Nucleus Pulposus Tissue Research Articles

445 Lumbar Intervertebral Disc mRNA Sequencing Identifies the Regulatory Pathway in Young Adult Patients with Disc Degeneration

INTRODUCTION: The process of intervertebral disc degeneration disease (DDD) is thought to be an age-related phenomenon that includes inflammation and cell apoptosis leading to decreased proteoglycan synthesis with resultant disc dehydration and loss of disc height. It has been reported that 42% of young adults have DDD characterized by a signal change on MRI, it seems beneficial to examine young adults to clarify the process of DDD. METHODS: We performed next generation RNA sequencing on 9 nucleus pulposus (NP) of the lumbar intervertebral disc from young adults (<40 years age, n = 4) and old adults (>40 years of age, n = 5) patients. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses were performed to identify the biological functions of differentially expressed mRNAs. RESULTS: A total of 98 genes were found to be significantly upregulated in young adults NP tissue compared to old adults NP tissue. These upregulated mRNAs were primarily involved in signal peptide, collagen, and extracellular matrix organization. Among the top upregulated genes such as LGALS1, COL6A2 and PTMS may have the key regulatory functions related to early age disc degeneration. In addition, we found 346 genes were significantly downregulated in young adults NP tissue compared to old adults NP tissue. They were primarily involved in glycoprotein, membrane, and immunoglobulin-like fold. CONCLUSION: This investigation provides a comprehensive overview of mRNA expression in the nucleus pulposus of intervertebral disc tissue in young adult patients compare with old adult patients. By applying computational analyses to our large dataset of human clinical specimens, we have been able to identify candidate gene regulatory networks that act in early age disc degeneration.

Restoring the dampened expression of the core clock molecule BMAL1 protects against compression-induced intervertebral disc degeneration

The circadian clock participates in maintaining homeostasis in peripheral tissues, including intervertebral discs (IVDs). Abnormal mechanical loading is a known risk factor for intervertebral disc degeneration (IDD). Based on the rhythmic daily loading pattern of rest and activity, we hypothesized that abnormal mechanical loading could dampen the IVD clock, contributing to IDD. Here, we investigated the effects of abnormal loading on the IVD clock and aimed to inhibit compression-induced IDD by targeting the core clock molecule brain and muscle Arnt-like protein-1 (BMAL1). In this study, we showed that BMAL1 KO mice exhibit radiographic features similar to those of human IDD and that BMAL1 expression was negatively correlated with IDD severity by systematic analysis based on 149 human IVD samples. The intrinsic circadian clock in the IVD was dampened by excessive loading, and BMAL1 overexpression by lentivirus attenuated compression-induced IDD. Inhibition of the RhoA/ROCK pathway by Y-27632 or melatonin attenuated the compression-induced decrease in BMAL1 expression. Finally, the two drugs partially restored BMAL1 expression and alleviated IDD in a diurnal compression model. Our results first show that excessive loading dampens the circadian clock of nucleus pulposus tissues via the RhoA/ROCK pathway, the inhibition of which potentially protects against compression-induced IDD by preserving BMAL1 expression. These findings underline the importance of the circadian clock for IVD homeostasis and provide a potentially effective therapeutic strategy for IDD.

Circular RNA hsa_circ_0083756 promotes intervertebral disc degeneration by sponging miR-558 and regulating TREM1 expression.

ObjectivesIntervertebral disc degeneration (IVDD) is a leading cause of low back pain. Circular RNAs (circRNAs) have been demonstrated to exert vital functions in IVDD. However, the role and mechanism of hsa_circ_0083756 in the development of IVDD remain unclear.Materials and methodsRT‐qPCR was performed to detect expressions of hsa_circ_0083756, miR‐558 and TREM1 in nucleus pulposus (NP) tissues and cells. CCK8 assay, flow cytometry, TUNEL assay, RT‐qPCR and WB were used to clarify the roles of hsa_circ_0083756 in NP cells proliferation and extracellular matrix (ECM) formation. Bioinformatics analyses, dual‐luciferase reporter gene experiment, RNA immunoprecipitation (RIP) assay and FISH assay were performed to predict and verify the targeting relationship between hsa_circ_0083756 and miR‐558, as well as that between miR‐558 and TREM1. Ultimately, the effect of hsa_circ_0083756 on IVDD was tested through anterior disc‐puncture IVDD animal model in rats.Resultshsa_circ_0083756 was upregulated in degenerative NP tissues and cells. In vitro loss‐of‐function and gain‐of‐function studies suggested that hsa_circ_0083756 knockdown promoted, whereas hsa_circ_0083756 overexpression inhibited NP cells proliferation and ECM formation. Mechanistically, hsa_circ_0083756 acted as a sponge of miR‐558 and subsequently promoted the expression of TREM1. Furthermore, in vivo study indicated that silencing of hsa_circ_0083756 could alleviate IVDD in rats.Conclusionshsa_circ_0083756 promoted IVDD via targeting the miR‐558/TREM1 axis, and hsa_circ_0083756 may serve as a potential therapeutic target for the treatment of IVDD.

