Degenerative Nucleus Pulposus Tissues Research Articles

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.

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

BackgroundIncreasing 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.MethodsWe 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.ResultsThe 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.ConclusionsSingle-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.

TNFα-reliant FSP1 up-regulation promotes intervertebral disc degeneration via caspase 3-dependent apoptosis

Intervertebral disc degeneration (IDD) is a common chronic inflammatory degenerative disease that causes lower back pain. However, the underlying mechanisms of IDD remain unclear. Ferroptosis suppressor protein 1 (FSP1) is a newly identified suppressor for ferroptosis. This study aims to investigate the role of FSP1 in IDD. Nucleus pulposus (NP) tissues in humans were collected and NP cells from rats were isolated to detect FSP1 expression pattern. The relationship between FSP1-mediated ferroptosis and apoptosis was identified using FSP1 inhibitor iFSP1. RNA sequencing was utilized to seek downstream molecules and related signaling pathways. Moreover, both exogenous recombinant FSP1 protein and endogenous small interfering RNA were implemented in this study to clarify the role of FSP1 in tumor necrosis factor-alpha (TNFα)-mediated NP cell apoptosis. Ultimately, the underlying mechanisms of FSP1-related signaling pathway in IDD were uncovered both in vitro and in vivo. As a result, FSP1 was up-regulated in human degenerative NP tissues and after TNFα stimulation. FSP1 inhibition by iFSP1 fails to trigger ferroptosis in NP cells while inhibiting TNFα-mediated apoptosis. Further experiments demonstrated that FSP1 was closely related to TNFα-reliant caspase 3 activation and mitochondrial damage. However, the exogenous addition of recombinant protein FSP1 does not induce cell death or intensify the efficacy of TNFα. Mechanically, FSP1 is involved in TNFα-mediated NF-κB signaling activation to accelerate the development of IDD. This study demonstrated that FSP1 promotes IDD through TNFα-reliant NF-κB signaling activation and caspase 3-dependent apoptosis. These findings suggested a novel therapeutic target for the treatment of IDD.

Adiponectin inhibits LPS-induced nucleus pulposus cell pyroptosis through the miR-135a-5p/TXNIP signaling pathway.

Pyroptosis, a newly discovered programmed cell death process, is characterized by NLRP3 inflammasome activation and pro-inflammatory mediator release. Nucleus pulposus (NP) cell pyroptosis is an important cause of intervertebral disc degeneration (IDD). Adiponectin (APN) is an adipokine and has an anti-inflammatory effect. However, whether and how APN protects against NP cell pyroptosis remains unexplored. Our results showed that human degenerated NP tissue displayed a significant increase in the protein levels of NLRP3, caspase-1 and GSDMD-N. APN expression was down-regulated in human degenerated NP tissue and NP cells challenged with lipopolysaccharide (LPS). Lentivirus-mediated overexpression of APN increased miR-135a-5p levels, decreased thioredoxin-interacting protein (TXNIP) expression and its interaction with NLRP3, and inhibited pyroptosis in human NP cells stimulated with LPS. TXNIP was identified as a direct target of miR-135a-5p. The inhibitory effects of APN on pyroptosis were reversed by pretreatment with miR-135a-5p inhibitor or lentiviral vector expressing TXNIP in LPS-treated human NP cells. In summary, these data suggest that APN restrains LPS-induced pyroptosis through the miR-135a-5p/TXNIP signaling pathway in human NP cells. Increasing APN levels could be a new approach to retard IDD.

