Dynamics of CD44+ bovine nucleus pulposus cells with inflammation.

Intervertebral Disc (IVD) degeneration has been associated with a chronic inflammatory response, but knowledge on the contribution of distinct IVD cells, namely CD44, to the progression of IVD degeneration remains elusive. Here, bovine nucleus pulposus (NP) CD44 cells were sorted and compared by gene expression and proteomics with the negative counterpart. NP cells were then stimulated with IL-1b (10 ng/ml) and dynamics of CD44 gene and protein expression was analyzed upon pro-inflammatory treatment. The results emphasize that CD44 has a multidimensional functional role in IVD metabolism, ECM synthesis and production of neuropermissive factors. CD44 widespread expression in NP was partially associated with CD14 and CD45, resulting in the identification of distinct cell subsets. In conclusion, this study points out CD44 and CD44-based cell subsets as relevant targets in the modulation of the IVD pro-inflammatory/degenerative cascade.

[Effects of Oxidative Stress on Mitochondrial Functions and Intervertebral Disc Cells]. / 氧化应激对线粒体功能及椎间盘细胞的影响.

Intervertebral disc degeneration is widely recognized as one of the main causes of lower back pain. Intervertebral disc cells are the primary cellular components of the discs, responsible for synthesizing and secreting collagen and proteoglycans to maintain the structural and functional stability of the discs. Additionally, intervertebral disc cells are involved in maintaining the nutritional and metabolic balance, as well as exerting antioxidant and anti-inflammatory effects within the intervertebral discs. Consequently, intervertebral disc cells play a crucial role in the process of disc degeneration. When these cells are exposed to oxidative stress, mitochondria can be damaged, which may disrupt normal cellular function and accelerate degenerative changes. Mitochondria serve as the powerhouse of cells, being the primary energy-producing organelles that control a number of vital processes, such as cell death. On the other hand, mitochondrial dysfunction may be associated with various degenerative pathophysiological conditions. Moreover, mitochondria are the key site for oxidation-reduction reactions. Excessive oxidative stress and reactive oxygen species can negatively impact on mitochondrial function, potentially leading to mitochondrial damage and impaired functionality. These factors, in turn, triggers inflammatory responses, mitochondrial DNA damage, and cell apoptosis, playing a significant role in the pathological processes of intervertebral disc cell degeneration. This review is focused on exploring the impact of oxidative stress and reactive oxygen species on mitochondria and the crucial roles played by oxidative stress and reactive oxygen species in the pathological processes of intervertebral disc cells. In addition, we discussed current cutting-edge treatments and introduced the use of mitochondrial antioxidants and protectants as a potential method to slow down oxidative stress in the treatment of disc degeneration.

Single-cell RNA-seq analysis reveals the Wnt/Ca2+ signaling pathway with inflammation, apoptosis in nucleus pulposus degeneration.

BACKGROUND: Increasing studies have shown degeneration of nucleus pulposus cells (NPCs) as an critical part of the progression of intervertebral disc degeneration (IVDD). However, there are relatively few studies on single-cell transcriptome contrasts in human degenerated NPCs. Moreover, differences in Wnt/Ca2+ signaling in human degenerated nucleus pulposus cells have not been elucidated. The aim of this study is to investigate the differential expression of Wnt/Ca2+ signaling pathway between normal and degenerated nucleus pulposus cells in humans and try to investigate its mechanism. METHODS: We performed bioinformatics analysis using our previously published findings to construct single cell expression profiles of normal and degenerated nucleus pulposus. Then, in-depth differential analysis was used to characterize the expression of Wnt/Ca2+ signaling pathway between normal and degenerated nucleus pulposus cells in humans. RESULTS: The obtained cell data were clustered into five different chondrocytes clusters, which chondrocyte 4 and chondrocyte 5 mainly accounted for a high proportion in degenerated nucleus pulposus tissues, but rarely in normal nucleus pulposus tissues. Genes associated within the Wnt/Ca2+ signaling pathway, such as Wnt5B, FZD1, PLC (PLCB1), CaN (PPP3CA) and NAFATC1 are mainly present in chondrocyte 3, chondrocyte 4 and chondrocyte 5 from degenerated nucleus pulposus tissues. In addition, as a receptor that activates Wnt signaling pathway, LRP5 is mainly highly expressed in chondrocyte 5 of degenerated nucleus pulposus cells. Six genes, ANGPTL4, PTGES, IGFBP3, GDF15, TRIB3 and TNFRSF10B, which are associated with apoptosis and inflammatory responses, and are widespread in chondrocyte 4 and chondrocyte 5, may be closely related to degenerative of nucleus pulposus cells. CONCLUSIONS: Single-cell RNA sequencing revealed differential expression of Wnt/Ca2+ signaling in human normal and degenerated nucleus pulposus cells, and this differential expression may be closely related to the abundance of chondrocyte 4 and chondrocyte 5 in degenerated nucleus pulposus cells. In degenerated nucleus pulposus cells, LRP5 activate Wnt5B, which promotes nucleus pulposus cell apoptosis and inflammatory response by regulating the Wnt/Ca2+ signaling pathway, thereby promoting disc degeneration. ANGPTL4, IGFBP3, PTGES in chondrocyte 4 and TRIB3, GDF15, TNFRSF10B in chondrocyte 5 may play an important role in this process.

