NPCs; H19, LINC00685, mir-185-5p, and mir-4306 were upregulated in degenerative NPCs; and the expression level of mir-663a did not change significantly in normal and degenerative NPCs. Conclusion: Our findings may provide new insights into the functional characteristics and mechanism of IDD and aid the development of IDD therapeutics.