Normal Nucleus Pulposus Cells Research Articles

Effect of PI3K/AKT/HIF-1α signaling pathway on oxidative stress and apoptosis in nucleus pulposus cells.

The aim of the study was to explore the expression of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT)/hypoxia-induced factor-1 alpha (HIF-1α) signaling pathway and the apoptosis of nucleus pulposus cells (NPCs) under different oxygen concentrations to clarify the biological characteristics of NPCs and the molecular mechanism of intervertebral disc degeneration (IVDD). Normal and degenerated human NPCs were collected. Leibovitz's medium with 100 μmol/L CoCl2 was used to establish a hypoxic culture environment, and 100 μmol/L H2O2 was used to establish an oxidative stress culture environment. Third-generation NPCs were divided into 6 groups: normal NPCs + hypoxia, normal NPCs + normoxia, normal NPCs + oxidative stress, degenerated NPCs + hypoxia, degenerated NPCs + normoxia, and degenerated NPCs + oxidative stress. Normal NPCs + hypoxia was used as the experimental control group. Cell viability and proliferation were detected by using the Cell Counting Kit-8 (CCK-8) method. Cell apoptosis rate was assessed by flow cytometry, and expression levels of PI3K, AKT, and HIF-1α were determined by Real-Time-Polymerase Chain Reaction (RT-PCR) and Western blotting. The cell proliferation rate of both normal and degenerated NPCs decreased with increasing oxygen concentration. Conversely, the apoptosis rate increased as the oxygen concentration increased (p<0.05). Compared with the control group, whether the cells degenerated had a very significant impact on the apoptosis rate (p<0.001), and oxygen concentration also had a highly significant impact on both the cell proliferation rate and apoptosis rate (both p<0.001). The interaction between cell degeneration and oxygen concentration significantly affected both cell proliferation and apoptosis rates (p<0.05). Considering the expression levels of PI3K, AKT, and HIF-1α, normal NPCs had the highest levels under low oxygen concentrations, followed by oxidative stress and normoxia. In degenerated NPCs, the expression levels also decrease as the oxygen concentration increases. The PI3K/Akt/HIF-1α signaling pathway plays a significant role in inhibiting oxidative stress, antagonizing NPC apoptosis, and consequently delaying IVDD.

Dysfunction of STING Autophagy Degradation in Senescent Nucleus Pulposus Cells Accelerates Intervertebral Disc Degeneration.

The pathogenesis of Intervertebral Disc Degeneration (IDD) is complex and multifactorial, with cellular senescence of nucleus pulposus (NP) cells and inflammation playing major roles in the progression of IDD. The stimulator of interferon genes (STING) axis is a key mediator of inflammation during infection, cellular stress, and tissue damage. Here, we present a progressive increase in STING in senescent NP cells with the degradation disorder. The STING degradation function in normal NP cells can prevent IDD. However, the dysfunction of STING degradation through autophagy causes the accumulation and high expression of STING in senescent NP cells as well as inflammation continuous activation together significantly promotes IDD. In senescent NP cells and intervertebral discs (IVDs), we found that STING autophagy degradation was significantly lower than that of normal NP cells and IVDs when STING was activated by 2'3'-cGAMP. Also, the above phenomenon was found in STINGgt/gt, cGAS-/- mice with models of age-induced, lumbar instability-induced IDD as well as found in the rat caudal IVD puncture models. Taken together, we suggested that the promotion of STING autophagy degradation in senescent NP Cells demonstrated a potential therapeutic modality for the treatment of IDD.

Identification of a potential novel biomarker in intervertebral disk degeneration by bioinformatics analysis and experimental validation.

