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.

Osteopontin deficiency promotes cartilaginous endplate degeneration by enhancing the NF-κB signaling to recruit macrophages and activate the NLRP3 inflammasome.

Intervertebral disc degeneration (IDD) is a major cause of discogenic pain, and is attributed to the dysfunction of nucleus pulposus, annulus fibrosus, and cartilaginous endplate (CEP). Osteopontin (OPN), a glycoprotein, is highly expressed in the CEP. However, little is known on how OPN regulates CEP homeostasis and degeneration, contributing to the pathogenesis of IDD. Here, we investigate the roles of OPN in CEP degeneration in a mouse IDD model induced by lumbar spine instability and its impact on the degeneration of endplate chondrocytes (EPCs) under pathological conditions. OPN is mainly expressed in the CEP and decreases with degeneration in mice and human patients with severe IDD. Conditional Spp1 knockout in EPCs of adult mice enhances age-related CEP degeneration and accelerates CEP remodeling during IDD. Mechanistically, OPN deficiency increases CCL2 and CCL5 production in EPCs to recruit macrophages and enhances the activation of NLRP3 inflammasome and NF-κB signaling by facilitating assembly of IRAK1-TRAF6 complex, deteriorating CEP degeneration in a spatiotemporal pattern. More importantly, pharmacological inhibition of the NF-κB/NLRP3 axis attenuates CEP degeneration in OPN-deficient IDD mice. Overall, this study highlights the importance of OPN in maintaining CEP and disc homeostasis, and proposes a promising therapeutic strategy for IDD by targeting the NF-κB/NLRP3 axis.

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.

Collagen II enrichment through scAAV6-RNAi-mediated inhibition of matrix-metalloproteinases 3 and 13 in degenerative nucleus-pulposus cells degenerative disc disease and biological treatment strategies.

Intervertebral disc (IVD) degeneration damaging the extracellular matrix (ECM) of IVDs is the main cause of spine-associated disorders. Degenerative disc disease (DDD) is a multifaceted disorder, where environmental factors, inflammatory cytokines and catabolic enzymes act together. DDD starts typically due to imbalance between ECM biosynthesis and degradation within IVDs, especially through unbalanced degradation of aggrecan and collagen II in nucleus pulposus (NP). Current treatment approaches are primarily based on conservative or surgical therapies, which are insufficient for biological regeneration. The disintegrins and metalloproteinases with thrombospondin motifs (ADAMTSs) and matrix metalloproteinases (MMPs) are the key proteolytic enzymes for degradation of aggrecan and collagens. Previously, high expression levels of ADAMTS4, ADAMTS5, MMP3 and MMP13, which are accompanied with low levels of aggrecan and collagen II, were demonstrated in degenerative human NP cells. Moreover, self-complementary adeno-associated virus type 6 (scAAV6) mediated inhibitions of ADAMTS4 and ADAMTS5 by RNA-interference (RNAi) could specifically enhance aggrecan level. Thus, MMPs are apparently the main degrading enzymes of collagen II in NP. Furthermore, scAAV6-mediated inhibitions of MMP3 and MMP13 have not yet been investigated. Therefore, we attempted to enhance the level of collagen II in degenerative NP cells by scAAV6-RNAi-mediated inhibitions of MMP3 and MMP13. MRI was used to determine preoperative grading of IVD degeneration in patients. After isolation and culturing of NP cells, cells were transduced with scAAV6-shRNAs targeting MMP3 or MMP13; and analysed by fluorescence microscopy, FACS, MTT assay, RT-qPCR, ELISA and western blotting. scAAV6-shRNRs have no impact on cell viability and proliferation, despite high transduction efficiencies (98.6%) and transduction units (1383 TU/Cell). Combined knockdown of MMP3 (92.8%) and MMP13 (90.9%) resulted in highest enhancement of collagen II (143.2%), whereby treatment effects were significant over 56 days (p < 0.001). Conclusively, scAAV6-RNAi-mediated inhibitions of MMP3 and MMP13 help to progress less immunogenic and enduring biological treatments in DDD.

