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.

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.

Regulation of endoplasmic reticulum stress on autophagy and apoptosis of nucleus pulposus cells in intervertebral disc degeneration and its related mechanisms.

Intervertebral disc degeneration (IVDD) is a common and frequent disease in orthopedics, which seriously affects the quality of life of patients. Endoplasmic reticulum stress (ERS)-regulated autophagy and apoptosis play an important role in nucleus pulposus (NP) cells in IVDD. Hypoxia and serum deprivation were used to induce NP cells. Cell counting kit-8 (CCK-8) assay was used to detect cell activity and immunofluorescence (IF) was applied for the appraisement of glucose regulated protein 78 (GRP78) and green fluorescent protein (GFP)-light chain 3 (LC3). Cell apoptosis was detected by flow cytometry and the expression of LC3II/I was detected by western blot. NP cells under hypoxia and serum deprivation were induced by lipopolysaccharide (LPS), and intervened by ERS inhibitor (4-phenylbutyric acid, 4-PBA) and activator (Thapsigargin, TP). Then, above functional experiments were conducted again and western blot was employed for the evaluation of autophagy-, apoptosis and ERS-related proteins. Finally, NP cells under hypoxia and serum deprivation were stimulated by LPS and intervened using apoptosis inhibitor z-Val-Ala-DL-Asp-fluoromethyl ketone (Z-VAD-FMK) and autophagy inhibitor 3-methyladenine (3-MA). CCK-8 assay, IF, flow cytometry and western blot were performed again. Besides, the levels of inflammatory cytokines were measured with enzyme-linked immunosorbent assay (ELISA) and the protein expressions of programmed death markers were estimated with western blot. It showed that serum deprivation induces autophagy and apoptosis. ERS was significantly activated by LPS in hypoxic and serum deprivation environment, and autophagy and apoptosis were significantly promoted. Overall, ERS affects the occurrence and development of IVDD by regulating autophagy, apoptosis and other programmed death.

Bioenergetic dysfunction in the pathogenesis of intervertebral disc degeneration.

Intervertebral disc (IVD) degeneration is a frequent cause of low back pain and is the most common cause of disability. Treatments for symptomatic IVD degeneration, including conservative treatments such as analgesics, physical therapy, anti-inflammatories and surgeries, are aimed at alleviating neurological symptoms. However, there are no effective treatments to prevent or delay IVD degeneration. Previous studies have identified risk factors for IVD degeneration such as aging, inflammation, genetic factors, mechanical overload, nutrient deprivation and smoking, but metabolic dysfunction has not been highlighted. IVDs are the largest avascular structures in the human body and determine the hypoxic and glycolytic features of nucleus pulposus (NP) cells. Accumulating evidence has demonstrated that intracellular metabolic dysfunction is associated with IVD degeneration, but a comprehensive review is lacking. Here, by reviewing the physiological features of IVDs, pathological processes and metabolic changes associated with IVD degeneration and the functions of metabolic genes in IVDs, we highlight that glycolytic pathway and intact mitochondrial function are essential for IVD homeostasis. In degenerated NPs, glycolysis and mitochondrial function are downregulated. Boosting glycolysis such as HIF1α overexpression protects against IVD degeneration. Moreover, the correlations between metabolic diseases such as diabetes, obesity and IVD degeneration and their underlying molecular mechanisms are discussed. Hyperglycemia in diabetic diseases leads to cell senescence, the senescence-associated phenotype (SASP), apoptosis and catabolism of extracellualr matrix in IVDs. Correcting the global metabolic disorders such as insulin or GLP-1 receptor agonist administration is beneficial for diabetes associated IVD degeneration. Overall, we summarized the recent progress of investigations on metabolic contributions to IVD degeneration and provide a new perspective that correcting metabolic dysfunction may be beneficial for treating IVD degeneration.

Rescuing Nucleus Pulposus Cells from ROS Toxic Microenvironment via Mitochondria-Targeted Carbon Dot-Supported Prussian Blue to Alleviate Intervertebral Disc Degeneration.