Mechanisms of inhibition of nucleus pulposus cells pyroptosis through SDF1/CXCR4-NFkB-NLRP3 axis in the treatment of intervertebral disc degeneration by Duhuo Jisheng Decoction

Intervertebral disc degeneration (IVDD) is one of the leading causes of lower back pain and the most common health problem in the world. Inflammasomes, which is mainly caused by NLRP3, mediated nucleus pulposus pyroptosis has been discovered to be strongly related to IVDD. In addition, Duhuo Jisheng Decoction (DHJSD) has anti-inflammatory and regulatory effects on NLRP3 inflammasome, but the molecular mechanism of whether DHJSD can regulate pyroptosis through NLRP3 to treat IVDD is unclear. In this study, we used a bioinformatics way to discover the molecular mechanism of DHJSD regulation of pyroptosis in IVDD, and validated our predictions through vitro and vivo experiments. Through bioinformatics, we found that NLRP3, GSDMD, IL-1βand other hub proteins of pyroptosis were highly expressed in IVDD SD rats, and network pharmacology discovered that DHJSD may control cellular senescence, apoptosis, and pyroptosis in order to treat IVDD. Additional findings demonstrated that DHJSD could successfully treat IVDD brought on by imaging and histomorphological analysis. Western blot showed that NLRP3, a key protein of pyroptosis, was elevated in rat degenerated nucleus pulposus tissue and lipopolysaccharide-treated Nucleus pulposus Cells (NPCs), and that DHJSD intervention was effective in reducing LPS-induced inflammatory responses and further suppressing the expression of pyroptosis related proteins to improve IVDD. The specific mechanism is that DHJSD inhibits NPCs pyroptosis via the SDF-1/CXCR4-NF-kB-NLRP3 axis. In conclusion, we revealed the intrinsic mechanism of DHJSD regulation of NPCs pyroptosis to improve IVDD and its intrinsic value for IVDD treatment.

Construction of circ_0071922-miR-15a-5p-mRNA network in intervertebral disc degeneration by RNA-sequencing.

Low back pain (LBP) is the main factor of global disease burden. Intervertebral disc degeneration (IVDD) has long been known as the leading reason of LBP. Increasing studies have verified that circular RNAs (circRNAs)-microRNAs (miRNAs)-mRNAs network is widely involved in the pathological processes of IVDD. However, no study was made to demonstrate the circRNAs-mediated ferroptosis, oxidative stress, extracellular matrix metabolism, and immune response in IVDD. We collected 3 normal and 3 degenerative nucleus pulposus tissues to conduct RNA-sequencing to identify the key circRNAs and miRNAs in IVDD. Bioinformatics analysis was then conducted to construct circRNAs-miRNAs-mRNAs interaction network associated with ferroptosis, oxidative stress, extracellular matrix metabolism, and immune response. We also performed animal experiments to validate the therapeutic effects of key circRNAs in IVDD. We found that circ_0015435 was most obviously upregulated and circ_0071922 was most obviously downregulated in IVDD using RNA-sequencing. Then we observed that hsa-miR-15a-5p was the key downstream of circ_0071922, and hsa-miR-15a-5p was the top upregulated miRNA in IVDD. Bioinformatics analysis was conducted to predict that 56 immunity-related genes, 29 ferroptosis-related genes, 23 oxidative stress-related genes and 8 ECM-related genes are the targets mRNAs of hsa-miR-15a-5p. Then we constructed a ceRNA network encompassing 24 circRNAs, 6 miRNAs, and 101 mRNAs. Additionally, we demonstrated that overexpression of circ_0071922 can alleviate IVDD progression in a rat model. The findings of this study suggested that circ_0071922-miR-15a-5p-mRNA signaling network might affect IVDD by modulating the nucleus pulposus cells ferroptosis, oxidative stress, ECM metabolism, and immune response, which is an effective therapeutic targets of IVDD.