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.

IL-32 aggravates metabolic disturbance in human nucleus pulposus cells by activating FAT4-mediated Hippo/YAP signaling.

Extracellular matrix (ECM) metabolism disorders in the inflammatory microenvironment play a key role in the pathogenesis of intervertebral disc degeneration (IDD). Interleukin-32 (IL-32) has been reported to be involved in the progression of various inflammatory diseases; however, it remains unclear whether it participates in the matrix metabolism of nucleus pulposus (NP) cells. Therefore, this study aimed to investigate the mechanism of IL-32 on regulating the ECM metabolism in the inflammatory microenvironment. RNA-seq was used to identify aberrantly expressed genes in NP cells in the inflammatory microenvironment. Western blotting, real-time quantitative PCR, immunohistochemistry and immunofluorescence analysis were performed to measure the expression of IL-32 and metabolic markers in human NP tissues or NP cells treated with or without tumor necrosis factor-α (TNF-α). In vivo, an adeno-associated virus overexpressing IL-32 was injected into the caudal intervertebral discs of rats to assess its effect on IDD. Proteins interacting with IL-32 were identified via immunoprecipitation and mass spectrometry. Lentivirus overexpressing IL-32 or knocking down Fat atypical cadherin 4 (FAT4), yes-associated protein (YAP) inhibitor-Verteporfin (VP) were used to treat human NP cells, to explore the pathogenesis of IL-32. Hippo/YAP signaling activity was verified in human NP tissues. IL-32 expression was significantly upregulated in degenerative NP tissues, as indicated in the clinical samples. Furthermore, IL-32 was remarkably overexpressed in TNF-α-induced degenerative NP cells. IL-32 overexpression induced IDD progression in the rat model. Mechanistically, the elevation of IL-32 in the inflammatory microenvironment enhanced its interactions with FAT4 and mammalian sterile 20-like kinase1/2 (MST1/2) proteins, prompting MST1/2 phosphorylation, and activating the Hippo/YAP signaling pathway, causing matrix metabolism disorder in NP cells. Our results suggest that IL-32 mediates matrix metabolism disorders in NP cells in the inflammatory micro-environment via the FAT4/MST/YAP axis, providing a theoretical basis for the precise treatment of IDD.

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.

A structured biomimetic nanoparticle as inflammatory factor sponge and autophagy-regulatory agent against intervertebral disc degeneration and discogenic pain.

Lower back pain (LBP) is a common condition closely associated with intervertebral disc degeneration (IDD), causing a significant socioeconomic burden. Inflammatory activation in degenerated discs involves pro-inflammatory cytokines, dysregulated regulatory cytokines, and increased levels of nerve growth factor (NGF), leading to further intervertebral disc destruction and pain sensitization. Macrophage polarization is closely related to autophagy. Based on these pathological features, a structured biomimetic nanoparticle coated with TrkA-overexpressing macrophage membranes (TMNP@SR) with a rapamycin-loaded mesoporous silica core is developed. TMNP@SR acted like sponges to adsorbe inflammatory cytokines and NGF and delivers the autophagy regulator rapamycin (RAPA) into macrophages through homologous targeting effects of the outer engineered cell membrane. By regulating autophagy activation, TMNP@SR promoted the M1-to-M2 switch of macrophages to avoid continuous activation of inflammation within the degenerated disc, which prevented the apoptosis of nucleus pulposus cells. In addition, TMNP@SR relieved mechanical and thermal hyperalgesia, reduced calcitonin gene-related peptide (CGRP) and substance P (SP) expression in the dorsal root ganglion, and downregulated GFAP and c-FOS signaling in the spinal cord in the rat IDD model. In summary, TMNP@SR spontaneously inhibits the aggravation of disc inflammation to alleviate disc degeneration and reduce the ingress of sensory nerves, presenting a promising treatment strategy for LBP induced by disc degeneration.

Magnetically attracting hydrogel reshapes iron metabolism for tissue repair.