SERPINA1 is a hub gene associated with intervertebral disc degeneration grade and affects the nucleus pulposus cell phenotype through the ADIRF-AS1/miR-214-3p axis

Long noncoding RNAs (lncRNAs) and miRNAs have been reported to participate in intervertebral disc degeneration (IDD) progression. However, the key lncRNA-miRNA axis and its corresponding affected hub genes in IDD remain unknown. In this study, weighted gene coexpression network analysis (WGCNA) was first used to determine the key gene cluster and hub genes implicated in IDD progression. The expression levels of ADIRF-AS1, miR-214-3p, and SERPINA1 in nucleus pulposus (NP) tissues were detected. The ADIRF-AS1/miR-214-3p/SERPINA1 axis was identified, and its effects on the proliferation, senescence, and apoptosis of NP cells were investigated in vitro and in vivo. SERPINA1 overexpression in NP cells promoted cell viability and inhibited cell apoptosis and senescence. Moreover, SERPINA1 regulated the IDD grade in rat models. The lncRNA ADIRF-AS1 was downregulated in high-grade degeneration NP tissues and positively correlated with SERPINA1. ADIRF-AS1 overexpression attenuated cellular degenerative changes in NP cells. miR-214-3p directly bound to SERPINA1 and ADIRF-AS1 and negatively regulated ADIRF-AS1 expression. miR-214-3p inhibition exerted similar effects on cellular degenerative changes in NP cells to SERPINA1 or ADIRF-AS1 overexpression. Furthermore, miR-214-3p overexpression partially reversed the effects of ADIRF-AS1 overexpression. Collectively, these data suggest that ADIRF-AS1 overexpression could mitigate IDD by binding to miR-214-3p to upregulate SERPINA1. Additional studies (especially those using an axial loading-induced IDD animal model) will be needed to further validate the role of the ADIRF-AS1/miR-214-3p/SERPINA1 signaling axis in IDD progression.

Long intergenic non-coding RNA -00917 regulates the proliferation, inflammation, and pyroptosis of nucleus pulposus cells via targeting miR-149-5p/NOD-like receptor protein 1 axis

ABSTRACT Intervertebral disc degeneration (IDD) has caused great trouble in people’s lives. Dysregulated long noncoding RNAs (lncRNAs) are closely linked to IDD progression. Our study aims to analyze the role of LINC00917 in the progression of IDD. Forty nucleus pulposus (NP) IDD tissues and 40 NP tissues of intervertebral discs without apparent degeneration were collected. TBHP was used to induce IDD. Cell proliferation was measured using the MTT and EdU assays. Pyroptosis was detected using flow cytometry. RT-qPCR and Western blot assays were performed to determine mRNA, miRNA, and protein expression. Dual-luciferase reporter and RNA pull-down assays were performed to verify the relationship between LINC00917 or NLRP1 and miR-149-5p. LINC00917 expression was enhanced in TBHP-treated nucleus pulposus cells (NPCs). The knockdown of LINC00917 promoted proliferation and inhibited cytotoxicity, inflammatory response, and pyroptosis of NPCs. LINC00917 functions as a sponge for miR-149-5p. Having silenced miR-149-5p, the effects of LINC00917 knockdown on NPC proliferation and inflammation-induced pyroptosis were alleviated. NLRP1 overexpression induced cellular dysfunction and pyroptosis of NPCs. LINC00917 knockdown restored NPC cellular functions and inhibited IDD progression by modulating the miR-149-5p/NLRP1 axis.