In situ forming macroporous biohybrid hydrogel for nucleus pulposus cell delivery

Degenerative intervertebral disc disease is a common source of chronic pain and reduced quality of life in people over the age of 40. While degeneration occurs throughout the disc, it most often initiates in the nucleus pulposus (NP). Minimally invasive delivery of NP cells within hydrogels that can restore and maintain the disc height while regenerating the damaged NP tissue is a promising treatment strategy for this condition. Towards this goal, a biohybrid ABA dimethacrylate triblock copolymer was synthesized, possessing a lower critical solution temperature below 37 °C and which contained as its central block an MMP-degradable peptide flanked by poly(trimethylene carbonate) blocks bearing pendant oligoethylene glycol groups. This triblock prepolymer was used to form macroporous NP cell-laden hydrogels via redox initiated (ammonium persulfate/sodium bisulfite) crosslinking, with or without the inclusion of thiolated chondroitin sulfate. The resulting macroporous hydrogels had water and mechanical properties similar to those of human NP tissue and were mechanically resilient. The hydrogels supported NP cell attachment and growth over 28 days in hypoxic culture. In hydrogels prepared with the triblock copolymer but without the chondroitin sulfate the NP cells were distributed homogeneously throughout in clusters and deposited collagen type II and sulfated glycosaminoglycans but not collagen type I. This hydrogel formulation warrants further investigation as a cell delivery vehicle to regenerate degenerated NP tissue. Statement of significanceThe intervertebral disc between the vertebral bones of the spine consists of three regions: a gel-like central nucleus pulposus (NP) within the annulus fibrosis, and bony endplates. Degeneration of the intervertebral disc is a source of chronic pain in the elderly and most commonly initiates in the NP. Replacement of degenerated NP tissue with a NP cell-laden hydrogel is a promising treatment strategy. Herein we demonstrate that a crosslinkable polymer with a lower critical solution temperature below 37 °C can be used to form macroporous hydrogels for this purpose. The hydrogels are capable of supporting NP cells, which deposit collagen II and sulfated glycosaminoglycans, while also possessing mechanical properties matching those of human NP tissue.

Screening of miR-15a-5p as a potential biomarker for intervertebral disc degeneration through RNA-sequencing

Low back pain (LBP) is a prevalent clinical condition that imposes substantial economic burdens on society. Intervertebral disc degeneration (IVDD) is recognized as a major contributing factor to LBP. Recent studies have highlighted the pivotal role of microRNAs (miRNAs) in regulating the onset and progression of IVDD. Understanding the involvement of miRNAs in IVDD will expand our knowledge of the underlying mechanisms and potentially identify novel therapeutic targets for managing LBP. However, the pathological process of IVDD and the miRNA-mediated pathomechanism in IVDD remain unclear. Herein, we comprehensively analyzed and divided the pathological process of IVDD into three stages based on the analysis by Risbud and colleagues. Results showed that IVDD was especially associated with cell death, oxidative stress, inflammatory and immune response, and extracellular matrix (ECM) metabolism. Subsequently, we obtained human normal and degenerative nucleus pulposus tissues, which were visually confirmed through histological staining techniques such as HE and TUNEL staining. RNA sequencing was then performed on these tissue samples. Additionally, miRNA (GSE116726) and mRNA (GSE56081/GSE70362/GSE23130/GSE34095) datasets were collected from the GEO database. Our analysis revealed that miR-15a-5p was significantly upregulated IVDD, as validated by both RNA sequencing and qRT-PCR experiments. To further refine our findings, bioinformatics analysis was conducted, merging the targets of miR-15a-5p and multiple mRNA datasets, ultimately identifying the overlapping IVDD-associated mRNAs. Notably, many cuproptosis-related genes (CRGs), ferroptosis-related genes, oxidative stress-related genes, and immunity-related genes were potential targets of miR-15a-5p. The miR-15a-5p-mRNA network was constructed using Cytoscape software. Additionally, PPI, functional, and pathway enrichment analyses of the CRGs were also performed. We found that MTF1, one of the CRGs, was highly expressed in IVDD and primarily localized in the nucleus of nucleus pulposus cells. These findings suggest that miR-15a-5p is a potential biomarker in IVDD, and targeting the miR-15a-5p-mRNA signaling pathway may be a promising strategy for treating IVDD diseases.

Identification and Verification of Key MiRNAs Associated with Intervertebral Disc Degeneration.

