Andrographolide influences IDD cell autophagy and oxidative stress under mechanical pressure via miR-9/FoxO3/PINK1/Parkin molecular axis.

Intervertebral disc degeneration (IDD) is characterized by the decreased function and number of nucleus pulposus cells (NPCs) caused by excessive intervertebral disc (IVD) pressure. This research aims to provide novel insights into IDD prevention and treatment by clarifying the effect of andrographolide (ANDR) on IDD cell autophagy and oxidative stress under mechanical stress. Human primary NPCs were extracted from the nucleus pulposus tissue of non-IDD trauma patients. An IDD cell model was established by posing mechanical traction on NPCs. Through the construction of an IDD rat model, the influence of ANDR on IDD pathological changes was explored in vivo. The proliferation and autophagy of NPCs were decreased while the apoptosis rate and oxidative stress reaction were increased by mechanical traction. ANDR intervention obviously alleviated this situation. MiR-9 showed upregulated expression in IDD cell model, while FoxO3 and PINK1/Parkin were downregulated. Decreased proliferation and autophagy as well as enhanced apoptosis and oxidative stress response of NPCs were observed following miR-9 mimics and H89 intervention, while the opposite trend was observed after FoxO3 overexpression. FoxO3 is a direct target downstream miR-9. The in vivo experiments revealed that after ANDR intervention, the number of apoptotic cells in rat IVD tissue decreased and the autophagy increased. In conclusion, ANDR improves NPC proliferation, and autophagy, inhibits apoptosis and oxidative stress, and alleviates the pathological changes of IDD via the miR-9/FoxO3/PINK1/Parkin axis, which may be a new and effective treatment for IDD in the future.

S100A6 Regulates nucleus pulposus cell apoptosis via Wnt/ß-catenin signaling pathway: an in vitro and in vivo study.

BACKGROUND: Intervertebral disc degeneration (IDD) is a common musculoskeletal degenerative disease, which often leads to low back pain and even disability, resulting in loss of labor ability and decreased quality of life. Although many progresses have been made in the current research, the underlying mechanism of IDD remains unclear. The apoptosis of nucleus pulposus (NP) cells (NPCs) is an important pathological mechanism in intervertebral disc degeneration (IDD). This study evaluated the relationship between S100A6 and NPCs and its underlying mechanism. METHODS: Mass spectrometry, bioinformatics, and quantitative real-time polymerase chain reaction (qRT-PCR) analyses were used to screen and verify hub genes for IDD in human IVD specimens with different degeneration degrees. Western blotting, immunohistochemistry (IHC), and/or immunofluorescence (IF) were used to detect the expression level of S100A6 in human NP tissues and NPCs. The apoptotic phenotype of NPCs and Wnt/ß-catenin signaling pathway were evaluated using flow cytometry, western blotting, and IF. S100A6 was overexpressed or knocked down in NPCs to determine its impact on apoptosis and Wnt/ß-catenin signaling pathway activity. Moreover, we used the XAV-939 to inhibit and SKL2001 to activate the Wnt/ß-catenin signaling pathway. The therapeutic effect of S100A6 inhibition on IDD was also evaluated. RESULTS: S100A6 expression increased in IDD. In vitro, increased S100A6 expression promoted apoptosis in interleukin (IL)-1ß-induced NPCs. In contrast, the inhibition of S100A6 expression partially alleviated the progression of annulus fibrosus (AF) puncture-induced IDD in rats. Mechanistic studies revealed that S100A6 regulates NPC apoptosis via Wnt/ß-catenin signaling pathway. CONCLUSIONS: This study showed that S100A6 expression increased during IDD and promoted NPCs apoptosis by regulating the Wnt/ß-catenin signaling pathway, suggesting that S100A6 is a promising new therapeutic target for IDD.

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

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

Phillyrin reduces ROS production to alleviate the progression of intervertebral disc degeneration by inhibiting NF-κB pathway.

