Degenerative Nucleus Pulposus Cells Research Articles

Protein Kinase C and Matrix Metalloproteinases Expression Using Phorbol Myristate Acetate in Degenerative Intervertebral Disc Cells.

Degeneration of nucleus pulposus (NP) cells involves multiple factors. The relationship between the canonical Wnt/β-catenin signaling pathway and matrix metalloproteinases (MMPs) is important in cellular senescence. Protein kinase C (PKC), an intermediate of the non-canonical Wnt pathway stimulated by phorbol myristate acetate (PMA), possibly prevents NP cell senescence, although not yet demonstrated in human-based studies. This study aimed to investigate the effect of PMA stimulation on the non-canonical and canonical Wnt pathways and MMP expression in human NP cells to ascertain its inhibitory effects on the senescence of NP cells. Human disc tissues of Pfirrmann grades 1 and 2 were collected from patients during spinal surgery and subsequently cultured. Protein and ribonucleic acid (RNA) were isolated from NP cells treated with PMA (400 nM) for 24 hours. Expression of MMP1, MMP13, tissue inhibitor of matrix metalloproteinase 1 (TIMP1), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), transient receptor potential vanilloid 4 (TRPV4), interleukin-6 (IL-6), and β-catenin were detected using western blot analysis. Messenger RNA (mRNA) expression of type II collagen and glycosaminoglycan (GAG) were analyzed using reverse transcription polymerase chain reaction. IL-6 and prostaglandin E2 (PGE2) levels were measured using enzyme-linked immunosorbent assay. Expression of PKC-δ (intermediate of the non-canonical Wnt pathway) and β-catenin (intermediate of the canonical Wnt pathway) was increased by PMA treatment. The mRNA levels of type II collagen and GAG increased; however, their protein levels were not altered. PMA treatment increased the expression of MMP1, TIMP1, ADAMTS5, IL-6, PGE2, and TRPV4; however, the expression of MMP13 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) was unaltered. PMA activated PKC-δ, affecting the non-canonical Wnt pathway; however, its effect on β-catenin in the canonical Wnt pathway was limited. β-catenin activation through the TRPV4 channel led to increased expression of MMP1 and ADAMTS5 and that of IL-6 and PGE2 owing to NF-κB expression. Consequently, the degeneration of NP cells was not prevented.

Mechanism of KMT2D-mediated epigenetic modification in IL-1β-induced nucleus pulposus cell degeneration.

Intervertebral disc (IVD) degeneration (IVDD) is characterized by structural destruction accompanied by accelerated signs of aging. This study aimed to investigate the mechanism of lysine methyltransferase 2D (KMT2D) in the proliferation, apoptosis, and inflammation of nucleus pulposus cells (NPCs) in IVDD. Mouse-derived NPCs were cultured and induced with interleukin-1 beta (IL-1β) to establish cell models. KMT2D expression was detected by western blot and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). KMT2D expression was interfered with, and the contents of IL-18, IL-6, and tumor necrosis factor (TNF) were detected by enzyme-linked immunosorbent assay. Cell proliferation, apoptosis, and the expression of miR-133a-5p and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2) were measured. The enrichment of KMT2D and Histone 3 Lysine 4 monomethylation/dimethylation (H3K4me1/2) on the miR-133a-5p promoter was analyzed by chromatin immunoprecipitation and qPCR. The binding of miR-133a-5p and PFKFB2 was analyzed by a dual-luciferase assay. IL-1β treatment promoted KMT2D expression in NPCs. KMT2D knockdown reduced inflammation and apoptosis and promoted the proliferation of IL-1β-induced NPCs. Mechanistically, KMT2D upregulated miR-133a-5p expression by increasing the level of H3K4me2 at the miR-133a-5p promoter, thereby promoting the binding between miR-133a-5p and PFKFB2 and downregulating the transcription of PFKFB2. miR-133a-5p overexpression or PFKFB2 knockdown alleviated the protective effect of KMT2D knockdown on IL-1β-induced NPCs. KMT2D promoted miR-133a-5p expression through H3K4me2 methylation, thereby promoting the binding of miR-133a-5p to PFKFB2, reducing the mRNA level of PFKFB2, promoting inflammation and apoptosis of IL-1β-induced NPCs, and inhibiting NPC proliferation.

