Self-powered triboelectric-responsive microneedles with controllable release of optogenetically engineered extracellular vesicles for intervertebral disc degeneration repair.

Excessive exercise is an etiological factor of intervertebral disc degeneration (IVDD). Engineered extracellular vesicles (EVs) exhibit excellent therapeutic potential for disease-modifying treatments. Herein, we fabricate an exercise self-powered triboelectric-responsive microneedle (MN) assay with the sustainable release of optogenetically engineered EVs for IVDD repair. Mechanically, exercise promotes cytosolic DNA sensing-mediated inflammatory activation in senescent nucleus pulposus (NP) cells (the master cell population for IVD homeostasis maintenance), which accelerates IVDD. TREX1 serves as a crucial nuclease, and disassembly of TRAM1-TREX1 complex disrupts the subcellular localization of TREX1, triggering TREX1-dependent genomic DNA damage during NP cell senescence. Optogenetically engineered EVs deliver TRAM1 protein into senescent NP cells, which effectively reconstructs the elimination function of TREX1. Triboelectric nanogenerator (TENG) harvests mechanical energy and triggers the controllable release of engineered EVs. Notably, an optogenetically engineered EV-based targeting treatment strategy is used for the treatment of IVDD, showing promising clinical potential for the treatment of degeneration-associated disorders.

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.

Exosomes derived from human urine-derived stem cells ameliorate IL-1ß-induced intervertebral disk degeneration.

BACKGROUND: Human intervertebral disk degeneration (IVDD) is a sophisticated degenerative pathological process. A key cause of IVDD progression is nucleus pulposus cell (NPC) degeneration, which contributes to excessive endoplasmic reticulum stress in the intervertebral disk. However, the mechanisms underlying IVDD and NPC degeneration remain unclear. METHODS: We used interleukin (IL)-1ß stimulation to establish an NPC-degenerated IVDD model and investigated whether human urine-derived stem cell (USC) exosomes could prevent IL-1ß-induced NPC degeneration using western blotting, quantitative real-time polymerase chain reaction, flow cytometry, and transcriptome sequencing techniques. RESULTS: We successfully extracted and identified USCs and exosomes from human urine. IL-1ß substantially downregulated NPC viability and induced NPC degeneration while modulating the expression of SOX-9, collagen II, and aggrecan. Exosomes from USCs could rescue IL-1ß-induced NPC degeneration and restore the expression levels of SOX-9, collagen II, and aggrecan. CONCLUSIONS: USC-derived exosomes can prevent NPCs from degeneration following IL-1ß stimulation. This finding can aid the development of a potential treatment strategy for IVDD.

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

Objective: The mechanisms of intervertebral disc degeneration (IVDD) in low back pain (LBP) patients are multiples. In this study, we attempt to investigate whether melatonergic system plays a potential role in IVDD patients with LBP by analyzing their clinical specimens. The fucus will be given to the correlation between the melatonin receptor expression and intervertebral disc tissue apoptosis. Methods: In this clinical study, 107 lumbar intervertebral disc nucleus pulposus (NP) specimens from patients with LBP were collected with patients' consents. The disc height (DH) discrepancy ratio, range of motion and sagittal parameters of the pathological plane were measured and Pfirrmann grade was used to classified the grades of IVDD level. Discs at grades 1-3 were served as normal control and grades 4-5 were considered as IVDD. The expression levels of melatonin receptor 1A (MT1) and 1B (MT2) were measured by immunohistochemistry. The apoptosis of NP was assessed using TUNEL staining. Their potential associations among MT1/2, DH, apoptosis, sagittal parameters with IVDD and LBP were evaluated with statistical analysis. Results: The incidence of IVDD was positively associated with age and negatively related to VAS scores for LBP (p < 0.001). Patients with higher degree of IVDD also have higher DH discrepancy ratio (p < 0.001), higher prevalence of lumbar instability (p = 0.003) and higher cell apoptosis compared to the control. Nevertheless, no statistically significant correlation was identified between Pfirrmann grade and lumbar sagittal parameters. MT1 and MT2 both were highly expressed in the NP tissues. Importantly, MT1 expression but not MT2 was significantly increased in the intervertebral disc tissue of patients with IVDD and its level correlated well with cell apoptosis level and the severity of IVDD as well as lower VAS scores for LBP. Conclusion: The highly elevated MT1 expression was found in NP tissues of patients with IVDD and LBP compared to the control. This phenomenon probably reflects the compensating response of the body to the pathological alteration of the IVDD and LBP. Therefore, these findings provide the novel information to use selective agonists of MT1 to target IVDD and LBP clinically.