BACKGROUND: Intervertebral disc degeneration (IDD) is an increasingly important cause of low back pain (LBP) that results in substantial health and economic burdens. Inflammatory pathway activation and the production of reactive oxygen species (ROS) play vital roles in the progression of IDD. Several studies have suggested that phillyrin has a protective role and inhibits inflammation and the production of ROS. However, the role of phillyrin in IDD has not been confirmed. PURPOSE: The purpose of this study was to investigate the role of phillyrin in IDD and its mechanisms. STUDY DESIGN: To establish IDD models in vivo, ex-vivo, and in vitro to verify the function of phillyrin in IDD. METHOD: The effects of phillyrin on extracellular matrix (ECM) degeneration, inflammation, and oxidation in nucleus pulposus (NP) cells were assessed using immunoblotting and immunofluorescence analysis. Additionally, the impact of phillyrin administration on acupuncture-mediated intervertebral disc degeneration (IDD) in rats was evaluated using various techniques such as MRI, HE staining, S-O staining, and immunohistochemistry (IHC). RESULT: Pretreatment with phillyrin significantly inhibited the IL-1ß-mediated reduction in the degeneration of ECM and apoptosis by alleviating activation of the NF-κB inflammatory pathway and the generation of ROS. In addition, in vivo and ex-vivo experiments verified the protective effect of phillyrin against IDD. CONCLUSION: Phillyrin can attenuate the progression of IDD by reducing ROS production and activating inflammatory pathways.

Chondrocyte-targeted exosome-mediated delivery of Nrf2 alleviates cartilaginous endplate degeneration by modulating mitochondrial fission.

BACKGROUND: Cartilaginous endplate (CEP) degeneration, which is an important contributor to intervertebral disc degeneration (IVDD), is characterized by chondrocyte death. Accumulating evidence has revealed that dynamin-related protein 1 (Drp1)-mediated mitochondrial fission and dysfunction lead to apoptosis during CEP degeneration and IVDD. Exosomes are promising agents for the treatment of many diseases, including osteoporosis, osteosarcoma, osteoarthritis and IVDD. Despite their major success in drug delivery, the full potential of exosomes remains untapped. MATERIALS AND METHODS: In vitro and in vivo models of CEP degeneration were established by using lipopolysaccharide (LPS). We designed genetically engineered exosomes (CAP-Nrf2-Exos) expressing chondrocyte-affinity peptide (CAP) on the surface and carrying the antioxidant transcription factor nuclear factor E2-related factor 2 (Nrf2). The affinity between CAP-Nrf2-Exos and CEP was evaluated by in vitro internalization assays and in vivo imaging assays. qRT‒PCR, Western blotting and immunofluorescence assays were performed to examine the expression level of Nrf2 and the subcellular localization of Nrf2 and Drp1. Mitochondrial function was measured by the JC-1 probe and MitoSOX Red. Mitochondrial morphology was visualized by MitoTracker staining and transmission electron microscopy (TEM). After subendplate injection of the engineered exosomes, the degree of CEP degeneration and IVDD was validated radiologically and histologically. RESULTS: We found that the cargo delivery efficiency of exosomes after cargo packaging was increased by surface modification. CAP-Nrf2-Exos facilitated chondrocyte-targeted delivery of Nrf2 and activated the endogenous antioxidant defence system in CEP cells. The engineered exosomes inhibited Drp1 S616 phosphorylation and mitochondrial translocation, thereby preventing mitochondrial fragmentation and dysfunction. LPS-induced CEP cell apoptosis was alleviated by CAP-Nrf2-Exo treatment. In a rat model of CEP degeneration, the engineered exosomes successfully attenuated CEP degeneration and IVDD and exhibited better repair capacity than natural exosomes. CONCLUSION: Collectively, our findings showed that exosome-mediated chondrocyte-targeted delivery of Nrf2 was an effective strategy for treating CEP degeneration.

Electroacupuncture improves apoptosis of nucleus pulposus cells via the IL-22/JAK2-STAT3 signaling pathway in a rat model of cervical intervertebral disk degeneration.