Intervertebral disc degeneration (IVDD) is invariably accompanied by excessive accumulation of reactive oxygen species (ROS), resulting in progressive deterioration of mitochondrial function and senescence in nucleus pulposus cells (NPCs). Significantly, the main ROS production site in non-immune cells is mitochondria, suggesting mitochondria is a feasible therapeutic target to reverse IVDD. Triphenylphosphine (TPP), which is known as mitochondrial-tropic ligands, is utilized to modify carbon dot-supported Prussian blue (CD-PB) to scavenge superfluous intro-cellular ROS and maintain NPCs at normal redox levels. CD-PB-TPP can effectively escape from lysosomal phagocytosis, permitting efficient mitochondrial targeting. After strikingly lessening the ROS in mitochondria via exerting antioxidant enzyme-like activities, such as superoxide dismutase, and catalase, CD-PB-TPP rescues damaged mitochondrial function and NPCs from senescence, catabolism, and inflammatory reaction in vitro. Imaging evaluation and tissue morphology assessment in vivo suggest that disc height index, mean grey values of nucleus pulposus tissue, and histological morphology are significantly improved in the IVDD model after CD-PB-TPP is locally performed. In conclusion, this study demonstrates that ROS-induced mitochondrial dysfunction and senescence of NPCs leads to IVDD and the CD-PB-TPP possesses enormous potential to rescue this pathological process through efficient removal of ROS via targeting mitochondria, supplying a neoteric strategy for IVDD treatment.

DNMT3a-mediated methylation of PPARγ promote intervertebral disc degeneration by regulating the NF-κB pathway.

Intervertebral disc degeneration (IVDD) is a common chronic musculoskeletal disease that causes chronic low back pain and imposes an immense financial strain on patients. The pathological mechanisms underlying IVDD have not been fully elucidated. The development of IVDD is closely associated with abnormal epigenetic changes, suggesting that IVDD progression may be controlled by epigenetic mechanisms. Consequently, this study aimed to investigate the role of epigenetic regulation, including DNA methyltransferase 3a (DNMT3a)-mediated methylation and peroxisome proliferator-activated receptor γ (PPARγ) inhibition, in IVDD development. The expression of DNMT3a and PPARγ in early and late IVDD of nucleus pulposus (NP) tissues was detected using immunohistochemistry and western blotting analyses. Cellularly, DNMT3a inhibition significantly inhibited IL-1ß-induced apoptosis and extracellular matrix (ECM) degradation in rat NP cells. Pretreatment with T0070907, a specific inhibitor of PPARγ, significantly reversed the anti-apoptotic and ECM degradation effects of DNMT3a inhibition. Mechanistically, DNMT3a modified PPARγ promoter hypermethylation to activate the nuclear factor-κB (NF-κB) pathway. DNMT3a inhibition alleviated IVDD progression. Conclusively, the results of this study show that DNMT3a activates the NF-κB pathway by modifying PPARγ promoter hypermethylation to promote apoptosis and ECM degradation. Therefore, we believe that the ability of DNMT3a to mediate the PPARγ/NF-κB axis may provide new ideas for the potential pathogenesis of IVDD and may become an attractive target for the treatment of IVDD.

Mesenchymal stem cell exosomes inhibit nucleus pulposus cell apoptosis via the miR-125b-5p/TRAF6/NF-κB pathway axis.

Intervertebral disc degeneration (IVDD) is the pathological basis of a range of degenerative spinal diseases and is the primary cause of lower back pain. Mesenchymal stem cell (MSC) transplantation inhibits IVDD progression. However, the specific mechanisms that underlie these effects remain unclear. In this study, candidate microRNAs (miRNAs) are screened using bioinformatics and high-throughput sequencing. TNF-α is used to induce nucleus pulposus cell (NPC) degeneration. MSC-derived exosomes (MSC-exosomes) are obtained using high-speed centrifugation and identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blot analysis. Cell viability is determined by CCK-8 assay. Flow cytometry and TUNEL assays are used to detect cell apoptosis. The expression levels of miR-125b-5p are detected by RT-qPCR, and a dual-luciferase gene reporter assay confirms the downstream target genes of miR-125b-5p. Protein expression is determined by western blot analysis. Rat models are used to validate the function of miR-125b-5p in MSC-exosomes. The results show that miR-125b-5p is expressed at low levels in degenerated disc tissues compared with that in normal disc tissues; however, it is highly expressed in MSC-exosomes. Furthermore, MSC-exosomes are efficiently taken up by NPCs while miR-125b-5p is delivered into NPCs; thus, MSC-exosomes act as inhibitors of apoptosis in NPCs. Overexpression of miR-125b-5p downregulates TRAF6 expression and inhibits NF-κB activation. However, TRAF6 overexpression reverses these effects of miR-125b-5p. We demonstrate that MSC-exosomes attenuate IVDD in vivo by delivering miR-125b-5p. MSC-exosomes can deliver miR-125b-5p to target TRAF6, inhibit NF-κB activation, and attenuate the progression of IVDD.