Intervertebral Disc Degeneration (IDD) is a heterogeneous spinal disease whose underlying molecular mechanism is unclear. This study aimed to identify, profile, and analyze microRNAs (miRNAs) related to IDD. Microarray Gene Expression IDD data (GSE63492) were downloaded from Gene Expression Omnibus datasets. We employed Weighted Gene Co-Expression Network Analysis (WGCNA) to construct a miRNA co-expression network, and the miRNAs related to the IDD stage were detected. The number of differentially expressed miRNAs between normal and degenerated nucleus pulposus tissues was calculated. Twenty-three clinical specimens were used to validate the expression of miRNAs using qRT-PCR. WGCNA identified 48 miRNAs significantly related to the IDD stage, and 94 miRNAs that were significantly different between normal and degenerated nucleus pulposus tissues. We selected 32 overlapping miRNAs and identified 347 corresponding target genes. The integrative analysis revealed the biological function and pathways of these targeted genes. Analysis of clinical specimens validated that hsa-miR-4534 was upregulated in IDD, whereas hsa-miR-1827 and hsa-miR- 185-5p were downregulated in IDD. This study has identified a subset of miRNAs that are related to IDD pathogenesis and hub miRNAs that are keys to the IDD co-expression network, which may potentially be utilized as indicators for treatment.

N6-methyladenosine hypomethylation of circGPATCH2L regulates DNA damage and apoptosis through TRIM28 in intervertebral disc degeneration.

Circular RNAs (circRNAs) are a class of noncoding RNAs that have been found to be involved in intervertebral disc degeneration (IVDD) progression, and N6-methyladenosine (m6A) broadly exists in circRNAs. Here, we identified circGPATCH2L with a low m6A methylation level to be upregulated in degenerative nucleus pulposus tissues. Mechanistically, as a protein decoy for tripartite motif containing 28 (TRIM28) within aa 402-452 region, circGPATCH2L abrogates the phosphorylation of TRIM28 and inhibits P53 degradation, which contributes to DNA damage accumulation and cellular apoptosis and leads to IVDD progression. Moreover, m6A-methylated circGPATCH2L is recognised and endoribonucleolytically cleaved by a YTHDF2-RPL10-RNase P/MRP complex to maintain the physiological state of nucleus pulposus cells. Thus, our data show the physiological significance of m6A modification in regulating circRNA abundance and provide a potentially effective therapeutic target for the treatment of IVDD.

CX3CL1/CX3CR1 axis alleviates inflammation and apoptosis in human nucleus pulpous cells via M2 macrophage polarization.

CX3C chemokine ligand 1 (CX3CL1) belongs to the CX3C chemokine family and is involved in various disease processes. However, its role in intervertebral disc degeneration (IDD) remains to be elucidated. In the present study, western blotting, reverse transcription-quantitative PCR and ELISA assays were used to assess target gene expression. In addition, immunofluorescence and TUNEL staining were used to assess macrophage infiltration, monocyte migration and apoptosis. The present study aimed to reveal if and how CX3CL1 regulates IDD progression by exploring its effect on macrophage polarization and apoptosis of human nucleus pulposus cells (HNPCs). The data showed that CX3CL1 bound to CX3C motif chemokine receptor 1 (CX3CR1) promoted the M2 phenotype polarization via JAK2/STAT3 signaling, followed by increasing the secretion of anti-inflammatory cytokines from HNPCs. In addition, HNPC-derived CX3CL1 promoted M2 macrophage-derived C-C motif chemokine ligand 17 release thereby reducing the apoptosis of HNPCs. In clinic, the reduction of mRNA and protein levels CX3CL1 in degenerative nucleus pulposus tissues (NPs) was measured. Increased M1 macrophages and pro-inflammatory cytokines were found in NPs of IDD patients with low CX3CL1 expression. Collectively, these findings suggested that the CX3CL1/CX3CR1 axis alleviates IDD by reducing inflammation and apoptosis of HNPCs via macrophages. Therefore, targeting CX3CL1/CX3CR1 axis is expected to produce a new therapeutic approach for IDD.

Brachyury positively regulates extracellular matrix synthesis via directly promoting aggrecan transcription in nucleus pulposus.