BACKGROUND: Cervical spondylosis (CS) is a prevalent disorder that can have a major negative impact on quality of life. Traditional conservative treatment has limited efficacy, and electroacupuncture (EA) is a novel treatment option. We investigated the application and molecular mechanism of EA treatment in a rat model of cervical intervertebral disk degeneration (CIDD). METHODS: The CIDD rat model was established, following which rats in the electroacupuncture (EA) group received EA. For overexpression of IL-22 or inhibition of JAK2-STAT3 signaling, the rats were injected intraperitoneally with recombinant IL-22 protein (p-IL-22) or the JAK2-STAT3 (Janus kinase 2-signal transducer and activator of transcription protein 3) inhibitor AG490 after model establishment. Rat nucleus pulposus (NP) cells were isolated and cultured. Cell counting kit-8 and flow cytometry were used to analyze the viability and apoptosis of the NP cells. Expression of IL-22, JAK2 and STAT3 was determined using RT-qPCR. Expression of IL-22/JAK2-STAT3 pathway and apoptosis related proteins was detected by Western blotting (WB). RESULTS: EA protected the NP tissues of CIDD rats by regulating the IL-22/JAK2-STAT3 pathway. Overexpression of IL-22 significantly promoted the expression of tumor necrosis factor (TNF)-α, IL-6, IL-1ß, matrix metalloproteinase (MMP)3 and MMP13 compared with the EA group. WB demonstrated that the expression of IL-22, p-JAK2, p-STAT3, caspase-3 and Bax in NP cells of the EA group was significantly reduced and Bcl-2 elevated compared with the model group. EA regulated cytokines and MMP through activation of IL-22/JAK2-STAT3 signaling in CIDD rat NP cells. CONCLUSION: We demonstrated that EA affected apoptosis by regulating the IL-22/JAK2-STAT3 pathway in NP cells and reducing inflammatory factors in the CIDD rat model. The results extend our knowledge of the mechanisms of action underlying the effects of EA as a potential treatment approach for CS in clinical practice.

Effective delivery of miR-150-5p with nucleus pulposus cell-specific nanoparticles attenuates intervertebral disc degeneration.

BACKGROUND: The use of gene therapy to deliver microRNAs (miRNAs) has gradually translated to preclinical application for the treatment of intervertebral disc degeneration (IDD). However, the effects of miRNAs are hindered by the short half-life time and the poor cellular uptake, owing to the lack of efficient delivery systems. Here, we investigated nucleus pulposus cell (NPC) specific aptamer-decorated polymeric nanoparticles that can load miR-150-5p for IDD treatment. METHODS: The role of miR-150-5p during disc development and degeneration was examined by miR-150-5p knockout (KO) mice. Histological analysis was undertaken in disc specimens. The functional mechanism of miR-150-5p in IDD development was investigated by qRT-PCR assay, Western blot, coimmunoprecipitation and immunofluorescence. NPC specific aptamer-decorated nanoparticles was designed, and its penetration, stability and safety were evaluated. IDD progression was assessed by radiological analysis including X-ray and MRI, after the annulus fibrosus needle puncture surgery with miR-150-5p manipulation by intradiscal injection of nanoparticles. The investigations into the interaction between aptamer and receptor were conducted using mass spectrometry, molecular docking and molecular dynamics simulations. RESULTS: We investigated NPC-specific aptamer-decorated polymeric nanoparticles that can bind to miR-150-5p for IDD treatment. Furthermore, we detected that nanoparticle-loaded miR-150-5p inhibitors alleviated NPC senescence in vitro, and the effects of the nanoparticles were sustained for more than 3 months in vivo. The microenvironment of NPCs improves the endo/lysosomal escape of miRNAs, greatly inhibiting the secretion of senescence-associated factors and the subsequent degeneration of NPCs. Importantly, nanoparticles delivering miR-150-5p inhibitors attenuated needle puncture-induced IDD in mouse models by targeting FBXW11 and inhibiting TAK1 ubiquitination, resulting in the downregulation of NF-kB signaling pathway activity. CONCLUSIONS: NPC-targeting nanoparticles delivering miR-150-5p show favorable therapeutic efficacy and safety and may constitute a promising treatment for IDD.

Lumbar instability remodels cartilage endplate to induce intervertebral disc degeneration by recruiting osteoclasts via Hippo-CCL3 signaling.

Degenerated endplate appears with cheese-like morphology and sensory innervation, contributing to low back pain and subsequently inducing intervertebral disc degeneration in the aged population.1 However, the origin and development mechanism of the cheese-like morphology remain unclear. Here in this study, we report lumbar instability induced cartilage endplate remodeling is responsible for this pathological change. Transcriptome sequencing of the endplate chondrocytes under abnormal stress revealed that the Hippo signaling was key for this process. Activation of Hippo signaling or knockout of the key gene Yap1 in the cartilage endplate severed the cheese-like morphological change and disc degeneration after lumbar spine instability (LSI) surgery, while blocking the Hippo signaling reversed this process. Meanwhile, transcriptome sequencing data also showed osteoclast differentiation related gene set expression was up regulated in the endplate chondrocytes under abnormal mechanical stress, which was activated after the Hippo signaling. Among the discovered osteoclast differentiation gene set, CCL3 was found to be largely released from the chondrocytes under abnormal stress, which functioned to recruit and promote osteoclasts formation for cartilage endplate remodeling. Over-expression of Yap1 inhibited CCL3 transcription by blocking its promoter, which then reversed the endplate from remodeling to the cheese-like morphology. Finally, LSI-induced cartilage endplate remodeling was successfully rescued by local injection of an AAV5 wrapped Yap1 over-expression plasmid at the site. These findings suggest that the Hippo signaling induced osteoclast gene set activation in the cartilage endplate is a potential new target for the management of instability induced low back pain and lumbar degeneration.