Sa12b-Modified Functional Self-Assembling Peptide Hydrogel Enhances the Biological Activity of Nucleus Pulposus Mesenchymal Stem Cells by Inhibiting Acid-Sensing Ion Channels

Various hydrogels have been studied for nucleus pulposus regeneration. However, they failed to overcome the changes in the acidic environment during intervertebral disc degeneration. Therefore, a new functionalized peptide RAD/SA1 was designed by conjugating Sa12b, an inhibitor of acid-sensing ion channels, onto the C-terminus of RADA16-I. Then, the material characteristics and biocompatibility of RAD/SA1, and the bioactivities and mechanisms of degenerated human nucleus pulposus mesenchymal stem cells (hNPMSCs) were evaluated. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) confirmed that RAD/SA1 self-assembling into three-dimensional (3D) nanofiber hydrogel scaffolds under acidic conditions. Analysis of the hNPMSCs cultured in the 3D scaffolds revealed that both RADA16-I and RAD/SA1 exhibited reliable attachment and extremely low cytotoxicity, which were verified by SEM and cytotoxicity assays, respectively. The results also showed that RAD/SA1 increased the proliferation of hNPMSCs compared to that in culture plates and pure RADA16-I. Quantitative reverse transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and western blotting demonstrated that the expression of collagen I was downregulated, while collagen II, aggrecan, and SOX-9 were upregulated. Furthermore, Ca2+ concentration measurement and western blotting showed that RAD/SA1 inhibited the expression of p-ERK through Ca2+-dependent p-ERK signaling pathways. Therefore, the functional self-assembling peptide nanofiber hydrogel designed with the short motif of Sa12b could be used as an excellent scaffold for nucleus pulposus tissue engineering. Moreover, RAD/SA1 exhibits great potential applications in the regeneration of mildly degenerated nucleus pulposus.

Sirtuin 1 participates in intervertebral disc degeneration via the nicotinamide phosphoribosyl transferase/nicotinamide adenine dinucleotide/sirtuin 1 pathway responsible for regulating autophagy of nucleus pulposus cells.

Disc degeneration is the main cause of discogenic low back pain, disc herniation, degenerative stenosis of spinal canal, lumbar spondylolisthesis and other diseases. In the process of intervertebral disc degeneration, water and extracellular matrix of nucleus pulposus tissues are lost, so the normal tension in the intervertebral disc cannot be maintained, which worsens the living environment of nucleus pulposus cells. Low back pain (LBP), with a high incidence rate of disability, has become an increasing health concern and a social and economic problem. The present study aimed to analyze the action mechanisms of nicotinamide phosphoribosyl transferase (Nampt) and sirtuin 1 (SIRT1) in intervertebral disc degeneration (IVDD). In total 26 patients with lumbar disc herniation who had surgical resection at The Third Affiliated Hospital of Jinzhou Medical University were recruited as the experimental group and their degenerative nucleus pulposus (DNP) tissues of intervertebral disc were collected. In addition, nucleus pulposus tissues of intervertebral disc were collected from 20 patients with burst fracture of lumbar spine at the same hospital (control). Nucleus pulposus cells from primary culturing were separated for subsequent experimentation. LC3 II/I, beclin-1, SIRT1 and NAMPT mRNA and protein expression levels were determined using reverse transcription-quantitative PCR and western blotting, respectively. Nicotinamide adenine dinucleotide (NAD) contents in nucleus pulposus cells was determined by NAD assay kit. The mRNA and protein expression levels of SIRT1 in DNP tissues were reduced compared with the control tissues and decreased with increasing disease severity. The expression of autophagy-associated LC3 II/I and beclin-1 in DNP tissues was reduced compared with control tissues. SIRT1 regulated the LC3 II/I and beclin-1 expression levels in nucleus pulposus cells. Treatment with resveratrol and inhibitor of SIRT1 showed that Nampt/NAD+/SIRT1 pathway participated in the process of IVDD by regulating autophagy of nucleus pulposus cells. SIRT1 serves a role in the process of IVDD through Nampt/NAD+/SIRT1 pathway that regulates autophagy of nucleus pulposus cells. SIRT1 may become a biological target for the treatment of IVDD.

Rac1 regulates nucleus pulposus cell degeneration by activating the Wnt/β-catenin signaling pathway and promotes the progression of intervertebral disc degeneration.