Nucleus pulposus (NP) degeneration is characterized by the decreased cellularity of nucleus pulposus cells (NPCs) and diminished content of hydrophilic extracellular matrix (ECM). Overexpression of brachyury has been reported to reverse the degenerated NPCs into healthy phenotypes. However, the direct correlation between brachyury and ECM has not been fully elucidated. This study revealed that brachyury expression decreased in human degenerated NP tissues and Lipopolysaccharide (LPS)-induced degenerated rat NPCs model. In vitro and in vivo experiments further showed that brachyury deficiency suppressed the synthesis of aggrecan and collagen II in NP. Mechanistically, ChIP-qPCR assays demonstrated that brachyury bound to the promoter region of aggrecan in NPCs. Furthermore, luciferase reporter assays revealed that brachyury transcriptionally activated aggrecan expression through binding with a novel specific motif. In rat in vivo model, brachyury overexpression partially reversed the degenerative phenotype. In conclusion, brachyury positively regulated ECM synthesis via directly promoting aggrecan transcription in NPCs. Accordingly, it may be helpful to be developed into a promising therapeutic target for NP degeneration.

The involvement of DDX3X in compression-induced nucleus pulposus pyroptosis.

A study has been conducted to investigate the relationship between DDX3X and nucleus pulposus (NP) pyroptosis. DDX3X and pyroptosis-related proteins (Caspase-1, Full-length GSDMD, Cleaved GSDMD) were measured in compression-induced human NP cells and tissue. DDX3X was overexpressed or knocked down by gene transfection. The expressions of NLRP3, ASC, and pyroptosis-related proteins were detected by Western blot assay. IL-1β and IL-18 were detected by ELISA. HE staining and immunohistochemistry were used to observe the expression of DDX3X, NLRP3, and Caspase-1 in the rat model of compression-induced disc degeneration. DDX3X, NLRP3, and Caspase-1 were highly expressed in degenerated NP tissue. Overexpression of DDX3X induced pyroptosis in NP cells and increased levels of NLRP3, IL-1β, IL-18, and pyroptosis-related proteins. Knockdown of DDX3X showed an opposite trend to overexpression of DDX3X. The NLRP3 inhibitor CY-09 effectively prevented the up-regulation of the expression of IL-1β, IL-18, ASC, Pro-caspase-1, Full-length GSDMD, and Cleaved GSDMD. Increased expression of DDX3X, NLRP3, and Caspase-1 was observed in the rat model of compression-induced disc degeneration. Our study showed that DDX3X mediates pyroptosis of NP cells by upregulating NLRP3 expression, which ultimately leads to intervertebral disc degeneration (IDD). This discovery deepens the understanding of IDD pathogenesis and provides a promising and novel therapeutic target for IDD.

LncRNA ZFAS1 promotes intervertebral disc degeneration by upregulating AAK1.

We investigated the function of lncRNA zinc finger antisense 1 (ZFAS1) in intervertebral disc degeneration (IDD) progression in vitro and in vivo. Nucleus pulposus (NP) tissues were obtained from 20 patients with IDD. IL-1β was used to stimulate primary NP cells to establish the IDD models in vitro. Gene expression was determined by RT-qPCR. 5-Ethynyl-2'-deoxyuridine and flow cytometry were performed to determine cell proliferation and apoptosis, and western blotting was conducted to measure the apoptosis- and extracellular matrix (ECM)-related protein expression. Luciferase reporter assay was used to examine the interactions between the genes. We also investigated the effect of ZFAS1 in a mouse model of IDD induced by needle punctures. Our results showed that ZFAS1 expression was elevated in degenerative NP tissues and IL-1β-treated NP cells. ZFAS1 knockdown inhibited NP cell apoptosis and ECM degradation induced by IL-1β. Mechanically, ZFAS1 sponged miR-4711-5p and adaptor-associated kinase 1 (AAK1) was targeted by miR-4711-5p. Furthermore, AAK1 overexpression partially eliminated the impact of ZFAS1 depletion on NP cell proliferation, apoptosis, and ECM degradation. More importantly, the results of the in vivo studies confirmed the effect of silencing ZFAS1 on alleviating the symptoms of IDD mice. Overall, silencing ZFAS1 inhibits IDD progression by reducing NP cell apoptosis and ECM degradation through the miR-4711-5p/AAK1 axis.