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

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

Inhibiting DNA methyltransferase DNMT3B confers protection against ferroptosis in nucleus pulposus and ameliorates intervertebral disc degeneration via upregulating SLC40A1.

Epigenetic changes are important considerations for degenerative diseases. DNA methylation regulates crucial genes by epigenetic mechanism, impacting cell function and fate. DNA presents hypermethylation in degenerated nucleus pulposus (NP) tissue, but its role in intervertebral disc degeneration (IVDD) remains elusive. This study aimed to demonstrate that methyltransferase mediated hypermethylation was responsible for IVDD by integrative bioinformatics and experimental verification. Methyltransferase DNMT3B was highly expressed in severely degenerated NP tissue (involving human and rats) and in-vitro degenerated human NP cells (NPCs). Bioinformatics elucidated that hypermethylated genes were enriched in oxidative stress and ferroptosis, and the ferroptosis suppressor gene SLC40A1 was identified with lower expression and higher methylation in severely degenerated human NP tissue. Cell culture using human NPCs showed that DNMT3B induced ferroptosis and oxidative stress in NPCs by downregulating SLC40A1, promoting a degenerative cell phenotype. An in-vivo rat IVDD model showed that DNA methyltransferase inhibitor 5-AZA alleviated puncture-induced IVDD. Taken together, DNA methyltransferase DNMT3B aggravates ferroptosis and oxidative stress in NPCs via regulating SLC40A1. Epigenetic mechanism within DNA methylation is a promising therapeutic biomarker for IVDD.

MIF inhibition attenuates intervertebral disc degeneration by reducing nucleus pulposus cell apoptosis and inflammation.

Nucleus pulposus cells (NPCs) apoptosis and inflammation are the extremely critical factors of intervertebral disc degeneration (IVDD). Nevertheless, the underlying procedure remains mysterious. Macrophage migration inhibitory factor (MIF) is a cytokine that promotes inflammation and has been demonstrated to have a significant impact on apoptosis and inflammation. For this research, we employed a model of NPCs degeneration stimulated by lipopolysaccharides (LPS) and a rat acupuncture IVDD model to examine the role of MIF in vitro and in vivo, respectively. Initially, we verified that there was a significant rise of MIF expression in the NP tissues of individuals with IVDD, as well as in rat models of IVDD. Furthermore, this augmented expression of MIF was similarly evident in degenerated NPCs. Afterwards, it was discovered that ISO-1, a MIF inhibitor, effectively decreased the quantity of cells undergoing apoptosis and inhibited the release of inflammatory molecules (TNF-α, IL-1ß, IL-6). Furthermore, it has been shown that the PI3K/Akt pathway plays a vital part in the regulation of NPCs degeneration by MIF. Ultimately, we showcased that the IVDD process was impacted by the MIF inhibitor in the rat model. In summary, our experimental results substantiate the significant involvement of MIF in the degeneration of NPCs, and inhibiting MIF activity can effectively mitigate IVDD.

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

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

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

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

MINK1 deficiency stimulates nucleus pulposus cell pyroptosis and exacerbates intervertebral disc degeneration.