Nucleus pulposus cell (NPC) dysfunction is considered as an important event related to intervertebral disc degeneration (IVDD). In the present study, tandem mass spectrometry (TMT) was used to detect total protein expression of nucleus pulposus (NP) in patients with IVDD and healthy controls. Bioinformatic analysis was performed to identify differentially expressed proteins that may be involved in the degeneration of NP. The results show that Rac1 may be a key protein involved in the degeneration of NP via Wnt/β-catenin pathway activation. We investigated the influence of Rac1 on IVDD degeneration and associated mechanisms. Rac1 expression increased in interleukin (IL)-1β-stimulated human NPCs, consistent with the results of TMT. The Rac1 inhibitor NSC23766 alleviated the degeneration of NPCs in vitro. Furthermore, Rac1 activated Wnt/β-catenin signaling, and the inhibition of this pathway significantly ameliorated the Rac1-mediated degenerative phenotype. NSC23766 exerted protective effects on IVDD in a puncture rat model. Taken together, these data suggest that Rac1 inhibition can delay NPC degeneration, probably through the regulation of the Wnt/β-catenin pathway. This study has the potential to advance understanding of the mechanism of occurrence of degenerative NP tissues and to provide novel strategies for slowing IVDD progression.

Cytosolic escape of mitochondrial DNA triggers cGAS-STING-NLRP3 axis-dependent nucleus pulposus cell pyroptosis

Low back pain (LBP) is a major musculoskeletal disorder and the socioeconomic problem with a high prevalence that mainly involves intervertebral disc (IVD) degeneration, characterized by progressive nucleus pulposus (NP) cell death and the development of an inflammatory microenvironment in NP tissue. Excessively accumulated cytosolic DNA acts as a damage-associated molecular pattern (DAMP) that is monitored by the cGAS-STING axis to trigger the immune response in many degenerative diseases. NLRP3 inflammasome-dependent pyroptosis is a type of inflammatory programmed death that promotes a chronic inflammatory response and tissue degeneration. However, the relationship between the cGAS-STING axis and NLRP3 inflammasome-induced pyroptosis in the pathogenesis of IVD degeneration remains unclear. Here, we used magnetic resonance imaging (MRI) and histopathology to demonstrate that cGAS, STING, and NLRP3 are associated with the degree of IVD degeneration. Oxidative stress induced cGAS-STING axis activation and NLRP3 inflammasome-mediated pyroptosis in a STING-dependent manner in human NP cells. Interestingly, the canonical morphological and functional characteristics of mitochondrial permeability transition pore (mPTP) opening with the cytosolic escape of mitochondrial DNA (mtDNA) were observed in human NP cells under oxidative stress. Furthermore, the administration of a specific pharmacological inhibitor of mPTP and self-mtDNA cytosolic leakage effectively reduced NLRP3 inflammasome-mediated pyroptotic NP cell death and microenvironmental inflammation in vitro and degenerative progression in a rat disc needle puncture model. Collectively, these data highlight the critical roles of the cGAS-STING-NLRP3 axis and pyroptosis in the progression of IVD degeneration and provide promising therapeutic approaches for discogenic LBP.

Bardoxolone Methyl Ameliorates Compression-Induced Oxidative Stress Damage of Nucleus Pulposus Cells and Intervertebral Disc Degeneration Ex Vivo.

Intervertebral disc degeneration (IDD) is the main cause of low back pain, and little is known about its molecular and pathological mechanisms. According to reports, excessive compression is a high-risk factor for IDD; compressive stress can induce oxidative stress in nucleus pulposus (NP) cells during IDD progression that, in turn, promotes cell apoptosis and extracellular matrix (ECM) degradation. Currently, NP tissue engineering is considered a potential method for IDD treatment. However, after transplantation, NP cells may experience oxidative stress and induce apoptosis and ECM degradation due to compressive stress. Therefore, the development of strategies to protect NP cells under excessive compressive stress, including pretreatment of NP cells with antioxidants, has important clinical significance. Among the various antioxidants, bardoxolone methyl (BARD) is used to protect NP cells from damage caused by compressive stress. Our results showed that BARD can protect the viability of NP cells under compression. BARD inhibits compression-induced oxidative stress in NP cells by reducing compression-induced overproduction of reactive oxygen species (ROS) and malondialdehyde. Thus, BARD has a protective effect on the compression-induced apoptosis of NP cells. This is also supported by changes in the expression levels of proteins related to the mitochondrial apoptosis pathway. In addition, BARD can inhibit ECM catabolism and promote ECM anabolism in NP cells. Finally, the experimental results of the mechanism show that the activation of the Nrf2 signaling pathway participates in the protection induced by BARD in compressed NP cells. Therefore, to improve the viability and biological functions of NP cells under compression, BARD should be used during transplantation.