A novel spherical GelMA-HAMA hydrogel encapsulating APET×2 polypeptide and CFIm25-targeting sgRNA for immune microenvironment modulation and nucleus pulposus regeneration in intervertebral discs

MethodsSingle-cell transcriptomics and high-throughput transcriptomics were used to screen factors significantly correlated with intervertebral disc degeneration (IDD). Expression changes of CFIm25 were determined via RT-qPCR and Western blot. NP cells were isolated from mouse intervertebral discs and induced to degrade with TNF-α and IL-1β. CFIm25 was knocked out using CRISPR-Cas9, and CFIm25 knockout and overexpressing nucleus pulposus (NP) cell lines were generated through lentiviral transfection. Proteoglycan expression, protein expression, inflammatory factor expression, cell viability, proliferation, migration, gene expression, and protein expression were analyzed using various assays (alcian blue staining, immunofluorescence, ELISA, CCK-8, EDU labeling, transwell migration, scratch assay, RT-qPCR, Western blot). The GelMA-HAMA hydrogel loaded with APET×2 polypeptide and sgRNA was designed, and its effects on NP regeneration were assessed through in vitro and mouse model experiments. The progression of IDD in mice was evaluated using X-ray, H&E staining, and Safranin O-Fast Green staining. Immunohistochemistry was performed to determine protein expression in NP tissue. Proteomic analysis combined with in vitro and in vivo experiments was conducted to elucidate the mechanisms of hydrogel action.ResultsCFIm25 was upregulated in IDD NP tissue and significantly correlated with disease progression. Inhibition of CFIm25 improved NP cell degeneration, enhanced cell proliferation, and migration. The hydrogel effectively knocked down CFIm25 expression, improved NP cell degeneration, promoted cell proliferation and migration, and mitigated IDD progression in a mouse model. The hydrogel inhibited inflammatory factor expression (IL-6, iNOS, IL-1β, TNF-α) by targeting the p38/NF-κB signaling pathway, increased collagen COLII and proteoglycan Aggrecan expression, and suppressed NP degeneration-related factors (COX-2, MMP-3).ConclusionThe study highlighted the crucial role of CFIm25 in IDD and introduced a promising therapeutic strategy using a porous spherical GelMA-HAMA hydrogel loaded with APET×2 polypeptide and sgRNA. This innovative approach offers new possibilities for treating degenerated intervertebral discs.Graphical Molecular mechanism of GCA targeting CFIm25 in NP cells to promote regeneration of degenerated intervertebral disc NP

Collagen II enrichment through scAAV6-RNAi-mediated inhibition of matrix-metalloproteinases 3 and 13 in degenerative nucleus-pulposus cells degenerative disc disease and biological treatment strategies.

Intervertebral disc (IVD) degeneration damaging the extracellular matrix (ECM) of IVDs is the main cause of spine-associated disorders. Degenerative disc disease (DDD) is a multifaceted disorder, where environmental factors, inflammatory cytokines and catabolic enzymes act together. DDD starts typically due to imbalance between ECM biosynthesis and degradation within IVDs, especially through unbalanced degradation of aggrecan and collagen II in nucleus pulposus (NP). Current treatment approaches are primarily based on conservative or surgical therapies, which are insufficient for biological regeneration. The disintegrins and metalloproteinases with thrombospondin motifs (ADAMTSs) and matrix metalloproteinases (MMPs) are the key proteolytic enzymes for degradation of aggrecan and collagens. Previously, high expression levels of ADAMTS4, ADAMTS5, MMP3 and MMP13, which are accompanied with low levels of aggrecan and collagen II, were demonstrated in degenerative human NP cells. Moreover, self-complementary adeno-associated virus type 6 (scAAV6) mediated inhibitions of ADAMTS4 and ADAMTS5 by RNA-interference (RNAi) could specifically enhance aggrecan level. Thus, MMPs are apparently the main degrading enzymes of collagen II in NP. Furthermore, scAAV6-mediated inhibitions of MMP3 and MMP13 have not yet been investigated. Therefore, we attempted to enhance the level of collagen II in degenerative NP cells by scAAV6-RNAi-mediated inhibitions of MMP3 and MMP13. MRI was used to determine preoperative grading of IVD degeneration in patients. After isolation and culturing of NP cells, cells were transduced with scAAV6-shRNAs targeting MMP3 or MMP13; and analysed by fluorescence microscopy, FACS, MTT assay, RT-qPCR, ELISA and western blotting. scAAV6-shRNRs have no impact on cell viability and proliferation, despite high transduction efficiencies (98.6%) and transduction units (1383 TU/Cell). Combined knockdown of MMP3 (92.8%) and MMP13 (90.9%) resulted in highest enhancement of collagen II (143.2%), whereby treatment effects were significant over 56days (p < 0.001). Conclusively, scAAV6-RNAi-mediated inhibitions of MMP3 and MMP13 help to progress less immunogenic and enduring biological treatments in DDD.