MiR-328-5p Induces Human Intervertebral Disc Degeneration by Targeting WWP2.

Intervertebral disc degeneration (IDD) development is regulated by miRNA, including inflammatory reactions, cell apoptosis, and degradation of extracellular matrix. Nucleus pulposus cells apoptosis has a absolute influence in the development of IDD. This experiment explores the mechanism of miR-328-5p regulating IDD. Through the analysis of miRNA and mRNA microarray database, we screened the target genes miR-328-5p and WWP2. We verified the expression of miR-328-5p, WWP2, and related apoptotic genes in normal and degenerative nucleus pulposus tissues by qRT-PCR. The expressions of WWP2, Bcl-2, and Bax were detected by qRT-PCR and western blot after transfection to nucleus pulposus cell. The nucleus pulposus cell proliferation and apoptosis after transfection were confirmed by CCK8 and flow cytometry. Luciferase reporter assay and bioinformatics analyzed the targeting relationship between miR-328-5p and WWP2. Firstly, the qRT-PCR experiments confirmed the significant increase of miR-328-5p expression, while significant reduction of WWP2 in a degenerative tissues compared to the normal tissues. Surprisingly, miR-328-5p expression was positively, while that of WWP2 negatively correlated with the degeneration grade of IDD. And we also identified the high expression of Bax and Caspase3, while low expression of Bcl-2 in a degenerative tissues. After miR-328-5p mimic transfected into nucleus pulposus cell, qRT-PCR and western blot confirmed that WWP2 and Bcl-2 expressions were downregulated, while Bax and Caspase3 expressions were upregulated, and the same results were obtained by knocking down WWP2. CCK8 and flow cytometry confirmed that miR-328-5p inhibited the proliferation and induced apoptosis of nucleus pulposus cells. WWP2 is a target gene of miR-328-5p by bioinformatics and luciferase reporter assay. In summary, miR-328-5p targets WWP2 to regulate nucleus pulposus cells apoptosis and then participates in the development of IDD. Furthermore, this study may provide new references and ideas for IDD treatment.

Comparative metagenomic analysis of human intervertebral disc nucleus pulposus and cartilaginous end plates.

