Mitochondrial 'Birth-Death' coordinator: An intelligent hydrogen nanogenerator to enhance intervertebral disc regeneration.

Currently, mitochondrial dysfunction caused by oxidative stress is a growing concern in degenerative diseases, notably intervertebral disc degeneration (IVDD). Dysregulation of the balance of mitochondrial quality control (MQC) has been considered the key contributor, while it's still challenging to effectively harmonize different MQC components in a simple and biologically safe way. Hydrogen gas (H2) is a promising mitochondrial therapeutic molecule due to its bio-reductivity and diffusibility across cellular membranes, yet its relationship with MQC regulation remains unknown. Herein, we propose a mitochondrial 'Birth-Death' coordinator achieved by an intelligent hydrogen nanogenerator (Fe@HP-OD), which can sustainably release H2 in response to the unique microenvironment in degenerated IVDs. Both in vitro and in vivo results prove alleviation of cellular oxidative stress and restoration of nucleus pulposus cells function, thereby facilitating successful IVD regeneration. Significantly, this study for the first time proposes the mitochondrial 'Birth-Death' coordination mechanism: 1) attenuation of overactivated mitochondrial 'Death' process (UPRmt and unselective mitophagy); and 2) activation of Adenosine 5'-monophosphate-activated protein kinase (AMPK) signaling pathway for mitochondrial 'Birth-Death' balance (mitochondrial biogenesis and controlled mitophagy). These pioneering findings can fill in the gaps in molecular mechanisms for H2 regulation on MQC homeostasis, and pave the way for future strategies towards restoring equilibrium of MQC system against degenerative diseases.

Machine learning and single-cell analysis identify the mitophagy-associated gene TOMM22 as a potential diagnostic biomarker for intervertebral disc degeneration.

Background: Mitophagy selectively eliminates potentially cytotoxic and damaged mitochondria and effectively prevents excessive cytotoxicity from damaged mitochondria, thereby attenuating inflammatory and oxidative responses. However, the potential role of mitophagy in intervertebral disc degeneration remains to be elucidated. Methods: The GSVA method, two machine learning methods (SVM-RFE algorithm and random forest), the CIBERSORT and MCPcounter methods, as well as the consensus clustering method and the WGCNA algorithm were used to analyze the involvement of mitophagy in intervertebral disc degeneration, the diagnostic value of mitophagy-associated genes in intervertebral disc degeneration, and the infiltration of immune cells, and identify the gene modules that were closely related to mitophagy. Single-cell analysis was used to detect mitophagy scores and TOMM22 expression, and pseudo-temporal analysis was used to explore the function of TOMM22 in nucleus pulposus cells. In addition, TOMM22 expression was compared between human normal and degenerated intervertebral disc tissue samples by immunohistochemistry and PCR. Results: This study identified that the mitophagy pathway score was elevated in intervertebral disc degeneration compared with the normal condition. A strong link was present between mitophagy genes and immune cells, which may be used to typify intervertebral disc degeneration. The single-cell level showed that mitophagy-associated gene TOMM22 was highly expressed in medullary cells of the disease group. Further investigations indicated the upregulation of TOMM22 expression in late-stage nucleus pulposus cells and its role in cellular communication. In addition, human intervertebral disc tissue samples established that TOMM22 levels were higher in disc degeneration samples than in normal samples. Conclusions: Our findings revealed that mitophagy may be used in the diagnosis of intervertebral disc degeneration and its typing, and TOMM22 is a molecule in this regard and may act as a potential diagnostic marker in intervertebral disc degeneration.

Quercetin relieves compression-induced cell death and lumbar disc degeneration by stabilizing HIF1A protein.