Nucleus Pulposus-Targeting Nanocarriers Facilitate Mirna-Based Therapeutics for Intervertebral Disc Degeneration.

Intervertebral disc degeneration (IDD) is a common cause of low back pain. Understanding its molecular mechanisms is the basis for developing specific treatment. To demonstrate that miR-22-3p is critical in the regulation of IDD, miRNA microarray analyses are conducted in conjunction with in vivo and in vitro experiments. The miR-22-3p knockout (KO) mice show a marked decrease in the histological scores. Bioinformatic analysis reveals that miR-22-3p plays a mechanistic role in the development of IDD by targeting SIRT1, which in turn activates the JAK1/STAT3 signaling pathway. This is confirmed by a luciferase reporter assay and western blot analysis. Therapeutically, the delivery of miR-22-3p inhibitors and mimics through the synthesized nanoparticles in the IDD model alleviates and aggravates IDD, respectively. The nanocarriers enhance transportation of miR-22-3p to nucleus pulposus cells, thus enabling the in vivo inhibition of miR-22-3p for therapeutic purposes and consequently promoting the development of miRNA-specific drugs for IDD.

Circ-STC2 promotes the ferroptosis of nucleus pulposus cells via targeting miR-486-3p/TFR2 axis.

BACKGROUND: Low back pain (LBP) has become the second leading cause of disability worldwide, which has brought great economic burden to people. It is generally believed that intervertebral disc degeneration (IDD) is the main cause of LBP. This study aimed to explore the role of circ-STC2 in the pathogenesis of IDD. METHODS: Nucleus pulposus cells (NPCs) were treated with T-Butyl Hydrogen Peroxide (TBHP) to establish IDD model in vitro. RT-qPCR was performed to detect mRNA expressions. The cell viability was detected with CCK-8 assay. The levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), Fe2+ and glutathione (GSH) of NPCs were measured by corresponding kits. The protein expressions were determined by western blot. Dual-luciferase reporter and RNA pull-down assays were conducted to verify the relationship between circ-STC2 or transferrin recepto 2 (TFR2) and miR-486-3p. RESULTS: Circ-STC2 and TFR2 expressions were up-regulated in IDD tissues, and miR-486-3p expression was down-regulated. Knockdown of circ-STC2 promoted the cell viability and inhibited the ferroptosis of the NPCs. The GSH levels, and glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) protein expressions were increased, the LDH, MDA and Fe2+ levels and achaete-scute complexlike 4 (ASCL4) protein expressions were decreased after circ-STC2 knockdown. Knockdown of miR-486-3p abrogated the si-circ-STC2 effects and overexpression of TFR2 reversed the miR-486-3p mimic effects. CONCLUSIONS: Circ-STC2 inhibits the cell viability, induced the ferroptosis of the TBHP treated NPCs via targeting miR-486-3p/TFR2 axis.

A Redox Homeostasis Modulatory Hydrogel with GLRX3+ Extracellular Vesicles Attenuates Disc Degeneration by Suppressing Nucleus Pulposus Cell Senescence.