Bone Marrow Mesenchymal Stem Cell-Derived Exosomes Alleviate Nuclear Pulposus Cells Degeneration Through the miR-145a-5p/USP31/HIF-1α Signaling Pathway.

Bone marrow mesenchymal stem cell (BMSC)-derived exosomes possess therapeutic potential against degenerative diseases. This study aimed to investigate the effects of BMSC-derived exosomes on intervertebral disc degeneration (IVDD) and explore the underlying molecular mechanisms. Through transcriptome sequencing and histological analysis, we observed a significant increase in HIF-1α expression in degenerative nucleus pulposus (NP) tissues. The addition of HIF-1α resulted in elevated expression of inflammatory factors IL-1β and IL-6, higher levels of matrix-degrading enzyme MMP13, and lower expression of aggrecan in NP cells. Co-culturing with BMSCs diminished the expression of HIF-1α, MMP13, IL-1β, and IL-6 in degenerative NP cells induced by overload pressure. miRNA chip analysis and PCR validation revealed that miR-145a-5p was the primary miRNA carried by BMSC-derived exosomes. Overexpression of miR-145a-5p was effective in minimizing the expression of HIF-1α, MMP13, IL-1β, and IL-6 in degenerative NP cells. Luciferase reporter assays confirmed USP31 as the target gene of miR-145a-5p, and the regulation of NP cells by BMSC-derived exosomes via miR-145a-5p was dependent on USP31. In conclusion, BMSC-derived exosomes alleviated IVDD through the miR-145a-5p/USP31/HIF-1α signaling pathway, providing valuable insights into the treatment of IVDD.

Overactivation of NF-kB pathway can induce apoptosis by down-regulating glycolysis in human degenerative nucleus pulposus cells

Intervertebral disc herniation, a prevalent condition in spinal surgery that frequently results in low back pain and lower limb dysfunction, significantly impacting patients' quality of life. Several factors, including spine biomechanics, biology, nutrition, injury, and abnormal inflammatory responses, have been associated with the development of intervertebral disc herniation. Among these factors, abnormal inflammatory responses have received considerable attention as a crucial mediator of both clinical symptoms and disease progression during the intervertebral disc herniation process. However, the underlying mechanisms of inflammation-induced intervertebral disc herniation remain inadequately explored. The NF-κB (Nuclear Factor-κB) pathway plays a central role in regulating the expression of proinflammatory cytokines. Research on intervertebral disc herniation has suggested that NF-κB can activate the NLRP3 inflammasome, thereby exacerbating intervertebral disc degeneration. Targeting the NF-κB pathway has shown promise in alleviating disc degeneration and associated pain. Previous research indicated that the upregulation of the NF-κB pathway, achieved through the inhibition of A20 (zinc finger protein A20), accelerated intervertebral disc herniation. In the present study, we observed that increased activation of NF-κB pathway activation suppressed the glycolysis process in nucleus pulposus cells (NPCs), leading to NPC apoptosis. Conversely, inhibition of the NF-κB pathway overactivated promoted the restoration of glycolysis and reversed NPC apoptosis, especially when treated with Lipopolysaccharide (LPS).