Background: Lumbar disc degeneration (LDD) is a prevalent condition characterized by the decreased viability and functional impairment of nucleus pulposus mesenchymal stem cells (NPMSCs). Shaoyao-Gancao decoction (SGD), a traditional Chinese medicine formula, has been used to treat LDD, but its active components and mechanisms are unclear. Methods: An integrative network pharmacology and transcriptome analysis were conducted to identify bioactive compounds in SGD that could target LDD. NPMSCs were cultured under mechanical compression as a cellular model of LDD. A rat model of annulus fibrosus needle-puncture was established to induce intervertebral disc degeneration. The effects of quercetin, a predicted active component, on alleviating compression-induced NPMSC death and LDD were evaluated in vitro and in vivo. Results: The analysis identified hypoxia-inducible factor 1-alpha (HIF1A) as a potential target of quercetin in LDD. HIF1A was upregulated in degenerated human disc samples and compression-treated NPMSCs. Quercetin treatment alleviated compression-induced oxidative stress, apoptosis, and loss of viability in NPMSCs by stabilizing HIF1A. The protective effects of quercetin were abrogated by HIF1A inhibition. In the rat model, quercetin ameliorated intervertebral disc degeneration. Conclusion: Our study identified HIF1A as a protective factor against compression-induced cell death in NPMSCs. Quercetin, a bioactive compound found in the traditional Chinese medicine formula SGD, improved the survival of NPMSCs and alleviated LDD progression by stabilizing HIF1A. Targeting the HIF1A pathway through natural compounds like quercetin could represent a promising strategy for the clinical management of LDD and potentially other degenerative disc diseases.

A novel spherical GelMA-HAMA hydrogel encapsulating APET×2 polypeptide and CFIm25-targeting sgRNA for immune microenvironment modulation and nucleus pulposus regeneration in intervertebral discs.

METHODS: Single-cell transcriptomics and high-throughput transcriptomics were used to screen factors significantly correlated with intervertebral disc degeneration (IDD). Expression changes of CFIm25 were determined via RT-qPCR and Western blot. NP cells were isolated from mouse intervertebral discs and induced to degrade with TNF-α and IL-1ß. CFIm25 was knocked out using CRISPR-Cas9, and CFIm25 knockout and overexpressing nucleus pulposus (NP) cell lines were generated through lentiviral transfection. Proteoglycan expression, protein expression, inflammatory factor expression, cell viability, proliferation, migration, gene expression, and protein expression were analyzed using various assays (alcian blue staining, immunofluorescence, ELISA, CCK-8, EDU labeling, transwell migration, scratch assay, RT-qPCR, Western blot). The GelMA-HAMA hydrogel loaded with APET×2 polypeptide and sgRNA was designed, and its effects on NP regeneration were assessed through in vitro and mouse model experiments. The progression of IDD in mice was evaluated using X-ray, H&E staining, and Safranin O-Fast Green staining. Immunohistochemistry was performed to determine protein expression in NP tissue. Proteomic analysis combined with in vitro and in vivo experiments was conducted to elucidate the mechanisms of hydrogel action. RESULTS: CFIm25 was upregulated in IDD NP tissue and significantly correlated with disease progression. Inhibition of CFIm25 improved NP cell degeneration, enhanced cell proliferation, and migration. The hydrogel effectively knocked down CFIm25 expression, improved NP cell degeneration, promoted cell proliferation and migration, and mitigated IDD progression in a mouse model. The hydrogel inhibited inflammatory factor expression (IL-6, iNOS, IL-1ß, TNF-α) by targeting the p38/NF-κB signaling pathway, increased collagen COLII and proteoglycan Aggrecan expression, and suppressed NP degeneration-related factors (COX-2, MMP-3). CONCLUSION: The study highlighted the crucial role of CFIm25 in IDD and introduced a promising therapeutic strategy using a porous spherical GelMA-HAMA hydrogel loaded with APET×2 polypeptide and sgRNA. This innovative approach offers new possibilities for treating degenerated intervertebral discs.

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

BACKGROUND: Limited 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. METHODS: RNA-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. RESULTS: We 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. CONCLUSIONS: We 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.

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.

Inhibition of KDM2/7 Promotes Notochordal Differentiation of hiPSCs.