Cholesterol Induces Pyroptosis and Matrix Degradation via mSREBP1-Driven Endoplasmic Reticulum Stress in Intervertebral Disc Degeneration.

Intervertebral disc degeneration (IDD) is closely associated with low back pain, but its underlying mechanism remains unclear. Cholesterol is an essential nutrient in mammalian cells. Alterations in cholesterol levels lead to impairments in cell physiology, such as cell proliferation and signal transduction. Previous clinical studies demonstrated that hypercholesterolemia could be a potential risk factor for IDD, but how cholesterol induces IDD remains unknown. The current study aimed to explore the regulatory role of cholesterol in IDD development and the potential underlying mechanisms. It was found that different forms of cholesterol levels were elevated in degenerative nucleus pulposus (NP) tissues in both humans and Sprague–Dawley rats. Rats fed a high cholesterol diet (HCD) exhibited degenerative features in the lumbar intervertebral disc compared with those fed a standard diet. Interestingly, this effect could be abolished by cholesterol-lowering drug atorvastatin. In NP cells treated with TNF-α and IL-1β, a significantly higher level of cholesterol was observed. These results suggested a pivotal role of cholesterol in the progression of IDD. We also observed accelerated pyroptosis in NP cells and extracellular matrix (ECM) degradation in the rat NP cells treated with exogenous cholesterol. We further demonstrated that endoplasmic reticulum stress was responsible for cholesterol-induced pyroptosis and ECM degradation. Moreover, RNA-seq analysis revealed that the mature form of SREBP1 (mSREBP1), an important regulator of lipid metabolism, is involved in regulating endoplasmic reticulum stress in knockdown experiments. In conclusion, this study demonstrated that cholesterol could induce pyroptosis in NP cells and ECM degradation by activating endoplasmic reticulum stress through stimulating mSREBP1 in IDD.

Electrical Stimulation-Mediated Tissue Healing in Porcine Intervertebral Disc Under Mechanically Dynamic Organ Culture Conditions.

Porcine intervertebral discs (IVDs) were excised and then drilled to simulate degeneration before being electrically stimulated for 21 days while undergoing mechanical loading. The discs were then analyzed for gene expression and morphology to assess regeneration. The purpose of this study was to investigate the effectiveness of the electrical stimulation of IVD treatment as an early intervention method in halting the progression of degenerative disc disease using an ex-vivo porcine model. Treatments for degenerative disc disease are limited in their efficacy and tend to treat the symptoms of the disease rather than repairing the degenerated disc itself. There is a dire need for an early intervention treatment that not only halts the progression of the disease but contributes to reviving the degenerated disc. Lumbar IVDs were extracted from a mature pig within 1 hour of death and were drilled with a 1.5 mm bit to simulate degenerative disc disease. Four IVDs at a time were then cultured in a dynamic bioreactor system under mechanical loading for 21 days, two with and two without the electrical stimulation treatment. The IVDs were assessed using histological analysis, magnetic resonance imaging, and quantitative reverse transcriptase polymerase chain reaction to quantify the effectiveness of the treatment on the degenerated discs. IVDs with electrical stimulation treatment exhibited extensive annular regeneration and prevented herniation of the nucleus pulposus (NP). In contrast, the untreated group of IVDs were unable to maintain tissue integrity and exhibited NP herniation through multiple layers of the annulus fibrosus. Gene expression showed an increase of extracellular matrix markers and antiinflammatory cytokine interleukin-4 (IL-4), while decreasing in pro-inflammatory markers and pain markers in electrically stimulated IVDs when compared to the untreated group. The direct electrical stimulation application in NP of damaged IVDs can be a viable option to regenerate damaged NP and annulus fibrosus tissues.

LncRNA nuclear receptor subfamily 2 group F member 1 antisense RNA 1 (NR2F1-AS1) aggravates nucleus pulposus cell apoptosis and extracellular matrix degradation