Rescue of nucleus pulposus cells from an oxidative stress microenvironment via glutathione-derived carbon dots to alleviate intervertebral disc degeneration.

The senescence of nucleus pulposus (NP) cells (NPCs), which is induced by the anomalous accumulation of reactive oxygen species (ROS), is a major cause of intervertebral disc degeneration (IVDD). In this research, glutathione-doped carbon dots (GSH-CDs), which are novel carbon dot antioxidant nanozymes, were successfully constructed to remove large amounts of ROS for the maintenance of NP tissue at the physical redox level. After significantly scavenging endogenous ROS via exerting antioxidant activities, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and total antioxidant capacity, GSH-CDs with good biocompatibility have been demonstrated to effectively improve mitochondrial dysfunction and rescue NPCs from senescence, catabolism, and inflammatory factors in vivo and in vitro. In vivo imaging data and histomorphological indicators, such as the disc height index (DHI) and Pfirrmann grade, demonstrated prominent improvements in the progression of IVDD after the topical application of GSH-CDs. In summary, this study investigated the GSH-CDs nanozyme, which possesses excellent potential to inhibit the senescence of NPCs with mitochondrial lesions induced by the excessive accumulation of ROS and improve the progression of IVDD, providing potential therapeutic options for clinical treatment.

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.

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.

Oxidative stress-induced EGR1 upregulation promotes NR4A3-mediated nucleus pulposus cells apoptosis in intervertebral disc degeneration.

This study aimed to reveal the specific role of early growth response protein 1 (EGR1) and nuclear receptor 4A3 (NR4A3) in nucleus pulposus cells (NPCs) and the related molecular mechanism and to identify a new strategy for treating intervertebral disc degeneration (IVDD). Bioinformatics analysis was used to explore and predict IVDD-related differentially expressed genes, and chromatin immunoprecipitation sequencing (ChIP-seq) revealed NR4A3 as the EGR1 target gene. An in vitro NPC model induced by tributyl hydrogen peroxide (TBHP) and a rat model induced by fibrous ring acupuncture were established. Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemical staining, immunofluorescence staining, and flow cytometry were used to detect the effects of EGR1 and NR4A3 knockdown and overexpression on NPC apoptosis and the expression of extracellular matrix (ECM) anabolism-related proteins. Interactions between EGR1 and NR4A3 were analyzed via ChIP-qPCR and dual luciferase assays. EGR1 and NR4A3 expression levels were significantly higher in severely degenerated discs (SDD) than in mildly degenerated discs (MDD), indicating that these genes are important risk factors in IVDD progression. ChIP-seq and RNA-seq revealed NR4A3 as a direct downstream target of EGR1, and this finding was verified by ChIP-qPCR and dual luciferase reporter experiments. Remarkably, the rescue experiments showed that EGR1 promotes TBHP-induced NPC apoptosis and impairs ECM anabolism, dependent on elevated NR4A3 expression. In summary, the EGR1-NR4A3 axis mediates the progression of NPC apoptosis and ECM impairment and is a potential therapeutic target in IVDD.

Hyperforin improves matrix stiffness induced nucleus pulposus inflammatory degeneration by activating mitochondrial fission.

OBJECTIVE: The continuously increasing extracellular matrix stiffness during intervertebral disc degeneration promotes disease progression. In an attempt to obtain novel treatment methods, this study aims to investigate the changes in nucleus pulposus cells under the stimulation of a stiff microenvironment. DESIGN: RNA sequencing and metabolomics experiments were combined to evaluate the primary nucleus pulposus and screen key targets under mechanical biological stimulation. Additionally, small molecules work in vitro were used to confirm the target regulatory effect and investigate the mechanism. In vivo, treatment effects were validated using a rat caudal vertebrae compression model. RESULTS: Our research results revealed that by activating TRPC6, hyperforin, a herbaceous extract can rescue the inflammatory phenotype caused by the stiff microenvironment, hence reducing intervertebral disc degeneration (IDD). Mechanically, it activates mitochondrial fission to inhibit PFKFB3. CONCLUSION: In summary, this study reveals the important bridging role of TRPC6 between mechanical stiffness, metabolism, and inflammation in the context of nucleus pulposus degeneration. TRPC6 activation with hyperforin may become a promising treatment for IDD.