The mitochondrial UPR induced by ATF5 attenuates intervertebral disc degeneration via cooperating with mitophagy.

Intervertebral disc degeneration (IVDD) is an aging disease that results in a low quality of life and heavy socioeconomic burden. The mitochondrial unfolded protein response (UPRmt) take part in various aging-related diseases. Our research intents to explore the role and underlying mechanism of UPRmt in IVDD. Nucleus pulposus (NP) cells were exposed to IL-1ß and nicotinamide riboside (NR) served as UPRmt inducer to treat NP cells. Detection of ATP, NAD + and NADH were used to determine the function of mitochondria. MRI, Safranin O-fast green and Immunohistochemical examination were used to determine the degree of IVDD in vivo. In this study, we discovered that UPRmt was increased markedly in the NP cells of human IVDD tissues than in healthy controls. In vitro, UPRmt and mitophagy levels were promoted in NP cells treated with IL-1ß. Upregulation of UPRmt by NR and Atf5 overexpression inhibited NP cell apoptosis and further improved mitophagy. Silencing of Pink1 reversed the protective effects of NR and inhibited mitophagy induced by the UPRmt. In vivo, NR might attenuate the degree of IDD by activating the UPRmt in rats. In summary, the UPRmt was involved in IVDD by regulating Pink1-induced mitophagy. Mitophagy induced by the UPRmt might be a latent treated target for IVDD.

Sirtuins in intervertebral disc degeneration: current understanding.

BACKGROUND: Intervertebral disc degeneration (IVDD) is one of the etiologic factors of degenerative spinal diseases, which can lead to a variety of pathological spinal conditions such as disc herniation, spinal stenosis, and scoliosis. IVDD is a leading cause of lower back pain, the prevalence of which increases with age. Recently, Sirtuins/SIRTs and their related activators have received attention for their activity in the treatment of IVDD. In this paper, a comprehensive systematic review of the literature on the role of SIRTs and their activators on IVDD in recent years is presented. The molecular pathways involved in the regulation of IVDD by SIRTs are summarized, and the effects of SIRTs on senescence, inflammatory responses, oxidative stress, and mitochondrial dysfunction in myeloid cells are discussed with a view to suggesting possible solutions for the current treatment of IVDD. PURPOSE: This paper focuses on the molecular mechanisms by which SIRTs and their activators act on IVDD. METHODS: A literature search was conducted in Pubmed and Web of Science databases over a 13-year period from 2011 to 2024 for the terms "SIRT", "Sirtuin", "IVDD", "IDD", "IVD", "NP", "Intervertebral disc degeneration", "Intervertebral disc" and "Nucleus pulposus". RESULTS: According to the results, SIRTs and a large number of activators showed positive effects against IVDD.SIRTs modulate autophagy, myeloid apoptosis, oxidative stress and extracellular matrix degradation. In addition, they attenuate inflammatory factor-induced disc damage and maintain homeostasis during disc degeneration. Several clinical studies have reported the protective effects of some SIRTs activators (e.g., resveratrol, melatonin, honokiol, and 1,4-dihydropyridine) against IVDD. CONCLUSION: The fact that SIRTs and their activators play a hundred different roles in IVDD helps to better understand their potential to develop further treatments for IVDD. NOVELTY: This review summarizes current information on the mechanisms of action of SIRTs in IVDD and the challenges and limitations of translating their basic research into therapy.