Ferroptosis, caused by disorders of iron metabolism, plays a critical role in various diseases, making the regulation of iron metabolism essential for tissue repair. In our analysis of degenerated intervertebral disc tissue, we observe a positive correlation between the concentration of extracellular iron ions (ex-iron) and the severity of ferroptosis in intervertebral disc degeneration (IVDD). Hence, inspired by magnets attracting metals, we combine polyether F127 diacrylate (FDA) with tannin (TA) to construct a magnetically attracting hydrogel (FDA-TA). This hydrogel demonstrates the capability to adsorb ex-iron and remodel the iron metabolism of cells. Furthermore, it exhibits good toughness and self-healing properties. Notably, it can activate the PI3K-AKT pathway to inhibit nuclear receptor coactivator 4-mediated ferritinophagy under ex-iron enrichment conditions. The curative effect and related mechanism are further confirmed in vivo. Consequently, on the basis of the pathological mechanism, a targeted hydrogel is designed to reshape iron metabolism, offering insights for tissue repair.

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.

Nucleotide polymorphism-based study utilizes human plasma liposomes to discover potential therapeutic targets for intervertebral disc disease.

Background: While intervertebral disc degeneration (IVDD) is crucial in numerous spinally related illnesses and is common among the elderly, the complete understanding of its pathogenic mechanisms is still an area of ongoing study. In recent years, it has revealed that liposomes are crucial in the initiation and progression of IVDD. However, their intrinsic mediators and related mechanisms remain unclear. With the development of genomics, an increasing amount of data points to the contribution of genetics in the etiology of disease. Accordingly, this study explored the causality between liposomes and IVDD by Mendelian randomization (MR) analysis and deeply investigated the intermediary roles of undetected metabolites. Methods: According to MR analysis, 179 liposomes and 1400 metabolites were evaluated for their causal association with IVDD. Single nucleotide polymorphisms (SNPs) are strongly associated with the concentrations of liposomes and metabolites. Consequently, they were employed as instrumental variables (IVs) to deduce if they constituted risk elements or protective elements for IVDD. Furthermore, mediation analysis was conducted to pinpoint possible metabolic mediators that link liposomes to IVDD. The inverse variance weighting (IVW) was the main analytical technique. Various confidence tests in the causality estimates were performed, including consistency, heterogeneity, pleiotropy, and sensitivity analyses. Inverse MR analysis was also utilized to estimate potential reverse causality. Results: MR analysis identified 13 liposomes and 79 metabolites markedly relevant to IVDD. Moreover, the mediation analysis was carried out by choosing the liposome, specifically the triacylglycerol (48:2) levels, which were found to be most notably associated with an increased risk of IVDD. In all, three metabolite-associated mediators were identified (3-methylcytidine levels, inosine 5'-monophosphate (IMP) to phosphate ratio, and adenosine 5'-diphosphate (ADP) to glycine ratio). Conclusion: The analysis's findings suggested possible causal connections between liposomes, metabolites, and IVDD, which could act as both forecast and prognosis clinical indicators, thereby aiding in the exploration of the pathogenesis behind IVDD.

The role of miR-155-5p in inflammation and mechanical loading during intervertebral disc degeneration.