ABSTRACT Emerging reports uncover that long noncoding RNAs (lncRNAs) help regulate intervertebral disc degeneration (IVDD). Here, we probe the function of lncRNA nuclear receptor subfamily 2 group F member 1 antisense RNA 1 (NR2F1-AS1) in IVDD. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was applied to verify the expression of NR2F1-AS1 and miR-145-5p in nucleus pulposus (NP) tissues from IVDD patients or NP cells dealt with IL-1β or TNF-α. Flow cytometry or the TdT-mediated dUTP nick end labeling (TUNEL) assay was performed to validate the apoptosis of NP cells with selective regulation of NR2F1-AS1 and miR-145-5p. ECM-related genes, FOXO1, Bax, and Bcl2 were evaluated by qRT-PCR or Western blot (WB). The targeted relationships between NR2F1-AS1 and miR-145-5p, miR-145-5p and FOXO1 were testified by the dual-luciferase reporter assay and the RNA immunoprecipitation (RIP) assay. Our outcomes substantiated that NR2F1-AS1 was up-regulated, while miR-145-5p was down-regulated in intervertebral disc tissues of IVDD patients or NP cells treated with IL-1β or TNF-α. Besides, overexpressing NR2F1-AS1 intensified ECM degradation and NP cell apoptosis induced by IL-1β, while knocking down NR2F1-AS1 or up-regulating miR-145-5p reversed IL-1β-mediated effects in NP cells. Meanwhile, NR2F1-AS1 choked miR-145-5p and abated its effects in NP cells. This study confirms that NR2F1-AS1 modulates IVDD progression by up-regulating the FOXO1 pathway through the sponge of miR-145-5p as a competitive endogenous RNA (ceRNA).

EZH2 upregulates the expression of MAPK1 to promote intervertebral disc degeneration via suppression of miR-129-5p.

This study was designed to verify whether enhancer of zeste homolog 2 (EZH2) affects intervertebral disc degeneration (IVDD) development through regulation of microRNA (miR)-129-5p/MAPK1. Initially, we collected lumbar nucleus pulposus (NP) tissue samples from patients with juvenile idiopathic scoliosis (n = 14) and IVDD (n = 34). We measured the expression of related genes in clinical IVDD tissues and a lipopolysaccharide (LPS)-induced NP cell model. After loss- and gain-of-function assays, NP cell proliferation and senescence were examined. The targeting relationship between miR-129-5p and MAPK1 was explored by dual luciferase reporter gene and RNA immunoprecipitation (RIP) assays. The enrichment of EZH2 and H3K27me3 in miR-129-5p promoter was verified by chromatin immunoprecipitation (ChIP). Finally, an IVDD rat model was established to test the effects of transduction with lentiviral vector carrying miR-129-5p agomir and/or oe-EZH2 in vivo. miR-129-5p was underexpressed, and EZH2 and MAPK1 levels were overexpressed in lumbar nucleus pulposus from human IVDD patients and in LPS-induced NP cells. miR-129-5p overexpression or silencing of MAPK1 promoted proliferation of NP cells, while inhibiting their senescence. EZH2 inhibited miR-129-5p through H3K27me3 modification in the miR-129-5p promoter. miR-129-5p could target the downregulation of MAPK1 expression. EZH2 overexpression increased the release of inflammatory factors and cell senescence factors, which was reversed by miR-129-5p agomir in vivo. Taken together, EZH2 inhibits miR-129-5p through H3K27me3 modification, which upregulates MAPK1, thereby promoting the development of IVDD.

In vitro and in vivo effects of hyperglycemia and diabetes mellitus on nucleus pulposus cell senescence.

Diabetes mellitus contributes to intervertebral disc degeneration. Nucleus pulposus cell senescence plays an important role in intervertebral disc degeneration. However, the effects of hyperglycemia on human nucleus pulposus cells and the underlying process remains poorly understood. In the current study, we evaluated the effects of high glucose levels on human nucleus pulposus cell senescence in vitro and the effects of hyperglycemia on rat nucleus pulposus aging in vivo. Human nucleus pulposus cells were cultured in high-glucose medium (200 mM glucose) for 48 h. Senescence-associated β-galactosidase staining, western blot analysis, and enzyme-linked immunosorbent assays were performed to evaluate human nucleus pulposus cell senescence. Flow cytometry and enzyme-linked immunosorbent assays were used to evaluate reactive oxygen species and advanced glycation end-product levels. Transcriptome sequencing followed by bioinformatics analysis was used to understand the abnormal biological processes of nucleus pulposus cells cultured in high-glucose medium. Diabetes mellitus rat models were established and histopathological and immunohistochemical analysis was conducted to examine nucleus pulposus tissue senescence in vivo. Exposure to a high glucose concentration promoted human nucleus pulposus cell senescence and increased the senescence-related secretion phenotype in human nucleus pulposus cells in vitro and in rat nucleus pulposus tissue in vivo. Bioinformatics analysis showed that hub genes were involved in nucleus pulposus cell cycle activities and cell senescence. The results suggest that appropriate blood glucose control may be key to preventing intervertebral disc degeneration in diabetic patients.