Study designThe diversity of microflora inhabiting endplate (EP) and nucleus pulposus (NP) tissues of human intervertebral disc (IVD) was profiled through NGS-supported 16S rRNA amplicon sequencing. Sixteen EP and their corresponding NP were excised from the brain-dead voluntary organ donors with no clinical history of low back pain, and 12 herniated and 8 degenerated NP tissues isolated from the patients undergoing spinal surgery were subjected to study the alteration in the microbial diversity.Objective(s)To understand in normal IVD, whether the colonization of bacteria to the NP is through the EP in discs with intact annulus fibrosus. To identify significantly differing microbial population(s) between normal and diseased IVD (NP).Background of the studyThere is increasing evidence for subclinical infection by fastidious low, growing bacteria to be a cause of disc degeneration. Although the presence of bacteria in NP has been reported well in literature, the source of bacteria is not clearly proved as the disc is avascular in healthy condition. Documentation of similar bacterial populations in the EP and NP may add proof that bacterial inoculation of NP occurs via the EP.Materials and methodsSixteen EP and their corresponding NP excised from brain-dead voluntary organ donors with no history of back pain and 20 diseased discs collected from patients undergoing microdiscectomy/fusion surgery were used for profiling microbiome through 16S rRNA amplicon sequencing using primers specific for V1-V9 hypervariable regions. Changes in bacterial diversity and abundance were analysed to identify the key microbial populations in normal IVD NP and EP tissues and those significantly altered in diseased IVD (NP).ResultsNP and EP shared a similar spectrum of microbiome but with varying abundance. The five dominant phyla identified were Proteobacteria, Firmicutes, Actinobacteria, OD1, and Bacteroidetes. Proteobacteria was found to be the most abundant phyla in both NP (62%) and EP (53%) of the normal IVD. This was followed by Firmicutes (16%), Actinobacteriota (11%), OD1 (Parcubacteria) (7.6%), and Bacteroidetes (2%) in NP and Firmicutes (23.4%), OD1 (Parcubacteria) (17.6%), Actinobacteriota (2.8%), and Bacteroidetes (2.6%) in EP, respectively. Under diseased conditions, Proteobacteria (68%) was dominant when compared with other phyla. However, there was no significant difference in the abundance of Proteobacteria between the normal and diseased discs. Interestingly, the other dominant phyla such as Firmicutes (Normal-NP: 16.2%; Diseased-NP: 4.02%) and Actinobacteria (Normal-NP: 11%; Diseased-NP: 0.99%) showed a significant reduction in degenerated discs. To understand the key microbial populations that are significantly altered during disease, correlation analysis was performed among the three phyla, which revealed a negative correlation in the ratio of Actinobacteria + Firmicutes vs. Proteobacteria (p = 0.001) in DD.ConclusionResults of our study clearly demonstrated a similar bacterial diversity but with varying abundance between the EP and NP, suggesting the existence of the endplate–nucleus pulposus axis in the normal IVD microbiome. Further, our results have indicated that the changes in the abundance of Actinobacteria + Firmicutes vs. Proteobacteria during DDD need further investigation.

Antioksidativno ponašanje a2-makroglobulina kod degeneracije intervertebralnog diska

Background: To clarify if a2-macroglobulin (a2M) has an antioxidative effect during the progression of the intervertebral disc degeneration (IVDD). Methods: The content of a2M and reactive oxygen species (ROS) were measured to compare mildly and severely degenerated human nucleus pulposus (NP) tissue by immunohistochemistry, mass spectrometry, and enzyme-linked immunosorbent assay (ELISA). Additionally, exogenic a2M was used to culture severely degenerated NP tissue in vitro. The effects of a2M on hypochlorite (HOCl)-treated NP cells were evaluated, containing antioxidative enzymes, ROS level, collagen II, and aggrecan expression, MMP3/13, and ADAMTS4/5. Results: ROS level increased in severely degenerated NP, accompanying with a decreased a2M content. Supplement of a2M could decrease the ROS level of cultured NP in vitro, meanwhile, the MMP13 and ADAMTS4 expression were also reduced. It was found that treatment of HOCl resulted in oxidative damage to NP cells and decreased a2M expression in a dose and time-dependent manner. Furthermore, exogenic a2M stimulation reversed the HOCl-triggered ROS accumulation. The promotion of SOD1/2, CAT, GPX1, collagen II, and aggrecan, and suppression of MMP3/13, ADAMTS4/5 expression caused by a2M were also observed. Conclusions: Our study indicates that a2M has an antioxidative ability in degenerated NP cells by promoting the antioxidative enzyme production.

Antioxidative behavior of a2-macroglobulin in intervertebral disc degeneration.

To clarify if a2-macroglobulin (α2M) has an antioxidative effect during the progression of the intervertebral disc degeneration (IVDD). The content of α2M and reactive oxygen species (ROS) were measured to compare mildly and severely degenerated human nucleus pulposus (NP) tissue by immunohistochemistry, mass spectrometry, and enzyme-linked immunosorbent assay (ELISA). Additionally, exogenic α2M was used to culture severely degenerated NP tissue in vitro. The effects of α2M on hypochlorite (HOCl)-treated NP cells were evaluated, containing antioxidative enzymes, ROS level, collagen II, and aggrecan expression, MMP3/13, and ADAMTS4/5. ROS level increased in severely degenerated NP, accompanying with a decreased α2M content. Supplement of α2M could decrease the ROS level of cultured NP in vitro, meanwhile, the MMP13 and ADAMTS4 expression were also reduced. It was found that treatment of HOCl resulted in oxidative damage to NP cells and decreased α2M expression in a dose and time-dependent manner. Furthermore, exogenic α2M stimulation reversed the HOCl-triggered ROS accumulation. The promotion of SOD1/2, CAT, GPX1, collagen II, and aggrecan, and suppression of MMP3/13, ADAMTS4/5 expression caused by α2M were also observed. Our study indicates that α2M has an antioxidative ability in degenerated NP cells by promoting the antioxidative enzyme production.