Laquinimod attenuates oxidative stress-induced mitochondrial injury and alleviates intervertebral disc degeneration by inhibiting the NF-κB signaling pathway.

BACKGROUND: Low back pain (LBP) caused by intervertebral disc degeneration (IVDD) is a significant global health concern. It is necessary to investigate the underlying pathological mechanisms leading to IVDD and develop precise treatment strategies for this condition. Considering the well-established anti-inflammatory properties and ability to reduce oxidative stress in various diseases, for the first time we aim to explore the potential of Laquinimod in alleviating IVDD. METHODS: We used hydrogen peroxide (H2O2) to simulate the oxidative stress microenvironment in IVDD, and Laquinimod for intervention purposes. Western blot analysis, quantitative real-time polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence assay were used to measure the expression levels of inflammatory cytokines, catabolic enzymes, and markers of extracellular matrix (ECM) synthesis in nucleus pulposus (NP) cells. In addition, dichlorofluorescin-diacetate (DCFH-DA) and JC-1 fluorescent probes, flow cytometry analysis, and qRT-PCR were used to measure mitochondrial function and apoptosis in NP cells under conditions of oxidative stress. An acupuncture-induced rat model of IVDD was established to further evaluate the efficacy of Laquinimod in alleviating IVDD in vivo. RESULTS: Our findings showed that Laquinimod significantly reduced the oxidative stress-induced inflammatory response in NP cells, downregulated the expression of catabolic enzymes, and markedly enhanced ECM degradation by inhibiting the NF-κB signaling pathway. The administration of Laquinimod concurrently improved the mitochondrial functional state and reduced apoptosis in NP cells. Additionally, in vivo experiments in rats showed that Laquinimod significantly alleviated acupuncture-induced IVDD. CONCLUSIONS: Collectively, the findings of this study provide new insights into the therapeutic potential of Laquinimod as a treatment for oxidative stress-induced IVDD.

Omilancor mitigates the senescence of nucleus pulposus cells induced by DDP through targeting MAP2K6.

PURPOSE: This study explores the potential of Omilancor in treating Intervertebral Disc Degeneration (IDD) through MAP2K6 targeting. METHODS: We analyzed mRNA microarray datasets to pinpoint MAP2K6 as a key regulator implicated in IDD progression. Follow-up studies demonstrated that cisplatin (DDP) could prompt cellular senescence in vitro by upregulating MAP2K6 expression. Through molecular docking and other analyses, we identified Omilancor as a compound capable of binding to MAP2K6. This interaction effectively impeded the cellular senescence induced by DDP. RESULTS: We further showed that administration of Omilancor could significantly alleviate the degeneration of IVDs in annulus fibrosus puncture-induced rat model. CONCLUSIONS: Omilancor shows promise as a treatment for IDD by targeting MAP2K6-mediated cellular senescence.

Involvement of DJ-1 in the pathogenesis of intervertebral disc degeneration via hexokinase 2-mediated mitophagy.

Intervertebral disc degeneration (IDD) is an important pathological basis for degenerative spinal diseases and is involved in mitophagy dysfunction. However, the molecular mechanisms underlying mitophagy regulation in IDD remain unclear. This study aimed to clarify the role of DJ-1 in regulating mitophagy during IDD pathogenesis. Here, we showed that the mitochondrial localization of DJ-1 in nucleus pulposus cells (NPCs) first increased and then decreased in response to oxidative stress. Subsequently, loss- and gain-of-function experiments revealed that overexpression of DJ-1 in NPCs inhibited oxidative stress-induced mitochondrial dysfunction and mitochondria-dependent apoptosis, whereas knockdown of DJ-1 had the opposite effect. Mechanistically, mitochondrial translocation of DJ-1 promoted the recruitment of hexokinase 2 (HK2) to damaged mitochondria by activating Akt and subsequently Parkin-dependent mitophagy to inhibit oxidative stress-induced apoptosis in NPCs. However, silencing Parkin, reducing mitochondrial recruitment of HK2, or inhibiting Akt activation suppressed DJ-1-mediated mitophagy. Furthermore, overexpression of DJ-1 ameliorated IDD in rats through HK2-mediated mitophagy. Taken together, these findings indicate that DJ-1 promotes HK2-mediated mitophagy under oxidative stress conditions to inhibit mitochondria-dependent apoptosis in NPCs and could be a therapeutic target for IDD.