A potential target gene CD63 for different degrees of intervertebral disc degeneration

Understanding molecular mechanisms of intervertebral disc degeneration (IDD) and providing a novel target for the treatment of IDD have important implications. We sought to explore a new promising gene target for the treatment of IDD. This study integrated 19,678 genes of 38 IDD patients from two gene datasets. Differentially Expressed Genes (DEGs) of annulus fibrosus were analyzed in groups with mild disc degeneration (MDD) and severe disc degeneration (SDD). We screened the hub gene through biological information technology (bioinformatic) methods. Then, we further validated the hub gene using annulus fibrosus and nucleus pulposus tissues from 12 patients with qRT-PCR. In addition, we explored its underlying molecular mechanism with GO, KEGG and GSEA. Through multiple screening bioinformatics methods, the hub gene CD63 was identified. The qRT-PCR explored that CD63 decreased significantly in SDD group compared to that in MDD group (P < 0.001). The GO, KEGG and GSEA of CD63 explored significant enrichment of the molecular features (P < 0.001), including the cellular component (Extracellular matrix, P < 0.001), the molecular function (collagen binding, P < 0.001), the biological processes (protein targeting, collagen fibril organization and platelet degranulation, P < 0.001) and the signaling pathways. Our research explored and validated a new regulatory gene, CD63 for different degrees of IDD. A new novel form of therapeutic target for IDD may be developed.

Oxidative Stress Aggravates Apoptosis of Nucleus Pulposus Cells through m6A Modification of MAT2A Pre-mRNA by METTL16.

The process of intervertebral disc degeneration (IVDD) is complex, and its mechanism is considered multifactorial. Apoptosis of oxidative stressed nucleus pulposus cells (NPCs) should be a fundamental element in the pathogenesis of IVDD. In our pilot study, we found that the expression of MAT2A decreased, and METTL16 increased in the degenerative nucleus pulposus tissues. Previous studies have shown that the balance of splicing, maturation, and degradation of MAT2A pre-mRNA is regulated by METTL16 m6A modification. In the current study, we aimed to figure out whether this mechanism was involved in the aberrant apoptosis of NPCs and IVDD. Human NPCs were isolated and cultured under oxidative stress. An IVDD animal model was established. It showed that significantly higher METTL16 expression and lower MAT2A expression were seen in either the NPCs under oxidative stress or the degenerative discs of the animal model. MAT2A was inhibited with siRNA in vitro or cycloleucine in vivo. METTL16 was overexpressed with lentivirus in vitro or in vivo. Downregulation of MAT2A or upregulation of METTL16 aggravated nucleus pulposus cell apoptosis and disc disorganization. The balance of splicing, maturation, and degradation of MAT2A pre-mRNA was significantly inclined to degradation in the NPCs with the overexpression of METTL16. Increased apoptosis of NPCs under oxidative stress could be rescued by reducing the expression of METTL16 using siRNA with more maturation of MAT2A pre-mRNA. Collectively, oxidative stress aggravates apoptosis of NPCs through disrupting the balance of splicing, maturation, and degradation of MAT2A pre-mRNA, which is m6A modified by METTL16.

Injectable decellularized nucleus pulposus tissue exhibits neuroinhibitory properties.

BackgroundChronic low back pain (LBP) is a leading cause of disability, but treatments for LBP are limited. Degeneration of the intervertebral disc due to loss of neuroinhibitory sulfated glycosaminoglycans (sGAGs) allows nerves from dorsal root ganglia to grow into the core of the disc. Treatment with a decellularized tissue hydrogel that contains sGAGs may inhibit nerve growth and prevent disc‐associated LBP.MethodsA protocol to decellularize porcine nucleus pulposus (NP) was adapted from previous methods. DNA, sGAG, α‐gal antigen, and collagen content were analyzed before and after decellularization. The decellularized tissue was then enzymatically modified to be injectable and form a gel at 37°C. Following this, the mechanical properties, microstructure, cytotoxicity, and neuroinhibitory properties were analyzed.ResultsThe decellularization process removed 99% of DNA and maintained 74% of sGAGs and 154% of collagen compared to the controls NPs. Rheology demonstrated that regelled NP exhibited properties similar to but slightly lower than collagen‐matched controls. Culture of NP cells in the regelled NP demonstrated an increase in metabolic activity and DNA content over 7 days. The collagen content of the regelled NP stayed relatively constant over 7 days. Analysis of the neuroinhibitory properties demonstrated regelled NP significantly inhibited neuronal growth compared to collagen controls.ConclusionsThe decellularization process developed here for porcine NP tissue was able to remove the antigenic material while maintaining the sGAG and collagen. This decellularized tissue was then able to be modified into a thermally forming gel that maintained the viability of cells and demonstrated robust neuroinhibitory properties in vitro. This biomaterial holds promise as an NP supplement to prevent nerve growth into the native disc and NP in vivo.