Intervertebral disc disease (IDD) is a debilitating spine condition that can be caused by intervertebral disc (IVD) damage which progresses towards IVD degeneration and dysfunction. Recently, human pluripotent stem cells (hPSCs) were recognized as a valuable resource for cell-based regenerative medicine in skeletal diseases. Therefore, adult somatic cells reprogrammed into human induced pluripotent stem cells (hiPSCs) represent an attractive cell source for the derivation of notochordal-like cells (NCs) as a first step towards the development of a regenerative therapy for IDD. Utilizing a differentiation method involving treatment with a four-factor cocktail targeting the BMP, FGF, retinoic acid, and Wnt signaling pathways, we differentiate CRISPR/Cas9-generated mCherry-reporter knock-in hiPSCs into notochordal-like cells. Comprehensive analysis of transcriptomic changes throughout the differentiation process identified regulation of histone methylation as a pivotal driver facilitating the differentiation of hiPSCs into notochordal-like cells. We further provide evidence that specific inhibition of histone demethylases KDM2A and KDM7A/B enhanced the lineage commitment of hiPSCs towards notochordal-like cells. Our results suggest that inhibition of KDMs could be leveraged to alter the epigenetic landscape of hiPSCs to control notochord-specific gene expression. Thus, our study highlights the importance of epigenetic regulators in stem cell-based regenerative approaches for the treatment of disc degeneration.

[Effects of electroacupuncture at "Jiaji" (EX-B 2) on extracellular matrix of chondrocytes and inflammatory reaction in rabbits with Modic changes of cartilage endplate].

OBJECTIVE: To observe the effects of electroacupuncture (EA) at "Jiaji" (EX-B 2) on extracellular matrix (ECM) of chondrocytes and inflammatory reaction in rabbits with Modic changes (MC) of cartilage endplate, and to explore the mechanism of EA in treating MC of endplate cartilage. METHODS: Eighteen male New Zealand white rabbits were randomly divided into a sham operation group, a model group and an EA group, 6 rabbits in each group. Based on the autoimmune theory, MC model was established by embedding autogenous nucleus pulposus in the rabbits of the model group and the EA group, based on autoimmunity. After successful modeling, EA was applied at bilateral "Jiaji" (EX-B 2) of L5 and L6 in the EA group, with disperse-dense wave, 2 Hz/15 Hz in frequency and 1 mA in current intensity, 20 min a time, once a day, 1-day interval was taken after continuous 6-day intervention, for 4 weeks totally. Before and after modeling, as well as before and after intervention, the comprehensive response score was observed. After modeling and intervention, magnetic resonance imaging (MRI) was used to observe the signal intensity of intervertebral disc and cartilage endplate. After intervention, the morphology of chondrocytes of cartilage endplate was observed by HE staining; the positive expression of a disintegrin and metalloproteinase with thrombospondin motif-5 (ADAMTS5) and Aggrecan in the cartilage endplate was detected by immunohistochemistry; the levels of inflammatory factors i.e. interleukin-1ß (1L-1ß) and tumor necrosis factor-α (TNF-α) in the cartilage endplate were detected by ELISA; the protein expression of ADAMTS5, Aggrecan, matrix metalloproteinase-13 (MMP-13), IL-1ß and TNF-α in the cartilage endplate was detected by Western blot. RESULTS: Compared with the sham operation group, in the model group, the comprehensive response score was decreased (P<0.01); L5/L6 intervertebral disc and the cancellous bones of endplate vertebral body showed low signal and unclear boundary; the chondrocytes of the cartilage endplate increased significantly, the cells were enlarged and hypertrophic, and the nuclei were wrinkled and clustered; the positive expression of ADAMTS5 as well as the levels of IL-1ß and TNF-α were increased (P<0.01), while the positive expression of Aggrecan was decreased (P<0.01) in the cartilage endplate; the protein expression of ADAMTS5, MMP-13, IL-1ß and TNF-α was increased (P<0.01), while that of Aggrecan was decreased (P<0.01) in the cartilage endplate. Compared with the model group, in the EA group, the comprehensive response score was increased (P<0.01); the signal of L5/L6 intervertebral disc and the cancellous bones of endplate vertebral body was enhanced; the chondrocytes of the cartilage endplate were reduced, the nuclei were slightly crumpled and scattered; the positive expression of ADAMTS5 as well as the levels of IL-1ß and TNF-α were decreased (P<0.05, P<0.01), while the positive expression of Aggrecan was increased (P<0.01) in the cartilage endplate; the protein expression of ADAMTS5, MMP-13, IL-1ß and TNF-α was decreased (P<0.05, P<0.01), while that of Aggrecan was increased (P<0.05) in the cartilage endplate. CONCLUSION: EA at "Jiaji" (EX-B 2) can delay the MC of cartilage endplate. The mechanism may be related to inhibiting the degradation of ECM of chondrocytes and the secretion of inflammatory factors, and repairing the degeneration of endplate cartilage.