Progenitor-like cells contributing to cellular heterogeneity in the nucleus pulposus are lost in intervertebral disc degeneration.

The nucleus pulposus (NP) in the intervertebral disc (IVD) arises from embryonic notochord. Loss of notochordal-like cells in humans correlates with onset of IVD degeneration, suggesting that they are critical for healthy NP homeostasis and function. Comparative transcriptomic analyses identified expression of progenitor-associated genes (GREM1, KRT18, and TAGLN) in the young mouse and non-degenerated human NP, with TAGLN expression reducing with aging. Lineage tracing using Tagln-CreERt2 mice identified peripherally located proliferative NP (PeriNP) cells in developing and postnatal NP that provide a continuous supply of cells to the entire NP. PeriNP cells were diminished in aged mice and absent in puncture-induced degenerated discs. Single-cell transcriptomes of postnatal Tagln-CreERt2 IVD cells indicate enrichment for TGF-ß signaling in Tagln descendant NP sub-populations. Notochord-specific removal of TGF-ß/BMP mediator Smad4 results in loss of Tagln+ cells and abnormal NP morphologies. We propose Tagln+ PeriNP cells are potential progenitors crucial for NP homeostasis.

Identifying the protective effects of miR-874-3p/ATF3 axis in intervertebral disc degeneration by single-cell RNA sequencing and validation.

Intervertebral disc degeneration (IVDD) severely affects the work and the quality of life of people. We previously demonstrated that silencing activation transcription factor 3 (ATF3) blocked the IVDD pathological process by regulating nucleus pulposus cell (NPC) ferroptosis, apoptosis, inflammation, and extracellular matrix (ECM) metabolism. Nevertheless, whether miR-874-3p mediated the IVDD pathological process by targeting ATF3 remains unclear. We performed single-cell RNA sequencing (scRNA-seq) and bioinformatics analysis to identify ATF3 as a key ferroptosis gene in IVDD. Then, Western blotting, flow cytometry, ELISA, and animal experiments were performed to validate the roles and regulatory mechanisms of miR-874-3p/ATF3 signalling axis in IVDD. ATF3 was highly expressed in IVDD patients and multiple cell types of IVDD rat, as revealed by scRNA-seq and bioinformatics analysis. GO analysis unveiled the involvement of ATF3 in regulating cell apoptosis and ECM metabolism. Furthermore, we verified that miR-874-3p might protect against IVDD by inhibiting NPC ferroptosis, apoptosis, ECM degradation, and inflammatory response by targeting ATF3. In vivo experiments displayed the protective effect of miR-874-3p/ATF3 axis on IVDD. These findings propose the potential of miR-874-3p and ATF3 as biomarkers of IVDD and suggest that targeting the miR-874-3p/ATF3 axis may be a therapeutic target for IVDD.

BMSC loaded photo-crosslinked hyaluronic acid/collagen hydrogel incorporating FG4592 for enhanced cell proliferation and nucleus pulposus differentiation.

Intervertebral disc degeneration arises from damage or degeneration of the nucleus pulposus (NP). In this study, we developed a photo-crosslinkable hydrogel incorporating FG4592 to support the growth and differentiation of bone-marrow-derived mesenchymal stem cells (BMSC). Initially, hyaluronic acid was modified with tyramine and combined with collagen to introduce riboflavin as a photo-crosslinker. This hydrogel transitioned from liquid to gel upon exposure to blue light in 3 min. The results showed that the hydrogel was biodegradable and had mechanical properties comparable to those of human NP tissues. Scanning electron microscopy after BMSC seeding in the hydrogel revealed an even distribution, and cells adhered to the collagen fibers in the hydrogel with minimal cell mortality. The effect of FG4592 on BMSC proliferation and differentiation was examined, revealing the capability of FG4592 to promote BMSC proliferation and direct differentiation resembling human NP cells. After cultivating BMSCs in the photo-crosslinked hydrogel, there was an upregulation in the expression of glycosaminoglycans, aggrecan, type II collagen, and keratin 19 proteins. Cross-species analyses of rat and human BMSCs revealed consistent results. For potential clinical applications, BMSC loaded with photo-crosslinked hydrogels can be injected into damaged intervertebral disc to facilitate NP regeneration.