Characterized by nucleus pulposus (NP) cell senescence and extracellular matrix (ECM) degradation, disc degeneration is a common pathology for various degenerative spinal disorders. To date, effective treatments for disc degeneration are absent. Here, we found that Glutaredoxin3 (GLRX3) is an important redox-regulating molecule associated with NP cell senescence and disc degeneration. Using a hypoxic preconditioning method, we developed GLRX3+ mesenchymal stem cell-derived extracellular vehicles (EVs-GLRX3), which enhanced the cellular antioxidant defense, thus preventing reactive oxygen species (ROS) accumulation and senescence cascade expansion in vitro. Further, a disc tissue-like biopolymer-based supramolecular hydrogel, which was injectable, degradable, and ROS-responsive, was proposed to deliver EVs-GLRX3 for treating disc degeneration. Using a rat model of disc degeneration, we demonstrated that the EVs-GLRX3-loaded hydrogel attenuated mitochondrial damage, alleviated the NP senescence state, and restored ECM deposition by modulating the redox homeostasis. Our findings suggested that modulation of redox homeostasis in the disc can rejuvenate NP cell senescence and thus attenuate disc degeneration.

Metabolic Biomarkers Differentiate a Surgical Intervertebral Disc from a Nonsurgical Intervertebral Disc.

BACKGROUND: Degeneration of the intervertebral disc (IVD) is caused by disturbances in metabolic processes, which lead to structural disorders. The aim of this report is to analyze metabolic disorders in the degeneration process by comparing control discs with degenerated discs. In our research on the nucleus pulposus (NP), we used NMR spectroscopy of extracts of hydrophilic and hydrophobic compounds of the tissue. METHODS: Nuclear magnetic resonance (NMR) spectroscopy allows the study of biochemistry and cellular metabolism in vitro. Hydrophilic and hydrophobic compounds were extracted from the NP of the intervertebral disc. In the NMR spectra, metabolites were identified and quantitatively analyzed. The results of our research indicate disturbances in the biosynthesis and metabolism of cholesterol, the biosynthesis and degradation of various fatty acid groups, ketone bodies, or lysine, and the metabolism of glycerophospholipids, purines, glycine, inositol, galactose, alanine, glutamate, and pyruvate in the biosynthesis of valine and isoleucine, leucine. All these disorders indicate pathomechanisms related to oxidative stress, energy, neurotransmission disturbances, and disturbances in the structure and functioning of cell membranes, inflammation, or chronic pain generators. CONCLUSIONS: NMR spectroscopy allows the identification of metabolites differentiating surgical from nonsurgical discs. These data may provide guidance in in vivo MRS studies in assessing the severity of lesions of the disc.

Roles of pyroptosis in intervertebral disc degeneration.

Intervertebral disc degeneration (IDD), the key pathological process in low back pain, is characterized by chronic inflammation and progressive cell death. Pyroptosis is a type of pro-inflammatory programmed necrosis mediated by inflammasomes that is dependent on the gasdermin family of proteins. An in-depth study of the pathological mechanisms of IDD has revealed that pyroptosis plays an important role in its occurrence and development. The molecular characteristics and activation signaling mechanisms of pyroptosis are reviewed in this paper. Moreover, the specific roles of pyroptosis in IDD pathology are outlined and various targeted drugs for its treatment are highlighted.

A Study Based on Network Pharmacology Decoding the Multi-Target Mechanism of Duhuo Jisheng Decoction for the Treatment of Intervertebral Disc Degeneration.

Intervertebral disc degeneration (IDD) poses a grim public health impact. Duhuo Jisheng Decoction (DJD), a traditional Chinese medicine formula, has recently received significant attention for its efficacy and safety in treating IDD. However, the pathological processes of IDD in which DJD interferes and molecular mechanism involved are poorly understood, which brings difficulties to the clinical practice of DJD for the treatment of IDD. This study systematically investigated the underlying mechanism of DJD treatment of IDD. Network pharmacology approaches were employed, integrating molecular docking and random walk with restart (RWR) algorithm, to identify key compounds and targets for DJD in the treatment of IDD. Bioinformatics approaches were used to further explore the biological insights in DJD treatment of IDD. The analysis identifies AKT1, PIK3R1, CHUK, ALB, TP53, MYC, NR3C1, IL1B, ERBB2, CAV1, CTNNB1, AR, IGF2, and ESR1 as key targets. Responses to mechanical stress, oxidative stress, cellular inflammatory responses, autophagy, and apoptosis are identified as the critical biological processes involved in DJD treatment of IDD. The regulation of DJD targets in extracellular matrix components, ion channel regulation, transcriptional regulation, synthesis and metabolic regulation of reactive oxygen products in the respiratory chain and mitochondria, fatty acid oxidation, the metabolism of Arachidonic acid, and regulation of Rho and Ras protein activation are found to be potential mechanisms in disc tissue response to mechanical stress and oxidative stress. MAPK, PI3K/AKT, and NF-κB signaling pathways are identified as vital signaling pathways for DJD to treat IDD. Quercetin and Kaempferol are assigned a central position in the treatment of IDD. This study contributes to a more comprehensive understanding of the mechanism of DJD in treating IDD. It provides a reference for applying natural products to delay the pathological process of IDD.