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.

Animal Models of Internal Endplate Injury-Induced Intervertebral Disc Degeneration: A Systematic Review.

OBJECTIVE: To systematically review relevant animal models of disk degeneration induced through the endplate injury pathway and to provide suitable animal models for exploring the intrinsic mechanisms and treatment of disk degeneration. DESIGN: PubMed, Web of Science, Cochrane and other databases were searched for literature related to animal models of disk degeneration induced by the endplate injury pathway from establishment to August 2024, and key contents in the literature were screened and extracted to analyze and evaluate each type of animal model using the literature induction method. RESULTS: Fifteen animal experimental studies were finally included in the literature, which can be categorized into direct injury models and indirect injury models, of which direct injury models include transvertebral injury models and transpedicular approach injury models, and indirect injury models include endplate ischemia models and vertebral fracture-induced endplate injury models. The direct injury models have a minimum observation period of 2 months and a maximum of 32 wk. All direct injury models were successful in causing disk degeneration, and the greater the number of interventions, the greater the degree of disk degeneration caused. The observation period for the indirect injury models varied from 4 wk to 70 wk. Of the 9 studies, only one study was unsuccessful in inducing disk degeneration, and this was the first animal study in this research to attempt to intervene on the endplate to cause disk degeneration. CONCLUSION: The damage to the direct injury model is more immediate and controllable in extent and can effectively lead to disk degeneration. The indirect injury models do not directly damage the endplate structure, making it easier to observe the physiological and pathological condition of the endplate and associated structures of the disk. None of them can completely simulate the corresponding process of endplate injury-induced disk degeneration in humans, and there is no uniform clinical judgment standard for this type of model. The most appropriate animal model still needs further exploration and discovery.

Hydrogel and Microgel Collaboration for Spatiotemporal Delivery of Biofactors to Awaken Nucleus Pulposus-Derived Stem Cells for Endogenous Repair of Disc.

Depletion of nucleus pulposus-derived stem cells (NPSCs) is a major contributing factor to the attenuation of endogenous regenerative capacity in intervertebral disc degeneration (IVDD). Introducing a hydrogel drug delivery system is a potential strategy for counteracting endogenous cell depletion. The present study proposes a delivery platform for the spatiotemporal release of multiple drugs by combining sodium alginate hydrogels with gelatin microgels (SCGP hydrogels). The SCGP hydrogels facilitated the initial release of chondroitin sulfate (ChS) and the gradual release of an independently developed parathyroid hormone-related peptide (P2). The combined action of these two small molecule drugs "awakened" the reserve NPSCs, mitigated cell damage induced by H2O2, significantly enhanced their biological activity, and promoted their differentiation toward nucleus pulposus cells. The mechanical and viscoelastic properties of the hydrogel are enhanced by physical and chemical dual cross-linking to adapt to the loading environment of the degenerated disc. A rat IVDD model is used to validate that the SCGP hydrogel can significantly inhibit the progression of IVDD and stimulate the endogenous repair of IVDD. Therefore, the spatiotemporal differential drug delivery system of the SCGP hydrogel holds promise as a convenient and efficacious therapeutic strategy for minimally invasive IVDD treatment.

Automated Three-Dimensional Imaging and Pfirrmann Classification of Intervertebral Disc Using a Graphical Neural Network in Sagittal Magnetic Resonance Imaging of the Lumbar Spine.