IL-32 aggravates metabolic disturbance in human nucleus pulposus cells by activating FAT4-mediated Hippo/YAP signaling

Extracellular matrix (ECM) metabolism disorders in the inflammatory microenvironment play a key role in the pathogenesis of intervertebral disc degeneration (IDD). Interleukin-32 (IL-32) has been reported to be involved in the progression of various inflammatory diseases; however, it remains unclear whether it participates in the matrix metabolism of nucleus pulposus (NP) cells. Therefore, this study aimed to investigate the mechanism of IL-32 on regulating the ECM metabolism in the inflammatory microenvironment. RNA-seq was used to identify aberrantly expressed genes in NP cells in the inflammatory microenvironment. Western blotting, real-time quantitative PCR, immunohistochemistry and immunofluorescence analysis were performed to measure the expression of IL-32 and metabolic markers in human NP tissues or NP cells treated with or without tumor necrosis factor-α (TNF-α). In vivo, an adeno-associated virus overexpressing IL-32 was injected into the caudal intervertebral discs of rats to assess its effect on IDD. Proteins interacting with IL-32 were identified via immunoprecipitation and mass spectrometry. Lentivirus overexpressing IL-32 or knocking down Fat atypical cadherin 4 (FAT4), yes-associated protein (YAP) inhibitor-Verteporfin (VP) were used to treat human NP cells, to explore the pathogenesis of IL-32. Hippo/YAP signaling activity was verified in human NP tissues. IL-32 expression was significantly upregulated in degenerative NP tissues, as indicated in the clinical samples. Furthermore, IL-32 was remarkably overexpressed in TNF-α-induced degenerative NP cells. IL-32 overexpression induced IDD progression in the rat model. Mechanistically, the elevation of IL-32 in the inflammatory microenvironment enhanced its interactions with FAT4 and mammalian sterile 20-like kinase1/2 (MST1/2) proteins, prompting MST1/2 phosphorylation, and activating the Hippo/YAP signaling pathway, causing matrix metabolism disorder in NP cells. Our results suggest that IL-32 mediates matrix metabolism disorders in NP cells in the inflammatory micro-environment via the FAT4/MST/YAP axis, providing a theoretical basis for the precise treatment of IDD.

Zhiqiao Gancao decoction regulated JAK2/STAT3/ macrophage M1 polarization to ameliorate intervertebral disc degeneration

BackgroundZhiqiao Gancao decoction (ZQGCD) was created by Professor Gong Zhengfeng, a renowned Chinese medicine expert. Clinical studies have shown its efficacy in alleviating pain and enhancing lumbar function in intervertebral disc degeneration (IDD) patients. However, the precise mechanism of ZQGCD in treating IDD remains unclear. MethodsThe active components of ZQGCD were identified using Liquid chromatography-tandem mass spectrometry (LC-MS/MS). A rat model of intervertebral disc degeneration was established, and rats in each group received ZQGCD for three weeks. Assessment parameters included hyperalgesia status, observation of intervertebral disc tissue degeneration and macrophage infiltration, and analysis of JAK2/STAT3 pathway protein expression in the intervertebral disc. Primary macrophage M1 polarization was induced using LPS, with cells treated using the JAK2 inhibitor (AZD1480) and ZQGCD to evaluate macrophage polarization, cellular supernatant inflammatory factors, and JAK2/STAT3 pathway expression. Macrophage supernatant served as a conditioned medium to observe its effects on the proliferation of nucleus pulposus cells (NPCs) and the expression of collagen II and MMP3 proteins. ResultsA total of 81 active components were identified in ZQGCD. Following ZQGCD treatment, infiltrating macrophages in intervertebral disc tissues of model rats decreased, the content of M1 macrophages decreased, while the content of M2 macrophages increased, the expression of proinflammatory factors and pain-inducing factors in serum decreased, and the expression of substance P in intervertebral disc tissue decreased. Consequently, the intervertebral disc degeneration and hyperalgesia of rats were improved. In vitro studies revealed that LPS induced M1 macrophage polarization. By inhibiting the JAK2/STAT3 pathway, both JAK2 inhibitors and ZQGCD effectively suppressed M1 polarization, resulting in decreased levels of IL-1β, IL-6, TNF-α, and various other inflammatory factors. Consequently, this inhibition led to a delay in the degeneration of NPCs. ConclusionThere is macrophage infiltration in the intervertebral disc tissue of IDD rats, and JAK2/STAT3 pathway is activated, macrophages are polarized to M1 type, resulting in inflammatory microenvironment, leading to intervertebral disc degeneration and hyperalgesia. ZQGCD exhibited a delaying effect on IDD and improved hyperalgesia by inhibiting the JAK2/STAT3/macrophage M1 polarization pathway.