Application of critical pathway of integrative medical service for lumbar herniated nucleus pulposus: A protocol for single-centered prospective observational study.

BACKGROUND: Lumbar herniated nucleus pulposus (L-HNP) is a condition in which fibroblasts escape due to degenerative changes or external forces in the intervertebral disc, causing neurological symptoms by compressing the dura mater or nerve root. OBJECTIVES: The purpose of this study is to analyze and compare the effectiveness, economic feasibility, and safety of using an integrated medical service critical pathway (CP) in L-HNP patients. METHODS: This single-center prospective observational study will be performed at Kyung Hee University Medicine Hospital and Kyung Hee University Korean Medicine Hospital. The inclusion criteria are a diagnosis of L-HNP on magnetic resonance imaging or computed tomography scans, age under 80 years, a visual analog scale score of 7 or higher for either lower back pain or lower extremity pain. The included 102 participants will be classified into 6 groups (n = 17 in each group): CP application with conservative treatment; CP application with open discectomy; CP application with intrabody fusion; conservative treatment without CP application; open discectomy without CP application; and interbody fusion without CP application. We will collect data on the visual analog scale, ODI, SF-36, and EQ-5D-3L scores; number of admission days; medical staff satisfaction; patients health service satisfaction; waiting time for consultations; use of pain relievers; and CP application and completion rates. CONCLUSION: In future, this study is expected to serve as a basis for follow-up studies on the development and application of CPs in integrated medical services for various diseases, including lumbar herniated nucleus pulposus.

N-Acetylcysteine-Derived Carbon Dots for Free Radical Scavenging in Intervertebral Disc Degeneration.

Intervertebral disc degeneration (IVDD) is associated with oxidative stress induced reactive oxygen species (ROS) dynamic equilibrium disturbance. Nanozymes, as nanomaterials with enzyme-like activity, can regulate intro-cellular ROS levels. In this study, a new carbon dots nanozyme, N-acetylcysteine-derived carbon dots (NAC-CDs), is developed and proved to be an ideal antioxidant and anti-senescent agent in IVDD management. The results confirmed the NAC-CDs have satisfactory biocompatibility and strong superoxide dismutase (250 U mg-1 ), catalase, glutathioneperoxidase-like activity, and total antioxidant capacity. Then, the powerful free radical scavenging and antioxidant ability of NAC-CDs are demonstrated in vitro as observing the reduced ROS in H2 O2 induced senescent nucleus pulposus cells (NPCs), in which the elimination efficiency of toxic ROS is more than 90%. NAC-CDs also maintained mitochondrial homeostasis and suppressed cellular senescence, subsequently inhibited the expression of inflammatory factors in NPCs. In vivo, evaluations of imaging and tissue morphology assessments suggested that disc height index, magnetic resonance imaging grade and histological score are significantly improved from the degenerative models when NAC-CDs is applied. In conclusion, the study developed a novel carbon dots nanozyme, which efficiently rescues IVDD from ROS induced NPCs senescence and provides a potential strategy in management of IVDD in clinic.

The effect of microRNA-663b in the inhibition of interleukin-1-induced nucleus pulposus cell apoptosis and inflammatory response.