This study aimed to develop a graph neural network (GNN) for automated three-dimensional (3D) magnetic resonance imaging (MRI) visualization and Pfirrmann grading of intervertebral discs (IVDs), and benchmark it against manual classifications. Lumbar IVD MRI data from 300 patients were retrospectively analyzed. Two clinicians assessed the manual segmentation and grading for inter-rater reliability using Cohen's kappa. The IVDs were then processed and classified using an automated convolutional neural network (CNN)-GNN pipeline, and their performance was evaluated using F1 scores. Manual Pfirrmann grading exhibited moderate agreement (κ = 0.455-0.565) among the clinicians, with higher exact match frequencies at lower lumbar levels. Single-grade discrepancies were prevalent except at L5/S1. Automated segmentation of IVDs using a pretrained U-Net model achieved an F1 score of 0.85, with a precision and recall of 0.83 and 0.88, respectively. Following 3D reconstruction of the automatically segmented IVD into a 3D point-cloud representation of the target intervertebral disc, the GNN model demonstrated moderate performance in Pfirrmann classification. The highest precision (0.81) and F1 score (0.71) were observed at L2/3, whereas the overall metrics indicated moderate performance (precision: 0.46, recall: 0.47, and F1 score: 0.46), with variability across spinal levels. The integration of CNN and GNN offers a new perspective for automating IVD analysis in MRI. Although the current performance highlights the need for further refinement, the moderate accuracy of the model, combined with its 3D visualization capabilities, establishes a promising foundation for more advanced grading systems.

An exploratory study of cervical disc degeneration model and mechanism of acupuncture therapy in rabbits.

Acupuncture is an important therapy method in traditional Chinese medicine for treating intervertebral disc degeneration (IVDD), offering a wide range of applications. It is based on the theory of Chinese veterinary medicine and combines the stage of the disease course and individual differences for syndrome differentiation and treatment. However, there are few studies on the acupuncture treatment of cervical disc degeneration (CDD) in rabbits. Treatment based on syndrome differentiation is the basic principle of Chinese veterinary treatment. The selection of acupoints for external treatment should be based on individual etiology and pathogenesis. Nevertheless, most current studies do not follow this guideline. In this study, we established the CDD model and explored the mechanism of acupuncture treatment in alleviating CDD in rabbits by selecting a group of main acupoints including cervical Jiaji, Fengchi, Tianzhu, Naohu, Dazhui, and Houxi acupoints, combined with Western medicine's understanding of the pathogenesis of cervical spondylosis, from the anti-inflammatory, analgesic, and tissue-repairing perspectives. Magnetic resonance imaging (MRI) confirmed the successful establishment of the rabbit CDD model. Acupuncture stimulation reduced the increase of average and maximum neck temperature due to CDD in rabbits. The acupuncture treatment relieved the spinal disc damage in the neck of the rabbit, which also decreased the expression level of pro-apoptotic factor Bax and increased the expression level of anti-apoptotic factor Bcl-2. In addition, it can alleviate the abnormal apoptosis of rabbit intervertebral disc, decrease the expression level of inflammatory factors such as TNF-α, IL-1ß, IL-2, and PGE2α, and alleviate the intense inflammation and pain response caused by CDD in rabbits. In conclusion, Acupuncture treatment can slow down the CDD of rabbits by regulating the inflammatory response and abnormal apoptosis of intervertebral disc tissue.

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.

NRF2 in age-related musculoskeletal diseases: Role and treatment prospects.

The NRF2 pathway is a metabolic- and redox-sensitive signaling axis in which the transcription factor controls the expression of a multitude of genes that enable cells to survive environmental stressors, such as oxidative stress, mainly by inducing the expression of cytoprotective genes. Basal NRF2 levels are maintained under normal physiological conditions, but when exposed to oxidative stress, cells activate the NRF2 pathway, which is crucial for supporting cell survival. Recently, the NRF2 pathway has been found to have novel functions in metabolic regulation and interplay with other signaling pathways, offering novel insights into the treatment of various diseases. Numerous studies have shown that targeting its pathway can effectively investigate the development and progression of age-related musculoskeletal diseases, such as sarcopenia, osteoporosis, osteoarthritis, and intervertebral disc degeneration. Appropriate regulation of the NRF2 pathway flux holds promise as a means to improve musculoskeletal function, thereby providing a new avenue for drug treatment of age-related musculoskeletal diseases in clinical settings. The review summarized an overview of the relationship between NRF2 and cellular processes such as oxidative stress, apoptosis, inflammation, mitochondrial dysfunction, ferroptosis, and autophagy, and explores the potential of targeted NRF2 regulation in the treatment of age-related musculoskeletal diseases.

Mapping knowledge structure and themes trends of non-surgical treatment in intervertebral disc degeneration.