Exosomes derived from human urine–derived stem cells ameliorate IL-1β-induced intervertebral disk degeneration

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

Hypoxia-Preconditioned BMSC-Derived Exosomes Induce Mitophagy via the BNIP3-ANAX2 Axis to Alleviate Intervertebral Disc Degeneration.

Intervertebral disc degeneration (IVDD) is a chronic degenerative disease involving the aging and loss of proliferative capacity of nucleus pulposus cells (NPCs), processes heavily dependent on mitochondrial dynamics and autophagic flux. This study finds that the absence of BCL2/adenovirus E1B 19kDa interacting protein 3 (BNIP3) is associated with senescence-related NPC degeneration, disrupting mitochondrial quality control. Bone marrow mesenchymal stem cells (BMSCs) have multidirectional differentiation potential and produce extracellular vesicles containing cellular activators. Therefore, in this study, BMSCs are induced under hypoxic stimulation to deliver BNIP3-rich extracellular vesicles to NPCs, thereby alleviating aging-associated mitochondrial autophagic flux, promoting damaged mitochondrial clearance, and restoring mitochondrial quality control. Mechanistically, BNIP3 is shown to interact with the membrane-bound protein annexin A2 (ANXA2), enabling the liberation of the transcription factor EB (TFEB) from the ANXA2-TFEB complex, promoting TFEB nuclear translocation, and regulating autophagy and lysosomal gene activation. Furthermore, a rat model of IVDD is established and verified the in vivo efficacy of the exosomes in repairing disc injuries, delaying NPC aging, and promoting extracellular matrix (ECM) synthesis. In summary, hypoxia-induced BMSC exosomes deliver BNIP3-rich vesicles to alleviate disc degeneration by activating the mitochondrial BNIP3/ANXA2/TFEB axis, providing a new target for IVDD treatment.

Dedifferentiation-like reprogramming of degenerative nucleus pulposus cells into notochordal-like cells by defined factors

The extensive degeneration of functional somatic cells and thedepletion of endogenous stem/progenitor populations present significant challenges to tissue regeneration in degenerative diseases. Currently, a cellular reprogramming approach enabling directly generating corresponding progenitor populations from degenerative somatic cells remains elusive. The present study focused on intervertebral disc degeneration (IVDD) and identified a three-factor combination (OCT4, FOXA2, TBXT [OFT]) that could induce the dedifferentiation-like reprogramming of degenerative nucleus pulposus cells (dNPCs) toward induced notochordal-like cells (iNCs). Single-cell transcriptomics dissected the transitions of cell identity during reprogramming. Further, OCT4 was found to directly interact with bromodomain PHD-finger transcription factor to remodel the chromatin during the early phases, which was crucial for initiating this dedifferentiation-like reprogramming. In rat models, intradiscal injection of adeno-associated virus carrying OFT generated iNCs from in situ dNPCs and reversed IVDD. These results collectively present a proof-of-concept for dedifferentiation-like reprogramming of degenerated somatic cells into corresponding progenitors through the development of a factor-based strategy, providing a promising approach for regeneration in degenerative disc diseases.

Tbxt alleviates senescence and apoptosis of nucleus pulposus cells through Atg7-mediated autophagy activation during intervertebral disk degeneration.