Regulatory mechanism of FCGR2A in macrophage polarization and its effects on intervertebral disc degeneration.

Intervertebral disc degeneration (IDD) poses a significant health burden, necessitating a deeper understanding of its molecular underpinnings. Transcriptomic analysis reveals 485 differentially expressed genes (DEGs) associated with IDD, underscoring the importance of immune regulation. Weighted gene co-expression network analysis (WGCNA) identifies a yellow module strongly correlated with IDD, intersecting with 197 DEGs. Protein-protein interaction (PPI) analysis identifies ITGAX, MMP9 and FCGR2A as hub genes, predominantly expressed in macrophages. Functional validation through in vitro and in vivo experiments demonstrates the pivotal role of FCGR2A in macrophage polarization and IDD progression. Mechanistically, FCGR2A knockdown suppresses M1 macrophage polarization and NF-κB phosphorylation while enhancing M2 polarization and STAT3 activation, leading to ameliorated IDD in animal models. This study sheds light on the regulatory function of FCGR2A in macrophage polarization, offering novel insights for IDD intervention strategies. KEY POINTS: This study unveils the role of FCGR2A in intervertebral disc (IVD) degeneration (IDD). FCGR2A knockdown mitigates IDD in cellular and animal models. Single-cell RNA-sequencing uncovers diverse macrophage subpopulations in degenerated IVDs. This study reveals the molecular mechanism of FCGR2A in regulating macrophage polarization. This study confirms the role of the NF-κB/STAT3 pathway in regulating macrophage polarization in IDD.

Inhibition of MAGL attenuates Intervertebral Disc Degeneration by Delaying nucleus pulposus senescence through STING.

Intervertebral disc degeneration (IVDD) stands as the primary cause of low back pain (LBP). A significant contributor to IVDD is nucleus pulposus cell (NPC) senescence. However, the precise mechanisms underlying NPC senescence remain unclear. Monoacylglycerol lipase (MAGL) serves as the primary enzyme responsible for the hydrolysis of 2-arachidonoylglycerol (2-AG), breaking down monoglycerides into glycerol and fatty acids. It plays a crucial role in various pathological processes, including pain, inflammation, and oxidative stress. In this study, we utilized a lipopolysaccharide (LPS)-induced NPC senescence model and a rat acupuncture-induced IVDD model to investigate the role of MAGL in IVDD both in vitro and in vivo. Initially, our results showed that MAGL expression was increased 2.41-fold and 1.52-fold within NP tissues from IVDD patients and rats induced with acupuncture, respectively. This increase in MAGL expression was accompanied by elevated expression of p16INK4α. Following this, it was noted that the suppression of MAGL resulted in a notable decrease in the quantity of SA-ß-gal-positive cells and hindered the manifestation of p16INK4α and the inflammatory factor IL-1ß in NPCs. MAGL inhibition promotes type II collagen (Col-2) expression and inhibits matrix metalloproteinase 13 (MMP13), thereby restoring the balance of extracellular matrix (ECM) metabolism both in vitro and in vivo. A significant role for STING has also been demonstrated in the regulation of NPC senescence by MAGL. The expression of the STING protein was reduced by 57% upon the inhibition of MAGL. STING activation can replicate the effects of MAGL and substantially increase LPS-induced inflammation while accelerating the senescence of NPCs. These results strongly indicate that the inhibition of MAGL can significantly suppress nucleus pulposus senescence via its interaction with STING, consequently restoring the balance of ECM metabolism. This insight provides new perspectives for potential treatments for IVDD.

Glutamine suppresses senescence and promotes autophagy through glycolysis inhibition-mediated AMPKα lactylation in intervertebral disc degeneration.

Regulating metabolic disorders has become a promising focus in treating intervertebral disc degeneration (IDD). A few drugs regulating metabolism, such as atorvastatin, metformin, and melatonin, show positive effects in treating IDD. Glutamine participates in multiple metabolic processes, including glutaminolysis and glycolysis; however, its impact on IDD is unclear. The current study reveals that glutamine levels are decreased in severely degenerated human nucleus pulposus (NP) tissues and aging Sprague-Dawley (SD) rat nucleus pulposus tissues, while lactate accumulation and lactylation are increased. Supplementary glutamine suppresses glycolysis and reduces lactate production, which downregulates adenosine-5'-monophosphate-activated protein kinase α (AMPKα) lactylation and upregulates AMPKα phosphorylation. Moreover, glutamine treatment reduces NP cell senescence and enhances autophagy and matrix synthesis via inhibition of glycolysis and AMPK lactylation, and glycolysis inhibition suppresses lactylation. Our results indicate that glutamine could prevent IDD by glycolysis inhibition-decreased AMPKα lactylation, which promotes autophagy and suppresses NP cell senescence.