Intervertebral disc (IVD) degeneration, induced by aging and irregular mechanical strain, is highly prevalent in the elderly population, serving as a leading cause of chronic low back pain and disability. Evolving evidence has revealed the involvement of nucleus pulposus (NP) pyroptosis in the pathogenesis of IVD degeneration, while the precise regulatory mechanisms of NP pyroptosis remain obscure. Misshapen/Nck-interacting kinase (NIK)-related kinase 1 (MINK1), a serine-threonine protein kinase, has the potential to modulate the activation of NLRP3 inflammasome, indicating its pivotal role in governing pyroptosis. In this study, to assess the significance of MINK1 in NP pyroptosis and IVD degeneration, NP tissues from patients with varying degrees of IVD degeneration, and IVD tissues from both aging-induced and lumbar spine instability (LSI) surgery-induced IVD degeneration mouse models, with or without MINK1 ablation, were meticulously evaluated. Our findings indicated a notable decline in MINK1 expression in NP tissues of patients with IVD degeneration and both mouse models as degeneration progresses, accompanied by heightened matrix degradation and increased NP pyroptosis. Moreover, MINK1 ablation led to substantial activation of NP pyroptosis in both mouse models, and accelerating ECM degradation and intensifying the degeneration phenotype in mechanically stress-induced mice. Mechanistically, MINK1 deficiency triggered NF-κB signaling in NP tissues. Overall, our data illustrate an inverse correlation between MINK1 expression and severity of IVD degeneration, and the absence of MINK1 stimulates NP pyroptosis, exacerbating IVD degeneration by activating NF-κB signaling, highlighting a potential innovative therapeutic target in treating IVD degeneration.

Screening of NSAIDs library identifies Tinoridine as a novel ferroptosis inhibitor for potential intervertebral disc degeneration therapy.

Low back pain (LBP) may profoundly impact the quality of life across the globe, and intervertebral disc degeneration (IVDD) is the major cause of LBP; however, targeted pharmaceutical interventions for IVDD are still lacking. Ferroptosis is a novel form of iron-dependent programmed cell death. Studies have showed that ferroptosis may closely associate with IVDD; thus, targeting ferroptosis may have great potential for IVDD therapy. Non-steroidal anti-inflammatory drugs (NSAIDs) are the first-line medications for LBP, while nuclear factor-erythroid 2-related factor-2 (Nrf2) is a key inhibitory protein for ferroptosis. In the current study, we conducted a molecular docking screening between NSAIDs library and Nrf2 protein. Tinoridine was shown to have a high binding affinity to Nrf2. The in vitro study in nucleus pulposus (NP) cells showed that Tinoridine may promote the expression and activity of Nrf2, it may also rescue RSL3-induced ferroptosis in NP cells. Knockdown of Nrf2 reverses the protective effect of Tinoridine on RSL3-induced ferroptosis in NP cells, suggesting that the inhibitory effect of Tinoridine on ferroptosis is through Nrf2. In vivo study demonstrated that Tinoridine may attenuate the progression of IVDD in rats. As NSAIDs are already clinically used for LBP therapy, the current study supports Tinoridine's application from the view of ferroptosis inhibition.

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

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

Evaluating lumbar disc degeneration by MRI quantitative metabolic indicators: the perspective of factor analysis.

PURPOSE: This study aimed to investigate an early diagnostic method for lumbar disc degeneration (LDD) and improve its diagnostic accuracy. METHODS: Quantitative biomarkers of the lumbar body (LB) and lumbar discs (LDs) were obtained using nuclear magnetic resonance (NMR) detection technology. The diagnostic weights of each biological metabolism indicator were screened using the factor analysis method. RESULTS: Through factor analysis, common factors such as the LB fat fraction, fat content, and T2* value of LDs were identified as covariates for the diagnostic model for the evaluation of LDD. This model can optimize the accuracy and reliability of LDD diagnosis. CONCLUSION: The application of biomarker quantification methods based on NMR detection technology combined with factor analysis provides an effective means for the early diagnosis of LDD, thereby improving diagnostic accuracy and reliability.

Brachyury promotes extracellular matrix synthesis through transcriptional regulation of Smad3 in nucleus pulposus.