Intervertebral disk degeneration (IDD) is a significant cause of low back pain, characterized by excessive senescence and apoptosis of nucleus pulposus cells (NPCs). However, the precise mechanisms behind this senescence and apoptosis remain unclear. This study aimed to investigate the role of T-box transcription factor T (Tbxt) in IDD both in vitro and in vivo, using a hydrogen peroxide (H2O2)-induced NPCs senescence and apoptosis model, as well as a rat acupuncture IDD model. First, the expression of p16 and cleaved-caspase 3 significantly increased in degenerated human NPCs, accompanied by a decrease in Tbxt expression. Knockdown of Tbxt exacerbated senescence and apoptosis in the H2O2-induced NPCs degeneration model. Conversely, upregulation of Tbxt alleviated these effects induced by H2O2. Mechanistically, bioinformatic analysis revealed that the direct downstream target genes of Tbxt were highly enriched in autophagy-related pathways, and overexpression of Tbxt significantly activated autophagy in NPCs. Moreover, the administration of the autophagy inhibitor, 3-methyladenine, impeded the impact of Tbxt on the processes of senescence and apoptosis in NPCs. Further investigation revealed that Tbxt enhances autophagy by facilitating the transcription of ATG7 through its interaction with a specific motif within the promoter region. In conclusion, this study suggests that Tbxt mitigates H2O2-induced senescence and apoptosis of NPCs by activating ATG7-mediated autophagy.NEW & NOTEWORTHY This study investigates the role of Tbxt in IDD. The results demonstrate that knockdown of Tbxt exacerbates H2O2-induced senescence and apoptosis in NPCs and IDD, whereas upregulation of Tbxt significantly protects against IDD both in vivo and in vitro. Mechanistically, in the nucleus, Tbxt enhances the transcription of ATG7, leading to increased expression of ATG7 protein levels. This, in turn, promotes elevated autophagy levels, ultimately alleviating IDD.

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

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

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

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

PHLDA2 overexpression facilitates senescence and apoptosis via the mitochondrial route in human nucleus pulposus cells by regulating Wnt/β-catenin signalling pathway.

Low back pain is a common clinical symptom of intervertebral disc degeneration (IVDD), which seriously affects the quality of life of the patients. The abnormal apoptosis and senescence of nucleus pulposus cells (NPCs) play important roles in the pathogenesis of IVDD. PHLDA2 is an imprinted gene related to cell apoptosis and tumour progression. However, its role in NPC degeneration is not yet clear. Therefore, this study was set to explore the effects of PHLDA2 on NPC senescence and apoptosis and the underlying mechanisms. The expression of PHLDA2 was examined in human nucleus pulposus (NP) tissues and NPCs. Immunohistochemical staining, magnetic resonance imaging imaging and western blot were performed to evaluate the phenotypes of intervertebral discs. Senescence and apoptosis of NPCs were assessed by SA-β-galactosidase, flow cytometry and western blot. Mitochondrial function was investigated by JC-1 staining and transmission electron microscopy. It was found that the expression level of PHLDA2 was abnormally elevated in degenerated human NP tissues and NPCs. Furthermore, knockdown of PHLDA2 can significantly inhibit senescence and apoptosis of NPCs, whereas overexpression of PHLDA2 can reverse senescence and apoptosis of NPCs in vitro. In vivo experiment further confirmed that PHLDA2 knockdown could alleviate IVDD in rats. Knockdown of PHLDA2 could also reverse senescence and apoptosis in IL-1β-treated NPCs. JC-1 staining indicated PHLDA2's knockdown impaired disruption of the mitochondrial membrane potential and also ameliorated superstructural destruction of NPCs as showed by transmission electron microscopy. Finally, we found the PHLDA2 knockdown promoted Collagen-II expression and suppressed MMP3 expression in NPCs by repressing wnt/β-catenin pathway. In conclusion, the results of the present study showed that PHLDA2 promotes IL-1β-induced apoptosis and senescence of NP cells via mitochondrial route by activating the Wnt/β-catenin pathway, and suggested that therapy targeting PHLDA2 may provide valuable insights into possible IVDD therapies.

POSTN knockdown suppresses IL-1β-induced inflammation and apoptosis of nucleus pulposus cells via inhibiting the NF-κB pathway and alleviates intervertebral disc degeneration.

The aim of this study is to investigate the effects of POSTN on IL-1β induced inflammation, apoptosis, NF-κB pathway and intervertebral disc degeneration (IVDD) in Nucleus pulposus (NP) cells (NPCs). NP tissue samples with different Pfirrmann grades were collected from patients with different degrees of IVDD. Western blot and immunohistochemical staining were used to compare the expression of POSTN protein in NP tissues. Using the IL-1β-induced IVDD model, NPCs were transfected with lentivirus-coated si-POSTN to down-regulate the expression of POSTN and treated with CU-T12-9 to evaluate the involvement of NF-κB pathway. Western blot, immunofluorescence, and TUNEL staining were used to detect the expression changes of inflammation, apoptosis and NF-κB pathway-related proteins in NPCs. To investigate the role of POSTN in vivo, a rat IVDD model was established by needle puncture of the intervertebral disc. Rats were injected with lentivirus-coated si-POSTN, and H&E staining and immunohistochemical staining were performed. POSTN expression is positively correlated with the severity of IVDD in human. POSTN expression was significantly increased in the IL-1β-induced NPCs degeneration model. Downregulation of POSTN protects NPCs from IL-1β-induced inflammation and apoptosis. CU-T12-9 treatment reversed the protective effect of si-POSTN on NPCs. Furthermore, lentivirus-coated si-POSTN injection partially reversed NP tissue damage in the IVDD model in vivo. POSTN knockdown reduces inflammation and apoptosis of NPCs by inhibiting NF-κB pathway, and ultimately prevents IVDD. Therefore, POSTN may be an effective target for the treatment of IVDD.