Silencing Gene-Engineered Injectable Hydrogel Microsphere for Regulation of Extracellular Matrix Metabolism Balance.

Extracellular matrix (ECM) metabolism balance is essential for maintaining tissue structure and function. However, the complex crosstalk between the ECM, resident cellular, and tissue microenvironment makes long-term maintenance of ECM metabolism balance in an abnormal microenvironment difficult to achieve. Herein, an injectable circRNA silencing-hydrogel microsphere (psh-circSTC2-lipo@MS) is constructed by grafting circSTC2 silencing genes-loaded 1,2-dioleoyl-3-trimethylammonium-propane/cholesterol/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOTAP/Chol/DOPE) cationic liposomes on methacrylated hyaluronic acid (HAMA) microspheres via amide bonds, which could silence pathological genes in nucleus pulposus (NP) cells to regulate ECM metabolism balance in the nutrient-restricted microenvironment, thereby inhibiting intervertebral disc (IVD) degeneration. HAMA microspheres prepared by microfluidics displayed good degradability, swellability, and injectability. And lipoplexes can be efficiently loaded and released for 27 d through chemical grafting. Cocultured under nutrient-restricted conditions for 72 h, psh-circSTC2-lipo@MS significantly promotes the synthesis of ECM-related proteins and inhibits the secretion of ECM catabolism-related proteases in NP cells. In the rat IVD nutrient-restricted model, local injection of psh-circSTC2-lipo@MS promotes ECM synthesis and restored NP tissue after 8 weeks. In summary, this study confirms that psh-circSTC2-lipo@MS as a safe and controllable targeted gene delivery system has great potential in regulating the ECM metabolism balance under an abnormal microenvironment.

Mechanism of Long Noncoding RNA HOTAIR in Nucleus Pulposus Cell Autophagy and Apoptosis in Intervertebral Disc Degeneration.

Objective Intervertebral disc degeneration (IDD) contributes to cervical and lumbar diseases. Long noncoding RNAs (lncRNAs) are implicated in IDD. This study explored the mechanism of lncRNA HOTAIR in IDD. Methods Normal and degenerative nucleus pulposus (NP) cells were isolated from NP tissues obtained in intervertebral disc surgery. Cell morphology was observed by immunocytochemistry staining and toluidine blue staining. NP cell markers were detected by RT-qPCR. Proliferation was detected by MTT assay. Autophagy-related proteins were detected by Western blot. Autophagosome was observed by monodansylcadaverine fluorescence staining. Apoptosis was detected by TUNEL staining and flow cytometry. si-HOTAIR and/or miR-148a inhibitor was introduced into degenerative NP cells. Binding relationships among HOTAIR, miR-148a, and PTEN were predicted and verified by dual-luciferase reporter assay and RNA pull-down. Finally, IDD rat models were established. Rat caudal intervertebral discs were assessed by HE staining. Expressions of HOTAIR, miR-148a, and PTEN were determined by RT-qPCR. Results HOTAIR was highly expressed in degenerative NP cells (p < 0.05). si-HOTAIR inhibited degenerative NP cell apoptosis and autophagy (p < 0.05). HOTAIR upregulated PTEN as a sponge of miR-148a. miR-148a was poorly expressed in degenerative NP cells. miR-148a deficiency partially reversed the inhibition of si-HOTAIR on degenerative NP cell autophagy and apoptosis (all p < 0.05). In vivo assay confirmed that si-HOTAIR impeded autophagy and apoptosis in intervertebral disc tissues, thus improving pathological injury in IDD rats (all p < 0.05). Conclusion LncRNA HOTAIR promoted NP cell autophagy and apoptosis via promoting PTEN expression as a ceRNA of miR-148a in IDD.