D-mannose alleviates intervertebral disc degeneration through glutamine metabolism.

BACKGROUND: Intervertebral disc degeneration (IVDD) is a multifaceted condition characterized by heterogeneity, wherein the balance between catabolism and anabolism in the extracellular matrix of nucleus pulposus (NP) cells plays a central role. Presently, the available treatments primarily focus on relieving symptoms associated with IVDD without offering an effective cure targeting its underlying pathophysiological processes. D-mannose (referred to as mannose) has demonstrated anti-catabolic properties in various diseases. Nevertheless, its therapeutic potential in IVDD has yet to be explored. METHODS: The study began with optimizing the mannose concentration for restoring NP cells. Transcriptomic analyses were employed to identify the mediators influenced by mannose, with the thioredoxin-interacting protein (Txnip) gene showing the most significant differences. Subsequently, small interfering RNA (siRNA) technology was used to demonstrate that Txnip is the key gene through which mannose exerts its effects. Techniques such as colocalization analysis, molecular docking, and overexpression assays further confirmed the direct regulatory relationship between mannose and TXNIP. To elucidate the mechanism of action of mannose, metabolomics techniques were employed to pinpoint glutamine as a core metabolite affected by mannose. Next, various methods, including integrated omics data and the Gene Expression Omnibus (GEO) database, were used to validate the one-way pathway through which TXNIP regulates glutamine. Finally, the therapeutic effect of mannose on IVDD was validated, elucidating the mechanistic role of TXNIP in glutamine metabolism in both intradiscal and orally treated rats. RESULTS: In both in vivo and in vitro experiments, it was discovered that mannose has potent efficacy in alleviating IVDD by inhibiting catabolism. From a mechanistic standpoint, it was shown that mannose exerts its anti-catabolic effects by directly targeting the transcription factor max-like protein X-interacting protein (MondoA), resulting in the upregulation of TXNIP. This upregulation, in turn, inhibits glutamine metabolism, ultimately accomplishing its anti-catabolic effects by suppressing the mitogen-activated protein kinase (MAPK) pathway. More importantly, in vivo experiments have further demonstrated that compared with intradiscal injections, oral administration of mannose at safe concentrations can achieve effective therapeutic outcomes. CONCLUSIONS: In summary, through integrated multiomics analysis, including both in vivo and in vitro experiments, this study demonstrated that mannose primarily exerts its anti-catabolic effects on IVDD through the TXNIP-glutamine axis. These findings provide strong evidence supporting the potential of the use of mannose in clinical applications for alleviating IVDD. Compared to existing clinically invasive or pain-relieving therapies for IVDD, the oral administration of mannose has characteristics that are more advantageous for clinical IVDD treatment.

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.

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.

Bradykinin protects nucleus pulposus cells from tert-butyl hydroperoxide-induced damage and delays intervertebral disc degeneration.

Intervertebral disc degeneration (IVDD) is a leading cause of degenerative spinal disorders, involving complex biological processes. This study investigates the role of the kallikrein-kinin system (KKS) in IVDD, focusing on the protective effects of bradykinin (BK) on nucleus pulposus cells (NPCs) under oxidative stress. Clinical specimens were collected, and experiments were conducted using human and rat primary NPCs to elucidate BK's impact on tert-butyl hydroperoxide (TBHP)-induced oxidative stress and damage. The results demonstrate that BK significantly inhibits TBHP-induced NPC apoptosis and restores mitochondrial function. Further analysis reveals that this protective effect is mediated through the BK receptor 2 (B2R) and its downstream PI3K/AKT pathway. Additionally, BK/PLGA sustained-release microspheres were developed and validated in a rat model, highlighting their potential therapeutic efficacy for IVDD. Overall, this study sheds light on the crucial role of the KKS in IVDD pathogenesis and suggests targeting the B2R as a promising therapeutic strategy to delay IVDD progression and promote disc regeneration.

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.

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.

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.

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.