The aim of this study was to explore the role and pathological mechanism of microRNA-663b in interleukin-1beta (IL-1ß)-induced inflammation and apoptosis of nucleus pulposus cells. First, the best concentration and time to construct the nucleus pulposus cell inflammation model was screen out. Overexpression or inhibition of miR-663b expression was performed by adding microRNA-663b mimic or microRNA-663b inhibitor. 293T cells were transfected according to experimental requirements. The luciferase activity of each group was detected to analyze the targeted regulation of microRNA-663b on interleukin-1 receptor (IL1R1). Compared with the mimic negative control (NC) group, the expression of inflammatory factors in the microRNA-663b overexpression group was inhibited (P<0.05), and the expression of type 2 collagen and polysaccharide protein increased (P<0.05), and the apoptosis of nucleus pulposus cells was inhibited (P<0.01), and the number of TUNEL-positive cells decreased significantly (P<0.01), and the microRNA and protein expression of IL1R1, the ratio of P-P65/P65 and phospho-nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (P-IκBα)/nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) protein expression were significantly decreased (P<0.05). The expression of inflammatory factors in the miR-663b inhibitor group was significantly higher than that in the inhibitor NC group (P<0.01), and the expression of type 2 collagen and polysaccharide protein was significantly decreased (P<0.01), and the number of apoptosis cells and TUNEL staining positive cells increased (p<0.01). The expression of IL1R1 gene and protein was significantly increased (P<0.01). The ratio of P-P65/P65 and P-IκBα/IκBα protein expression increased (P<0.05). IL1R1 is a downstream target gene of microRNA-663b. MicroRNA-663b may down-regulate the expression of IL1R1 at the transcriptional level by targeting IL1R1, inhibit the inflammatory response of nucleus pulposus cells, and slow down the degeneration of nucleus pulposus cells.

Role of lncRNA MAGI2-AS3 in lipopolysaccharide-induced nucleus pulposus cells injury by regulating miR-374b-5p/interleukin-10 axis.

BACKGROUND: Intervertebral disc degeneration (IDD) is a pathological process that occurs during the natural aging of intervertebral discs. Accumulating evidence suggests that noncoding RNAs (ncRNAs), including microRNAs and long ncRNAs (lncRNAs), participate in the pathogenesis and development of IDD. Herein, we examined the role of lncRNA MAGI2-AS3 in the pathogenic mechanism of IDD. MATERIAL AND METHODS: To develop an IDD in vitro model, we treated human nucleus pulposus (NP) cells with lipopolysaccharide (LPS). Aberrant levels of lncRNA MAGI2-AS3, miR-374b-5p, interleukin (IL)-10 and extracellular matrix (ECM)-related proteins in NP cells were examined using reverse transcription-quantitative PCR and western blot analysis. LPS-induced NP cell injury and inflammatory response were confirmed using the MTT assay, flow cytometry, Caspase3 activity, and enzyme-linked immunosorbent assay. Dual-luciferase reporter assay and rescue experiments were performed to confirm targets between lncRNA MAGI2-AS3 and miR-374b-5p or miR-374b-5p and IL-10. RESULTS: LPS-induced NP cells exhibited low levels of lncRNA MAGI2-AS3 and IL-10 expression, along with high miR-374b-5p expression. miR-374b-5p was a target of lncRNA MAGI2-AS3 and IL-10. LncRNA MAGI2-AS3 ameliorated injury, inflammatory response, and ECM degradation in LPS-treated NP cells by downregulating miR-374b-5p to upregulate IL-10 expression. CONCLUSIONS: LncRNA MAGI2-AS3 increased IL-10 expression levels by sponging miR-374b-5p, which, in turn, alleviated LPS-triggered decreased NP cell proliferation and increased apoptosis, inflammatory response, and ECM degradation. Therefore, lncRNA MAGI2-AS3 may be a potential therapeutic target for IDD.

The deubiquitinase USP11 ameliorates intervertebral disc degeneration by regulating oxidative stress-induced ferroptosis via deubiquitinating and stabilizing Sirt3.