Disassembly of the TRIM56-ATR complex promotes cytoDNA/cGAS/STING axis-dependent intervertebral disc inflammatory degeneration.

As the leading cause of disability worldwide, low back pain (LBP) is recognized as a pivotal socioeconomic challenge to the aging population and is largely attributed to intervertebral disc degeneration (IVDD). Elastic nucleus pulposus (NP) tissue is essential for the maintenance of IVD structural and functional integrity. The accumulation of senescent NP cells with an inflammatory hypersecretory phenotype due to aging and other damaging factors is a distinctive hallmark of IVDD initiation and progression. In this study, we reveal a mechanism of IVDD progression in which aberrant genomic DNA damage promoted NP cell inflammatory senescence via activation of the cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) axis but not of absent in melanoma 2 (AIM2) inflammasome assembly. Ataxia-telangiectasia-mutated and Rad3-related protein (ATR) deficiency destroyed genomic integrity and led to cytosolic mislocalization of genomic DNA, which acted as a powerful driver of cGAS/STING axis-dependent inflammatory phenotype acquisition during NP cell senescence. Mechanistically, disassembly of the ATR-tripartite motif-containing 56 (ATR-TRIM56) complex with the enzymatic liberation of ubiquitin-specific peptidase 5 (USP5) and TRIM25 drove changes in ATR ubiquitination, with ATR switching from K63- to K48-linked modification, c thereby promoting ubiquitin-proteasome-dependent dynamic instability of ATR protein during NP cell senescence progression. Importantly, an engineered extracellular vesicle-based strategy for delivering ATR-overexpressing plasmid cargo efficiently diminished DNA damage-associated NP cell senescence and substantially mitigated IVDD progression, indicating promising targets and effective approaches to ameliorate the chronic pain and disabling effects of IVDD.

The Sanbi Decoction alleviates intervertebral disc degeneration in rats through intestinal flora and serum metabolic homeostasis modulation.

BACKGROUND: Intervertebral disc degeneration (IVDD) is an essential cause of low back pain (LBP), the incidence of which has risen in recent years and is progressively younger, but treatment options are limited, placing a serious economic burden on society. Sanbi decoction (SBD) is an important classical formula for the treatment of IVDD, which can significantly improve patients' symptoms and is a promising alternative therapy. PURPOSE: The aim of this study is to investigate the safety and efficacy of SBD in the treatment of IVDD and to explore the underlying mechanisms by using an integrated analytical approach of microbiomics and serum metabolomics, as well as by using molecular biology. METHODS: A rat IVDD puncture model was established and treated by gavage with different concentrations of SBD, and clean faeces, serum, liver, kidney, and intervertebral disc (IVD) were collected after 4 weeks. We assessed the safety by liver and kidney weighing, functional tests and tissue staining, the expression of tumor necrosis factor-alpha (TNF-ɑ), interleukin 1ß (IL-1ß) and interleukin 6 (IL-6) inflammatory factors in serum was detected by ELISA kits, and X-ray test, magnetic resonance imaging (MRI) examination, immunohistochemistry (IHC), western blotting (WB), hematoxylin-eosin (HE) staining and safranin O-fast green (SO/FG) staining were used to assess the efficacy. Finally, we performed 16S rRNA sequencing analysis on the faeces of different groups and untargeted metabolomics on serum and analyzed the association between them. RESULTS: SBD can effectively reduce the inflammatory response, regulate the metabolic balance of extracellular matrix (ECM), improve symptoms, and restore IVD function. In addition, SBD can significantly improve the diversity of intestinal flora and maintain the balance. At the phylum level, SBD greatly increased the relative abundance of Patescibacteria and Actinobacteriota and decreased the relative abundance of Bacteroidota. At the genus level, SBD significantly increased the relative abundance of Clostridia_UCG-014, Enterorhabdus, and Adlercreutzia, and decreased the relative abundance of Ruminococcaceae_UCG-005 (p < 0.05). Untargeted metabolomics indicated that SBD significantly improved serum metabolites and altered serum expression of 4alpha-phorbol 12,13-didecanoate (4alphaPDD), euscaphic acid (EA), alpha-muricholic acid (α-MCA), 5-hydroxyindoleacetic acid (5-HIAA), and kynurenine (Kyn) (p < 0.05), and the metabolic pathways were mainly lipid metabolism and amino acid metabolism. CONCLUSIONS: This study demonstrated that SBD can extensively regulate intestinal flora and serum metabolic homeostasis to reduce inflammatory response, inhibit the degradation of ECM, restore IVD height and water content to achieve apparent therapeutic effect for IVDD.