Irisin Ameliorates Intervertebral Disc Degeneration by Activating LATS/YAP/CTGF Signaling.

Unbalanced metabolism of an extracellular matrix (ECM) in nucleus pulposus cells (NPCs) is widely acknowledged as the primary cause of intervertebral disc degeneration (IDD). Irisin, a novel myokine, is cleaved from fibronectin type III domain-containing 5 (FNDC5) and has recently been proven to regulate the metabolism of ECM. However, little is known about its potential on NPCs and the development of IDD. Therefore, this study sought to examine the protective effects and molecular mechanism of irisin on IDD in vivo and in vitro. Decreased expression levels of FNDC5 and anabolism markers (COL2A1 and ACAN) but increased levels of catabolism markers (ADAMTS4) were found in degenerative nucleus pulposus (NP) tissues. In a punctured-induced rat IDD model, irisin treatment was found to significantly slow the development of IDD, and in TNF-α-stimulated NPCs, irisin treatment partly reversed the disorder of ECM metabolism. In mechanism, RNA-seq results suggested that irisin treatment affected the Hippo signaling pathway. Further studies revealed that with irisin treatment, the phosphorylation levels of key factors (LATS and YAP) were downregulated, while the expression level of CTGF was upregulated. Moreover, CTGF knockdown partially eliminated the protective effects of irisin on the metabolism of ECM in NPCs, including inhibiting the anabolism and promoting the catabolism. Taken together, this study demonstrated that the expression levels of FNDC5 were decreased in degenerative NP tissues, while irisin treatment promoted the anabolism, inhibited the catabolism of the ECM in NPCs, and delayed the progression of IDD via LATS/YAP/CTGF signaling. These results shed light on the protective actions of irisin on NPCs, leading to the development of a novel therapeutic target for treating IDD.

Microglia and macrophages contribute to the development and maintenance of sciatica in lumbar disc herniation.

Lumbar disc herniation (LDH) is a major cause of sciatica. Emerging evidence indicated that inflammation induced by the herniated nucleus pulposus (NP) tissues plays a major role in the pathogenesis of sciatica. However, the underlying mechanisms are still elusive. Although microglia and macrophages have been implicated in nerve injury-induced neuropathic pain, their roles in LDH-induced sciatica largely remain unknown. This study successfully established and modified a mouse model of LDH. We found that nerve root compression using degenerated NP tissues can initiate remarkable and persistent sciatica, with increased and prolonged macrophage infiltration in dorsal root ganglia (DRG) and significant activation of microglia in the spinal dorsal horn. Instead, compression of the nerve root with nondegenerated NP tissues only led to transient sciatica, with transient infiltration and activation of macrophages and microglia. Moreover, continuous treatment of PLX5622, a specific colony-stimulating factor 1 receptor antagonist, ablated both macrophages and microglia, which effectively alleviated LDH-induced sciatica. However, mechanical allodynia reoccurred along with the repopulation of macrophages and microglia after the withdrawal of PLX5622. Using RNA sequencing analysis, the current study depicted transcriptional profile changes of DRG after LDH and identified several macrophage-related potential target candidates. Our results suggested that microglia and macrophages may play an essential role in the development and maintenance of LDH-induced sciatica. Targeting microglia and macrophages may be a promising treatment for chronic LDH-induced sciatica.