BACKGROUND: Intervertebral disc (IVD) degeneration is a multifactorial pathological process resulting in the dysregulation of IVD cell activity. The catabolic shift observed in IVD cells during degeneration leads to increased inflammation, extracellular matrix (ECM) degradation, aberrant intracellular signaling and cell loss. Importantly, these pathological processes are known to be interconnected and to collectively contribute to the progression of the disease. MicroRNAs (miRNAs) are known as strong post-transcriptional regulators, targeting multiple genes simultaneously and regulating numerous intracellular pathways. Specifically, miR-155-5p has been of particular interest since it is known as a pro-inflammatory mediator and contributing factor to diseases like cancer and osteoarthritis. This study investigated the role of miR-155-5p in IVD degeneration with a specific focus on inflammation and mechanosensing. METHODS: Gain- and loss-of-function studies were performed through transfection of human Nucleus pulposus (NP) and Annulus fibrosus (AF) cells isolated from degenerated IVDs with miR-155-5p mimics, inhibitors or their corresponding non-targeting control. Transfected cells were then subjected to an inflammatory environment or mechanical loading. Conditioned media and cell lysates were collected for phosphorylation and cytokine secretion arrays as well as gene expression analysis. RESULTS: Increased expression of miR-155-5p in AF cells resulted in significant upregulation of interleukin (IL)-8 cytokine secretion during cyclic stretching and a similar trend in IL-6 secretion during inflammation. Furthermore, miR-155-5p mimics increased the expression of the brain-derived neurotrophic factor (BDNF) in AF cells undergoing cyclic stretching. In NP cells, miR-155-5p gain-of-function resulted in the activation of the mitogen-activated protein kinase (MAPK) signaling pathway through increased phosphorylation of p38 and p53. Lastly, miR-155-5p inhibition caused a significant increase in the anti-inflammatory cytokine IL-10 in AF cells and the tissue inhibitor of metalloproteinases (TIMP)-4 in NP cells respectively. CONCLUSION: Overall, these results show that miR-155-5p contributes to IVD degeneration by enhancing inflammation through pro-inflammatory cytokines and MAPK signaling, as well as by promoting the catabolic shift of AF cells during mechanical loading. The inhibition of miR-155-5p may constitute a potential therapeutic approach for IVD degeneration and low back pain.

A2AR regulate inflammation through PKA/NF-κB signaling pathways in intervertebral disc degeneration.

BACKGROUND: Reduction of inflammatory damage and inhibition of nucleus pulposus (NP) apoptosis are considered to be the main effective therapy idea to reverse the intervertebral disc degeneration (IDD) and alleviate the chronic low back pain. The adenosine A2A receptor (A2AR), as a member of G protein-coupled receptor families, plays an important role in the anti-inflammation and relieving pain. So far, the impact of A2AR on IDD therapy is unclear. The aim of this study was to explore the role of Adenosine A2A receptor (A2AR) in the intervertebral disc degeneration (IDD) and clarify potential mechanism. MATERIALS AND METHODS: IL-1ß and acupuncture was used to establish IDD model rats. A2AR agonist CGS-21680 and A2AR antagonist SCH442416 were used to investigate the therapeutical effects for IDD. Histological examination, western blotting analysis and RT-PCR were employed to evaluate the the association between A2AR and cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway. RESULTS: A2AR activity of the intervertebral disc tissues was up-regulated in feedback way, and cAMP, PKA and CREB expression were also increased. But in general, IL-1ß-induced IDD promoted the significant up-regulation the expression of inflammatory factors. The nucleus pulposus (NP) inflammation was exacerbated in result of MMP3 and Col-II decline through activating NF-κB signaling pathway. A2AR agonist CGS-21680 exhibited a disc protective effect through significantly increasing A2AR activity, then further activated cAMP/PKA signaling pathway with attenuating the release of TNF-α and IL-6 via down-regulating NF-κB. In contrast, SCH442416 inhibited A2AR activation, consistent with lower expression levels of cAMP and PKA, further leading to the acceleration of IDD. CONCLUSIONS: The activation of A2AR can prevent inflammatory responses and mitigates degradation of IDD thus suggest a potential novel therapeutic strategy of IDD.

Static magnetic field-modulated mesenchymal stem cell-derived mitochondria-containing microvesicles for enhanced intervertebral disc degeneration therapy.