Mitochondrial-Targeted Metal-Phenolic Nanoparticles to Attenuate Intervertebral Disc Degeneration: Alleviating Oxidative Stress and Mitochondrial Dysfunction.

As intervertebral disc degeneration (IVDD) proceeds, the dysfunctional mitochondria disrupt the viability of nucleus pulposus cells, initiating the degradation of the extracellular matrix. To date, there is a lack of effective therapies targeting the mitochondria of nucleus pulposus cells. Here, we synthesized polygallic acid-manganese (PGA-Mn) nanoparticles via self-assembly polymerization of gallic acid in an aqueous medium and introduced a mitochondrial targeting peptide (TP04) onto the nanoparticles using a Schiff base linkage, resulting in PGA-Mn-TP04 nanoparticles. With a size smaller than 50 nm, PGA-Mn-TP04 possesses pH-buffering capacity, avoiding lysosomal confinement and selectively accumulating within mitochondria through electrostatic interactions. The rapid electron exchange between manganese ions and gallic acid enhances the redox capability of PGA-Mn-TP04, effectively reducing mitochondrial damage caused by mitochondrial reactive oxygen species. Moreover, PGA-Mn-TP04 restores mitochondrial function by facilitating the fusion of mitochondria and minimizing their fission, thereby sustaining the vitality of nucleus pulposus cells. In the rat IVDD model, PGA-Mn-TP04 maintained intervertebral disc height and nucleus pulposus tissue hydration. It offers a nonoperative treatment approach for IVDD and other skeletal muscle diseases resulting from mitochondrial dysfunction, presenting an alternative to traditional surgical interventions.

Comprehensive analysis of gene expression profiles of annulus fibrosus subtypes and hub genes in intervertebral disc degeneration.

Intervertebral disc degeneration (IVDD) has been considered a major cause of low back pain. Therefore, further molecular subtypes of IVDD and identification of potential critical genes are urgently needed. First, consensus clustering was used to classify patients with IVDD into two subtypes and key module genes for subtyping were identified using weighted gene co-expression network analysis (WGCNA). Then, key module genes for the disease were identified by WGCNA. Subsequently, SVM and GLM were used to identify hub genes. Based on the above genes, a nomogram was constructed to predict the subtypes of IVDD. Finally, we find that ROM1 is lowered in IVDD and is linked to various cancer prognoses. The present work offers innovative diagnostic and therapeutic biomarkers for molecular subtypes of IVDD.

Ginsenoside Rg1 relieves rat intervertebral disc degeneration and inhibits IL-1ß-induced nucleus pulposus cell apoptosis and inflammation via NF-κB signaling pathway.

The study aimed to investigate the effect of ginsenoside Rg1 on intervertebral disc degeneration (IVDD) in rats and IL-1ß-induced nucleus pulposus (NP) cells, and explore its underlying mechanism. Forty IVDD rat models were divided into the IVDD group, low-dose (L-Rg1) group (intraperitoneal injection of 20 mg/kg/d ginsenoside Rg1), medium-dose (M-Rg1) group (intraperitoneal injection of 40 mg/kg/d ginsenoside Rg1), and high-dose (H-Rg1) group (intraperitoneal injection of 80 mg/kg/d ginsenoside Rg1). The pathological change was observed by HE and safranin O-fast green staining. The expression of IL-1ß, IL-6, TNF-α, MMP3, aggrecan, and collagen II was detected. The expression of NF-κB p65 in IVD tissues was detected. Rat NP cells were induced by IL-1ß to simulate IVDD environment and divided into the control group, IL-1ß group, and 20, 50, and 100 µmol/L Rg1 groups. The cell proliferation activity, the apoptosis, and the expression of IL-6, TNF-α, MMP3, aggrecan, collagen II, and NF-κB pathway-related protein were detected. In IVDD rats, ginsenoside Rg1 improved the pathology of IVD tissues; suppressed the expression of IL-1ß, IL-6, TNF-α, aggrecan, and collagen II; and inhibited the expression of p-p65/p65 and nuclear translocation of p65, to alleviate the IVDD progression. In the IL-1ß-induced NP cells, ginsenoside Rg1 also improved the cell proliferation and inhibited the apoptosis and the expression of IL-6, TNF-α, aggrecan, collagen II, p-p65/p65, and IκK in a dose-dependent manner. Ginsenoside Rg1 alleviated IVDD in rats and inhibited apoptosis, inflammatory response, and ECM degradation in IL-1ß-induced NP cells. And Rg1 may exert its effect via inhibiting the activation of NF-κB signaling pathway.