Metabolic dysfunction of the extracellular matrix (ECM) is one of the primary causes of intervertebral disc degeneration (IVDD). Previous studies have demonstrated that the transcription factor Brachyury (Bry) has the potential to promote the synthesis of collagen II and aggrecan, while the specific mechanism is still unknown. In this study, we used a lipopolysaccharide (LPS)-induced model of nucleus pulposus cell (NPC) degeneration and a rat acupuncture IVDD model to elucidate the precise mechanism through which Bry affects collagen II and aggrecan synthesis in vitro and in vivo. First, we confirmed Bry expression decreased in degenerated human nucleus pulposus (NP) cells (NPCs). Knockdown of Bry exacerbated the decrease in collagen II and aggrecan expression in the lipopolysaccharide (LPS)-induced NPCs degeneration in vitro model. Bioinformatic analysis indicated that Smad3 may participate in the regulatory pathway of ECM synthesis regulated by Bry. Chromatin immunoprecipitation followed by quantitative polymerase chain reaction (ChIP-qPCR) and luciferase reporter gene assays demonstrated that Bry enhances the transcription of Smad3 by interacting with a specific motif on the promoter region. In addition, Western blot and reverse transcription-qPCR assays demonstrated that Smad3 positively regulates the expression of aggrecan and collagen II in NPCs. The following rescue experiments revealed that Bry-mediated regulation of ECM synthesis is partially dependent on Smad3 phosphorylation. Finally, the findings from the in vivo rat acupuncture-induced IVDD model were consistent with those obtained from in vitro assays. In conclusion, this study reveals that Bry positively regulates the synthesis of collagen II and aggrecan in NP through transcriptional activation of Smad3.NEW & NOTEWORTHY Mechanically, in the nucleus, Bry enhances the transcription of Smad3, leading to increased expression of Smad3 protein levels; in the cytoplasm, elevated substrate levels further lead to an increase in the phosphorylation of Smad3, thereby regulating collagen II and aggrecan expression. Further in vivo experiments provide additional evidence that Bry can alleviate IVDD through this mechanism.

Palmitic acid induces lipid droplet accumulation and senescence in nucleus pulposus cells via ER-stress pathway.

Intervertebral disc degeneration (IDD) is a highly prevalent musculoskeletal disorder affecting millions of adults worldwide, but a poor understanding of its pathogenesis has limited the effectiveness of therapy. In the current study, we integrated untargeted LC/MS metabolomics and magnetic resonance spectroscopy data to investigate metabolic profile alterations during IDD. Combined with validation via a large-cohort analysis, we found excessive lipid droplet accumulation in the nucleus pulposus cells of advanced-stage IDD samples. We also found abnormal palmitic acid (PA) accumulation in IDD nucleus pulposus cells, and PA exposure resulted in lipid droplet accumulation and cell senescence in an endoplasmic reticulum stress-dependent manner. Complementary transcriptome and proteome profiles enabled us to identify solute carrier transporter (SLC) 43A3 involvement in the regulation of the intracellular PA level. SLC43A3 was expressed at low levels and negatively correlated with intracellular lipid content in IDD nucleus pulposus cells. Overexpression of SLC43A3 significantly alleviated PA-induced endoplasmic reticulum stress, lipid droplet accumulation and cell senescence by inhibiting PA uptake. This work provides novel integration analysis-based insight into the metabolic profile alterations in IDD and further reveals new therapeutic targets for IDD treatment.

Mrgprb2-mediated mast cell activation exacerbates Modic changes by regulating immune niches.

Modic changes are radiographic features associated with microfracture, low-virulence organism infection and chronic inflammation with inflammatory cell infiltration in the vertebral endplate region. Mast cells, as innate immune cells similar to macrophages, are present in painful degenerated intervertebral discs. However, the involvement and mechanisms of mast cells in the development of Modic changes remain unclear. Herein, we found increased mast cell infiltration in samples from patients with Modic changes and in mouse models of Modic changes. To clarify the role of mast cells in the progression of Modic changes, we used mast cell-deficient (KITW-SH/W-SH) mice to construct a model of Modic changes and found that the severity of Modic changes in KITW-SH/W-SH mice was significantly lower than that in WT mice. These findings were further supported by the use of a mast cell-specific activator (compound 48/80) and a stabilizer (cromolyn). Furthermore, we found that mast cells were not activated via the classic IgE pathway in the Modic change models and that Mrgprb2 is the specific receptor for mast cell activation reported in recent studies. Then, we utilized Mrgprb2 knockout mice to demonstrate that Mrgprb2 knockout inhibited mast cell activation and thus reduced the degree of Modic changes. Transcriptomic sequencing revealed aberrant PI3K-AKT and MAPK pathway activation in the Mrgprb2-deficient mast cells. Additionally, Mrgpbrb2-activated mast cells regulate immune niches by recruiting macrophages, promoting M1 polarization and reducing M2 polarization, thereby promoting the progression of Modic changes. These findings suggest that mast cells may serve as a novel therapeutic target for addressing Modic changes.