Intervertebral disc degeneration (IVDD) is characterized by the senescence and declining vitality of nucleus pulposus cells (NPCs), often driven by mitochondrial dysfunction. This study elucidates that mesenchymal stem cells (MSCs) play a crucial role in attenuating NPC senescence by secreting mitochondria-containing microvesicles (mitoMVs). Moreover, it demonstrates that static magnetic fields (SMF) enhance the secretion of mitoMVs by MSCs. By distinguishing mitoMV generation from exosomes, this study shifts focus to understanding the molecular mechanisms of SMF intervention, emphasizing cargo transport and plasma membrane budding processes, with RNA sequencing indicating the potential involvement of the microtubule-based transport protein Kif5b. The study further confirms the interaction between Rab22a and Kif5b, revealing Rab22a's role in sorting mitoMVs into microvesicles (MVs) and potentially mediating subsequent plasma membrane budding. Subsequent construction of a gelatin methacrylate (GelMA) hydrogel delivery system further addresses the challenges of in vivo application and verifies the substantial potential of mitoMVs in delaying IVDD. This research not only sheds light on the molecular intricacies of SMF-enhanced mitoMV secretion but also provides innovative perspectives for future IVDD therapeutic strategies.

Multiomics analysis reveals the potential of LPCAT1-PC axis as a therapeutic target for human intervertebral disc degeneration.

Intervertebral disc degeneration (IDD) is a highly prevalent musculoskeletal disorder that is associated with considerable morbidity. However, there is currently no drug available that has a definitive therapeutic effect on IDD. In this study, we aimed to identify the molecular features and potential therapeutic targets of IDD through a comprehensive multiomics profiling approach. By integrating transcriptomics, proteomics, and ultrastructural analyses, we discovered dysfunctions in various organelles, including mitochondria, the endoplasmic reticulum, the Golgi apparatus, and lysosomes. Metabolomics analysis revealed a reduction in total phosphatidylcholine (PC) content in IDD. Through integration of multiple omics techniques with disease phenotypes, a pivotal pathway regulated by the lysophosphatidylcholine acyltransferase 1 (LPCAT1)-PC axis was identified. LPCAT1 exhibited low expression levels and exhibited a positive correlation with PC content in IDD. Suppression of LPCAT1 resulted in inhibition of PC synthesis in nucleus pulposus cells, leading to a notable increase in nucleus pulposus cell senescence and damage to cellular organelles. Consequently, PC exhibits potential as a therapeutic agent, as it facilitates the repair of the biomembrane system and alleviates senescence in nucleus pulposus cells via reversal of downregulation of the LPCAT1-PC axis.

The regulatory mechanism of cyclic GMP-AMP synthase on inflammatory senescence of nucleus pulposus cell.

BACKGROUND: Cellular senescence features irreversible growth arrest and secretion of multiple proinflammatory cytokines. Cyclic GMP-AMP synthase (cGAS) detects DNA damage and activates the DNA-sensing pathway, resulting in the upregulation of inflammatory genes and induction of cellular senescence. This study aimed to investigate the effect of cGAS in regulating senescence of nucleus pulposus (NP) cells under inflammatory microenvironment. METHODS: The expression of cGAS was evaluated by immunohistochemical staining in rat intervertebral disc (IVD) degeneration model induced by annulus stabbing. NP cells were harvested from rat lumbar IVD and cultured with 10ng/ml IL-1ß for 48 h to induce premature senescence. cGAS was silenced by cGAS specific siRNA in NP cells and cultured with IL-1ß. Cellular senescence was evaluated by senescence-associated beta-galactosidase (SA-ß-gal) staining and flow cytometry. The expression of senescence-associated secretory phenotype including IL-6, IL-8, and TNF-a was evaluated by ELISA and western blotting. RESULTS: cGAS was detected in rat NP cells in cytoplasm and the expression was significantly increased in degenerated IVD. Culturing in 10ng/ml IL-1ß for 48 h induced cellular senescence in NP cells with attenuation of G1-S phase transition. In senescent NP cells the expression of cGAS, p53, p16, NF-kB, IL-6, IL-8, TNF-α was significantly increased while aggrecan and collagen type II was reduced than in normal NP cells. In NP cells with silenced cGAS, the expression of p53, p16, NF-kB, IL-6, IL-8, and TNF-α was reduced in inflammatory culturing with IL-1ß. CONCLUSION: cGAS was increased by NP cells in degenerated IVD promoting cellular senescence and senescent inflammatory phenotypes. Targeting cGAS may alleviate IVD degeneration by reducing NP cell senescence.