CircEYA3 aggravates intervertebral disc degeneration through the miR-196a-5p/EBF1 axis and NF-κB signaling.

Intervertebral disc degeneration (IDD) is a well-established cause of disability, and extensive evidence has identified the important role played by regulatory noncoding RNAs, specifically circular RNAs (circRNAs) and microRNAs (miRNAs), in the progression of IDD. To elucidate the molecular mechanism underlying IDD, we established a circRNA/miRNA/mRNA network in IDD through standardized analyses of all expression matrices. Our studies confirmed the differential expression of the transcription factors early B-cell factor 1 (EBF1), circEYA3, and miR-196a-5p in the nucleus pulposus (NP) tissues of controls and IDD patients. Cell proliferation, apoptosis, and extracellular mechanisms of degradation in NP cells (NPC) are mediated by circEYA3. MiR-196a-5p is a direct target of circEYA3 and EBF1. Functional analysis showed that miR-196a-5p reversed the effects of circEYA3 and EBF1 on ECM degradation, apoptosis, and proliferation in NPCs. EBF1 regulates the nuclear factor kappa beta (NF-кB) signalling pathway by activating the IKKß promoter region. This study demonstrates that circEYA3 plays an important role in exacerbating the progression of IDD by modulating the NF-κB signalling pathway through regulation of the miR196a-5p/EBF1 axis. Consequently, a novel molecular mechanism underlying IDD development was elucidated, thereby identifying a potential therapeutic target for future exploration.

Platelet-rich plasma-derived extracellular vesicles inhibit NF-κB/NLRP3 pathway-mediated pyroptosis in intervertebral disc degeneration via the MALAT1/microRNA-217/SIRT1 axis.

BACKGROUND: Intervertebral disc degeneration (IDD) is a main contributor to lower back pain, and compression stress-induced apoptosis of nucleus pulposus (NP) cells and extracellular matrix (ECM) degradation has been implicated in the IDD progression. The functions of platelet-rich plasma (PRP)-derived extracellular vesicles (PRP-EVs) in regulating these biological processes remain unclear in IDD. Here, we aimed to investigate the key role of long noncoding RNA (lncRNA) MALAT1 incorporated in PRP-EVs in IDD. METHODS: Tert-butyl hydroperoxide (TBHP)-induced damage in NP cells was treated with PRP-EVs extracted from healthy volunteers, followed by MTT, EdU, TUNEL, and Western blot assays. IDD mice were also treated with PRP-EVs. Histomorphological and pathological changes were evaluated. The pyroptosis of cells and the degradation of ECM were detected by ELISA and immunohistochemistry. We screened the differentially expressed lncRNAs in NP cells after PRP-EVs treatment by microarray analysis. The downstream targets of MALAT1 in NP cells were predicted and validated by rescue experiments. FINDINGS: TBHP induction reduced cell proliferation and exacerbated pyroptosis and ECM degradation, and PRP-EVs inhibited TBHP-induced cell damage. PRP-EVs-treated mice with IDD had reduced Thompson scores, increased NP tissue content, and restored ECM. PRP-EVs upregulated MALAT1 expression in vivo and in vitro, whereas MALAT1 downregulation exacerbated NP cell pyroptosis and ECM degradation. MALAT1 upregulated SIRT1 expression by downregulating microRNA (miR)-217 in NP cells. SIRT1 blocked the NF-κB/NLRP3 pathway-mediated pyroptosis, thereby alleviating IDD. INTERPRETATION: PRP-EVs deliver MALAT1 to regulate miR-217/SIRT1, thereby controlling NP cell pyroptosis in IDD.