Increasing studies have reported that intervertebral disc degeneration (IVDD) is the main contributor and independent risk factor for low back pain (LBP), it would be, therefore, enlightening that investigating the exact pathogenesis of IVDD and developing target-specific molecular drugs in the future. Ferroptosis is a new form of programmed cell death characterized by glutathione (GSH) depletion, and inactivation of the regulatory core of the antioxidant system (glutathione system) GPX4. The close relationship of oxidative stress and ferroptosis has been studied in various of diseases, but the crosstalk between of oxidative stress and ferroptosis has not been explored in IVDD. At the beginning of the current study, we proved that Sirt3 decreases and ferroptosis occurs after IVDD. Next, we found that knockout of Sirt3 (Sirt3-/-) promoted IVDD and poor pain-related behavioral scores via increasing oxidative stress-induced ferroptosis. The (immunoprecipitation coupled with mass spectrometry) IP/MS and co-IP demonstrated that USP11 was identified to stabilize Sirt3 via directly binding to Sirt3 and deubiquitinating Sirt3. Overexpression of USP11 significantly ameliorate oxidative stress-induced ferroptosis, thus relieving IVDD by increasing Sirt3. Moreover, knockout of USP11 in vivo (USP11-/-) resulted in exacerbated IVDD and poor pain-related behavioral scores, which could be reversed by overexpression of Sirt3 in intervertebral disc. In conclusion, the current study emphasized the importance of the interaction of USP11 and Sirt3 in the pathological process of IVDD via regulating oxidative stress-induced ferroptosis, and USP11-mediated oxidative stress-induced ferroptosis is identified as a promising target for treating IVDD.

Dynamic changes of enhancer and super enhancer landscape in degenerated nucleus pulposus cells.

Inflammatory cascade and extracellular matrix remodeling have been identified as pivotal pathological factors in the progression of intervertebral disc degeneration (IDD), but the mechanisms underlying the aberrant activation of transcription during nucleus pulposus (NP) cell degeneration remain elusive. Super-enhancers (SEs) are large clusters of adjacent lone enhancers, which control expression modes of cellular fate and pathogenic genes. Here, we showed that SEs underwent tremendous remodeling during NP cell degeneration and that SE-related transcripts were most abundant in inflammatory cascade and extracellular matrix remodeling processes. Inhibition of cyclin-dependent kinase 7, a transcriptional kinase-mediated transcriptional initiation in trans-acting SE complex, constricted the transcription of inflammatory cascades, and extracellular matrix remodeling-related genes such as IL1ß and MMP3 in NP cells, meanwhile, also restrained the transcription of Mmp16, Tnfrsf21, and Il11ra1 to retard IDD in rats. In summary, our findings clarify SEs control the transcription of genes associated with inflammatory cascade and extracellular matrix remodeling during NP cell degeneration and identify inhibition of the cyclin-dependent kinase 7, required for SE-mediated transcriptional activation, as a therapeutic option for IDD.

Stiff Substrate Induces Nucleus Pulposus Cell Ferroptosis via YAP and N-Cadherin Mediated Mechanotransduction.

Increased tissue stiffness is associated with various pathological processes, such as fibrosis, inflammation, and aging. The matrix stiffness of the nucleus pulposus (NP) tissues increases gradually during intervertebral disc degeneration (IDD), while the mechanism through which NP cells sense and react to matrix stiffness remains unclear. In this study, the results indicate that ferroptosis is involved in stiff substrate-induced NP cell death. The expression of acyl-CoA synthetase long-chain family member 4 (ACSL4) increases in NP cells of the stiff group, which mediates lipid peroxidation and ferroptosis in NP cells. In addition, stiff substrate activates the hippo signaling cascade and induces the nuclear translocation of yes-associated protein (YAP). Interestingly, inhibition of YAP is efficient to reverse the increase of ACSL4 expression caused by matrix stiffness. Furthermore, stiff substrate suppresses the expression of N-cadherin in NP cells. N-cadherin overexpression can inhibit YAP nuclear translocation via the formation of the N-cadherin/ß-catenin/YAP complex, and reverse matrix stiffness-induced ferroptosis in NP cells. Finally, the effects of YAP inhibition and N-cadherin overexpression on IDD progression are further illustrated in animal models. These findings reveal a new mechanism of mechanotransduction in NP cells, providing novel insights into the development of therapies for the treatment of IDD.