Metformin prevents the onset and progression of intervertebral disc degeneration: New insights and potential mechanisms (Review).

Metformin has been the go­to medical treatment for addressing type 2 diabetes mellitus (T2DM) as a frontline oral antidiabetic. Obesity, cancer and bone deterioration are linked to T2DM, which is considered a metabolic illness. Numerous diseases associated with T2DM, such as tumours, cardiovascular disease and bone deterioration, may be treated with metformin. Intervertebral disc degeneration (IVDD) is distinguished by degeneration of the spinal disc, accompanied by the gradual depletion of proteoglycans and water in the nucleus pulposus (NP) of the IVD, resulting in lower back pain. The therapeutic effect of metformin on IVDD has also attracted much attention. By stimulating AMP­activated kinase, metformin could enhance autophagy and suppress cell senescence, apoptosis and inflammation, thus effectively delaying IVDD. The present review aimed to systematically explain the development of IVDD and mechanism of metformin in the treatment and prevention of IVDD to provide a reference for the clinical application of metformin as adjuvant therapy in the treatment of IVDD.

Needle-scalpel therapy inhibits the apoptosis of nucleus pulposus cells via the SDF-1/CXCR4 axis in a rat degenerative cervical intervertebral disc model.

As a common disease, cervical spondylosis (CS) results from the degeneration of the cervical intervertebral disc. However, there are still no effective clinical strategies for the treatment of this disease. Needle-scalpel (Ns), a therapy guided by traditional Chinese medicine theory, alleviates intervertebral disc degradation and is widely used in the clinic to treat CS. Stromal cell-derived factor-1 (SDF-1) and its receptor CXC receptor 4 (CXCR4) in nucleus pulposus cells play an important role in CS onset and development. This study aimed to explore whether Ns can relieve pain and regulate the SDF-1/CXCR4 axis in nucleus pulposus cells to inhibit apoptosis, thereby delaying cervical intervertebral disc degradation in a rat model of CS. It was found that the Ns-treated groups exhibited higher mechanical allodynia scores than the model group, and H&E staining, MRI, and scanning electron microscopy revealed that Ns therapy inhibited intervertebral disc degeneration. Additionally, Ns therapy significantly inhibited increases in the RNA and protein expression levels of SDF-1 and CXCR4. Furthermore, these treatments alleviated the apoptosis of nucleus pulposus cells, which manifested as a decline in the proportion of apoptotic nucleus pulposus cells and inhibition of the decrease in the levels of Bcl-2/Bax. These findings indicated that Ns mitigated CS-induced pain, inhibited the apoptosis of nucleus pulposus cells, and alleviated intervertebral disc degeneration in CS rats. These effects may be mediated by specifically regulating the SDF-1/CXCR4 signaling axis. Based on these findings, we conclude that Ns might serve as a promising therapy for the treatment of CS.

Isorhapontigenin delays senescence and matrix degradation of nucleus pulposus cells via PI3K/AKT/mTOR-mediated autophagy pathway in vitro and alleviates intervertebral disc degeneration in vivo.

Intervertebral disc degeneration (IVDD), a common degenerative disc disease, is a major etiological factor for back pain, affecting a significant number of middle-aged and elderly individuals worldwide. Thus, IVDD is a major socio-economic burden. The factors contributing to the complex IVDD etiology, which has not been elucidated, include inflammation, oxidative stress, and natural aging. In particular, inflammation and aging of nucleus pulposus cells are considered primary pathogenic factors. Isorhapontigenin (ISO) is a polyphenolic compound commonly found in traditional Chinese herbs and grapes. We have demonstrated that ISO exerts anti-inflammatory and anti-aging effects and mitigates extracellular matrix (ECM) degradation. In this study, in vitro experiments revealed that, ISO delays aging and ECM degradation by promoting PI3K/AKT/mTOR-mediated autophagy. Meanwhile, in vivo experiments affirmed that ISO delays the progression of IVDD.