Network Pharmacology with Metabolomics Study to Reveal the Mechanisms of Bushen Huoxue Formula in Intervertebral Disc Degeneration Treatment.

Background: Intervertebral disc degeneration (IVDD) is a pathophysiological process that leads to severe back pain or neurological deficits. The Bushen Huoxue Formula (BSHXF) is a traditional herbal remedy widely used to treat diseases related to IVDD. However, its pharmacological mechanism needs further exploration. Objective: This study aimed to elucidate the mechanisms through which BSHXF treats IVDD-related diseases by integrating metabolomics with network pharmacology. Methods: Network pharmacology was utilized to identify potential targets of BSHXF against IVDD. Additionally, an animal model of needle puncture-induced disc degeneration was established to assess the effect of BSHXF. Mice were randomly assigned to the sham group, model group, and BSHXF group. Various techniques, including PCR, CCK-8 assay, MRI, histological examinations, and immunohistochemical analyses, were employed to evaluate degenerative and oxidative stress conditions in mouse disc tissue and cultured nucleus pulposus (NP) cells. UHPLC-HRMS/MS was used to differential distinct metabolites in the disc tissue from different groups, and MetaboAnalyst 5.0 was employed to enrich the metabolic pathways. Results: Through network pharmacology, 15 core proteins were identified through protein-protein interaction (PPI) network construction. Functional enrichment analysis highlighted the critical role of BSHXF in addressing IVDD by influencing the response to oxidative stress. Furthermore, experimental evidence demonstrated that BSHXF significantly improved the pathological progression of IVDD and increased oxidative stress markers SOD-1 and GPX1, both in the disc degeneration model and cultured NP cells. Metabolomics identified differential metabolites among the three groups, revealing 15 metabolic pathways between the sham and model groups, and 13 metabolic pathways enriched between the model and BSHXF groups. Conclusion: This study, integrating network pharmacology and metabolomics, suggests that BSHXF can alleviate IVDD progression by modulating oxidative stress. Key metabolic pathways associated with BSHXF-mediated reduction of oxidative stress include the citrate cycle, cysteine and methionine metabolism, alanine, aspartate and glutamate metabolism, glycine, serine and threonine metabolism, D-glutamine and D-glutamate metabolism, glutathione metabolism, and tryptophan metabolism. While this research demonstrates the therapeutic potential of BSHXF in reducing oxidative stress levels in IVDD, further research is needed to thoroughly understand its underlying mechanisms.

Comparative Analysis of Autophagy and Apoptosis in Disc Degeneration: Understanding the Dynamics of Temporary-Compression-Induced Early Autophagy and Sustained-Compression-Triggered Apoptosis.

The purpose of this study was to investigate the initiation of autophagy activation and apoptosis in nucleus pulposus cells under temporary compression (TC) and sustained compression (SC) to identify ideal research approaches in intervertebral disc degeneration. Various techniques were used: radiography (X-ray), magnetic resonance imaging (MRI), transmission electron microscope (TEM), H&E staining, Masson's trichrome staining, immunohistochemistry (IHC) (LC3, beclin-1, and cleaved caspase-3), and real-time polymerase chain reaction (RT-qPCR) for autophagy-related (beclin-1, LC3, and P62) and apoptosis-related (caspase-3 and PARP) gene expression analysis. X-ray and MRI revealed varying degrees of disc degeneration, ranging from moderate to severe in both groups. The severity was directly linked to compression duration, with SC resulting in notably severe central NP cell degeneration. Surprisingly, TC also caused similar, though less severe, degeneration. Elevated expression of LC3 and beclin-1 was identified after 6 weeks, but it notably declined after 12 weeks. Central NP cells in both groups exhibited increased expression of cleaved caspase-3 that was positively correlated with the duration of SC. TC showed fewer apoptotic markers compared to SC. LC3, beclin-1, and P62 mRNA expression peaked after 6 weeks and declined after 12 weeks in both groups. Cleaved caspase-3 and PARP expression peaked in SC, positively correlating with longer compression duration, while TC showed lower levels of apoptosis gene expression. Furthermore, TEM results revealed different events of the autophagic degradation process after 2 weeks of compression. TCmay be ideal for studying early triggered autophagy-mediated degeneration, while SC may be ideal for studying late or slower-triggered apoptosis-mediated degeneration.