Background: Intervertebral disc degeneration (IDD) is a chronic disabling disease caused by degeneration of nucleus pulposus cells, decreased activity and the number of nucleus pulposus cells, decreased extracellular matrix, and infiltration of inflammatory factors, resulting in low back and leg pain. Recent studies have shown that non-surgical treatment is of great significance in reversing the progression of degenerative disc disease, and there are more relevant literature reports. However, there is no bibliometric analysis in this area. This study aimed to describe the knowledge structure and thematic trends of non-surgical treatment methods for IDD through bibliometrics. Methods: Articles and reviews on non-surgical treatment of disc degeneration from 1998 to 2022 were collected on the Web of Science. VOSviewer 1.6.18, CiteSpace 6.1.R3, R package "bibliometrix" and two online analysis platforms were used for bibliometric and visual literature analysis. Results: 961 articles were screened for inclusion, including 821 articles and 140 reviews. The analysis of our study shows that publications in the non-surgical treatment of disc degeneration are increasing annually, with publications coming mainly from North America and Asia, with China and the United States dominating. Huazhong Univ Sci & Technol and Wang K are the most prolific institutions and authors, respectively, and Le Maitre CL is the most co-cited author. However, there is less collaboration between institutions in different countries. Spine is both the most published and the most cited journal. According to the co-citation and co-occurrence analysis results, "mesenchymal stem cells," "exosomes," "medication," and "tissue engineering" are the current research hotspots in this field. Conclusions: This study employs bibliometric analysis to explore the knowledge structure and trends of non-surgical treatments for IDD from 2013 to 2022. Key research hotspots include mesenchymal stem cells, exosomes, medication, and tissue engineering. The number of publications, especially from China and the USA, has increased significantly, though international collaboration needs improvement. Influential contributors include Wang K and the journal Spine. These findings provide a comprehensive overview and highlight important future directions for the field.

The causality between smoking and intervertebral disc degeneration mediated by IL-1ß secreted by macrophage: A Mendelian randomization study.

There is still a lack of high-level evidence regarding the causal relationship between smoking and intervertebral disc degenerative diseases. This study utilized data from genome wide analysis studies and conducted two-sample Mendelian randomization analyses across multiple heterogeneous datasets. We evaluated the causal relationships between smoking behavior, serum inflammatory factors, serum chemokines, and intervertebral disc degeneration. Sensitivity analysis was performed to examine data heterogeneity and the pleiotropy of causal effects. The results indicated that smokers were liable to develop intervertebral disc degeneration (OR 1.770; 95 % CI, 1.519-2.064; p = 2.992 × 10-13), and long-term smoking behavior increased the risk of intervertebral disc degeneration (OR 1.715; 95 % CI 1.475-1.994; P = 2.220 × 10-12). Additionally, a causal relationship was confirmed between serum IL-1ß level and intervertebral disc degeneration (OR 1.087; 95 % CI, 1.023-1.154; p = 0.007). The "smoking index" representing lifelong smoking habit was also found to be causally related to serum MCP-3 level(ß = 0.292; SE = 0.093; p = 0.002). All of the causality mentioned above remained stable in sensitivity tests. Based on the analysis results and fundamental medicine theories around macrophage-induced inflammation in degenerative intervertebral discs, we have constructed a new mechanism that long-term smoking could induce an increase in serum MCP-3 level, promoting the gathering and activation of monocyte macrophages. Furthermore, the recruited macrophages led to an increase in local IL-1ß within the intervertebral disc, ultimately exacerbating the process of intervertebral disc degeneration. What we have found is expected to accelerate the development of prevention and treatment of intervertebral disc degeneration.

Gait assessment in a female rat Sprague Dawley model of disc-associated low back pain.