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

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

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.

Ephrin B2 (EFNB2) potentially protects against intervertebral disc degeneration through inhibiting nucleus pulposus cell apoptosis

Nucleus pulposus (NP) cell apoptosis is a significant indication of accelerated intervertebral disc degeneration; however, the precise mechanism is unelucidated as of yet. Ephrin B2 (EFNB2), the only gene down-regulated in the three degraded intervertebral disc tissue microarray groups (GSE70362, GSE147383 and GSE56081), was screened for examination in this study. Subsequently, EFNB2 was verified to be down-regulated in degraded NP tissue samples. Interleukin-1 (IL-1β) treatment of NP cells to simulate the IDD environment indicated that IL-1β treatment decreased EFNB2 expression. In degenerative NP cells stimulated by IL-1β, EFNB2 knockdown significantly increased the rate of apoptosis as well as the apoptosis-related molecules cleaved-caspase-3 and the Bax to Bcl-2 ratio. EFNB2 was found to promote AKT, PI3K, and mTOR phosphorylation; the PI3K/AKT signaling role was investigated using the PI3K inhibitor LY294002. EFNB2 overexpression significantly increased PI3K/AKT pathway activity in IL-1β-stimulated NP cells than the normal control. Moreover, EFNB2 partially alleviated NP cell apoptosis induced by IL-1β, reduced the cleaved-cas3 level, and decreased the Bax/Bcl-2 ratio after the addition of the inhibitor LY294002. Additionally, EFNB2 overexpression inhibited the ERK1/2 phosphorylation; the effects of EFNB2 overexpression on ERK1/2 phosphorylation, degenerative NP cell viability, and cell apoptosis were partially reversed by ERK signaling activator Ceramide C6. EFNB2 comprehensively inhibited the apoptosis of NP cells by activating the PI3K/AKT signaling and inhibiting the ERK signaling, obviating the exacerbation of IDD. EFNB2 could be a potential target to protect against degenerative disc changes.

Activation of Nuclear Factor Erythroid 2-Related Factor 2 Antagonizes the Reactive Oxygen Species Modulator 1-Induced Oxidative Stress in Nucleus Pulposus Cells

This study investigates the role of Reactive Oxygen Species Modulator 1 (ROMO1) in oxidative stress within nucleus pulposus (NP) cells and its potential regulation by Nuclear factor erythroid 2-related factor 2 (Nrf2). Intervertebral disc samples from patients were collected, and ROMO1, Nrf2, collagen I/II levels were analyzed to establish their potential connection. Human NP cells were cultured and exposed to H2O2 to induce oxidative stress. To elucidate ROMO1’s impact on NP cell metabolism, NP cells were transfected with ROMO1. Concurrently, Nrf2 activators and inhibitors were used to modulate Nrf2 expression during culturing. Oxidative stress was assessed through CAT and SOD1 gene expression analysis and measurement of cellular reactive oxygen species (ROS) production. NP cell status was determined by evaluating cell viability and collagen I/II expression. Results indicated elevated ROMO1 expression in severe intervertebral disc degeneration (IDD) and after H2O2 treatment. ROMO1 overexpression increased ROS production, suppressed CAT, SOD, and collagen II expression, while elevating collagen I and negatively affecting cell viability. However, Nrf2 activation effectively suppressed ROMO1 expression and protected NP cells from oxidative stress induced by H2O2 or ROMO1. In conclusion, ROMO1 exacerbates oxidative stress and contributes to NP cell degeneration, a process mitigated by Nrf2 activation.