PURPOSE: Gait disturbances are common in human low back pain (LBP) patients, suggesting potential applicability to rodent LBP models. This study aims to assess the influence of disc-associated LBP on gait in female Sprague Dawley rats and explore the utility of the open-source Gait Analysis Instrumentation and Technology Optimized for Rodents (GAITOR) suite as a potential alternative tool for spontaneous pain assessment in a previously established LBP model. MATERIALS AND METHODS: Disc degeneration was surgically induced using a one-level disc scrape injury method, and microcomputed tomography was used to assess disc volume loss. After disc injury, axial hypersensitivity was evaluated using the grip strength assay, and an open field test was used to detect spontaneous pain-like behavior. RESULTS: Results demonstrated that injured animals exhibit a significant loss in disc volume and reduced grip strength. Open field test did not detect significant differences in distance traveled between sham and injured animals. Concurrently, animals with injured discs did not display significant gait abnormalities in stance time imbalance, temporal symmetry, spatial symmetry, step width, stride length, and duty factor compared to sham. However, comparisons with reference values of normal gait reported in prior literature reveal that injured animals exhibit mild deviations in forelimb and hindlimb stance time imbalance, forelimb temporal symmetry, and hindlimb spatial symmetry at some time points. CONCLUSIONS: This study concludes that the disc injury may have very mild effects on gait in female rats within 9 weeks post-injury and recommends future in depth dynamic gait analysis and longer studies beyond 9 weeks to potentially detect gait.

Protective effects of alpinetin against interleukin-1ß-exposed nucleus pulposus cells: Involvement of the TLR4/MyD88 pathway in a cellular model of intervertebral disc degeneration.

Intervertebral disc degeneration (IDD) causes a variety of symptoms such as low back pain, disc herniation, and spinal stenosis, which can lead to high social and economic costs. Alpinetin has an anti-inflammatory potential, but its effect on IDD is unclear. Herein, we investigated the effect of alpinetin on IDD. To mimic an in vitro model of IDD, nucleus pulposus cells (NPCs) were exposed to interleukin 1ß (IL-1ß). The viability of NPCs was assessed by CCK-8 assay. The expression of Toll-like receptor 4 (TLR4), myeloid differentiation primary response protein 88 (MyD88), aggrecan, collagen-2, and matrix metalloproteinase-3 (MMP-3) was examined by qRT-PCR and western blotting. The protein levels of B cell lymphoma-2 (Bcl-2), Bcl-2-associated protein X (Bax), and cleaved caspase-3 were scrutinized by western blotting. The flow cytometry assay was performed to assess apoptosis of NPCs. The contents of inflammatory factors were examined by ELISA kits. Results showed that alpinetin repressed IL-1ß-tempted activation of the TLR4/MyD88 pathway and apoptosis in NPCs. Alpinetin alleviated IL-1ß-tempted inflammatory responses and oxidative stress in NPCs. Moreover, alpinetin lessened IL-1ß-tempted extracellular matrix (ECM) degeneration in NPCs by enhancing the expression of aggrecan and collagen-2 and reducing the expression of MMP-3. The effects of alpinetin on IL-1ß-exposed NPCs were neutralized by TLR4 upregulation. In conclusion, alpinetin repressed IL-1ß-tempted apoptosis, inflammatory responses, oxidative stress, and ECM degradation in NPCs through the inactivation of the TLR4/MyD88 pathway.

Identification of extracellular matrix proteins in plasma as a potential biomarker for intervertebral disc degeneration.

PURPOSE: Recently, there has been significant focus on extracellular matrix proteolysis due to its importance in the pathological progression of intervertebral disc degeneration (IVDD). The present study investigates the circulating levels of extracellular matrix proteins in the plasma of IVDD and determines their potential relevance as biomarkers in disc degeneration. METHODS: Global proteomic analysis was performed in the plasma samples of 10 healthy volunteers (HV) and 10 diseased subjects (DS) after depletion of highly abundant proteins such as albumin and IgG. RESULTS: We identified 144 and 135 matrix-associated proteins in plasma samples from healthy volunteers (HV) and patients with disc degeneration (DS), respectively. Among these, 49 of the matrix-associated proteins were identical to the proteins found in intervertebral disc (IVD) tissues retrieved from the in-house library. Applying stringent parameters, we selected 28 proteins, with 26 present in DS and 21 in HV. 19 proteins were found common between the groups, two of which-aggrecan (ACAN) and fibulin 1 (FBLN1) - showed statistically significant differences. Specifically, ACAN was up-regulated and FBLN1 was down-regulated in the DS-plasma. In particular, DS-plasma exhibited specific expression of collagen type 2a1 (COL2A1), native to the nucleus pulposus. CONCLUSION: The distinct presence of collagen type 2a1 and the elevated expression of aggrecan in IVDD plasma may serve as the basis for the development of a potential biomarker for monitoring the progression of disc degeneration.