Intervertebral disk degeneration (IVDD) is a major cause of low back pain and one of the most common health problems all over the world. However, the early diagnosis of IVDD is still restricted. The purpose of this study is to identify and validate the key characteristic gene of IVDD and analyze its correlation with immune cell infiltration. 3 IVDD-related gene expression profiles were downloaded from the Gene Expression Omnibus database to screen for differentially expressed genes (DEGs). Gene Ontology (GO) and gene set enrichment analysis (GSEA) were conducted to explore the biological functions. Two machine learning algorithms were used to identify characteristic genes, which were tested to further find the key characteristic gene. The receiver operating characteristic curve was performed to estimate the clinical diagnostic value of the key characteristic gene. The excised human intervertebral disks were obtained, and the normal nucleus pulposus (NP) and degenerative NP were carefully separated and cultured in vitro. The expression of the key characteristic gene was validated by real-time quantitative PCR (qRT-PCR). The related protein expression in NP cells was detected by Western blot. Finally, the correlation was investigated between the key characteristic gene and immune cell infiltration. A total of 5 DEGs, including 3 upregulated genes and 2 downregulated genes, were screened between IVDD and control samples. GO enrichment analysis showed that DEGs were enriched to 4 items in BP, 6 items in CC, and 13 items in MF. They mainly included the regulation of ion transmembrane transport, transporter complex, and channel activity. GSEA suggested that the cell cycle, DNA replication, graft versus host disease, and nucleotide excision repair were enriched in control samples, while complement and coagulation cascades, Fc γ R-mediated phagocytosis, neuroactive ligand-receptor interaction, the NOD-like receptor signaling pathway, gap junctions, etc., were enriched in IVDD samples. Furthermore, ZNF542P was identified and tested as key characteristic gene in IVDD samples through machine learning algorithms and showed a good diagnostic value. The results of qRT-PCR showed that compared with normal NP cells, the expression of ZNF542P gene was decreased in degenerated NP cells. The results of Western blot suggested that compared with normal NP cells, the expression of NLRP3 and pro Caspase-1 was increased in degenerated NP cells. Finally, we found that the expression of ZNF542P was positively related to the proportions of T cells gamma delta (γδT cells). ZNF542P is a potential biomarker in the early diagnosis of IVDD and may be associated with the NOD-like receptor signaling pathway and the infiltration of γδT cells.

PLK1 Mitigates Intervertebral Disc Degeneration by Delaying Senescence of Nucleus Pulposus Cells.

Intervertebral disc degeneration (IVDD) is the primary cause of low back pain; however, the molecular mechanisms involved in the pathogenesis of IVDD are not fully understood. Polo-like kinase 1 (PLK1) plays numerous roles in the cell cycle, including in cell proliferation and senescence. To investigate the involvement of PLK1 in IVDD, we used patient tissues and an animal model of IVDD. Samples were analyzed via immunoblotting, quantitative real-time polymerase chain reaction (qPCR), immunofluorescence, and immunohistochemistry. Our results demonstrated that PLK1 expression was decreased in nucleus pulposus cells (NPCs) of degenerative IVDs. The inhibition of PLK1 kinase activity in normal NPCs increased the expression of p53 protein, inhibited cell proliferation, and induced senescence. Our results suggest that PLK1 regulates the degeneration of the IVD through p53, revealing the function and mechanism of PLK1 in IVDD and providing a theoretical basis and experimental evidence for the potential treatment of low back pain.

LncRNA HOTAIR influences cell proliferation via miR-130b/PTEN/AKT axis in IDD

ABSTRACT Intervertebral disc degeneration (IDD) constitutes the pathological foundation of most musculoskeletal disorders of the spine. Previous studies have noted that cell proliferation is a common feature of IDD. Bioinformatics indicated that aberrantly expressed long non-coding RNAs (lncRNAs) were involved in the development of IDD. In this study, we aimed to investigate the function of lncRNA HOTAIR in the proliferation of human nucleus pulposus (NP) cells of IDD in vitro and further clarified its mechanism. The expression of HOTAIR and miR-130b was quantified by qRT-PCR in nucleus pulposus (NP) tissues. Furthermore, NP cells proliferation were assayed by CCK8 and Immunostaining. Dual-luciferase reporter and RIP assay were used to examine the expression of HOTAIR, PTEN, and their co-target gene miR-130b. Western blotting was used to test AKT expression. Our in vitro experiments on human normal NP cells observed that HOTAIR was significantly dysregulated in IDD. Further, HOTAIR can suppress proliferation by directly targeting miR-130b. In addition, Both HOTAIR and PTEN were confirmed to target miR-130b, and miR-130b upregulation reversed the phenomenon of ectopic expression of HOTAIR. More importantly, HOTAIR upregulation significantly reduced CyclinD1 protein expression by PTEN/AKT signaling pathway. Our findings suggest that HOTAIR may bind to miR-130b and subsequently increased CyclinD1 expression via PTEN/Akt pathway. Thereby, HOTAIR could become a potential target for the treatment of IDD. Abbreviations : IDD; intervertebral disc degenerationncRNAs; non-coding RNAslncRNAs; long non-coding RNAsmiRNAs; microRNAsNP; nucleus pulposusqRT-PCR; quantitative reverse transcription–PCR LBP; Low back pain ORF; open reading frame HOTAIR; Hox transcript antisense intergenic RNA FAF1; Fas-associated protein factor-1Erk; extracellular signal-regulated kinase TUG1; Taurine Up-regulated Gene 1 HIF1A hypoxia-inducible factor 1-alpha PI3K; phosphoinositide-3 kinase AIS; adolescent idiopathic scoliosis ECM; extracellular matrix LN;lupus nephritis CT;computed tomography MRI; magnetic resonance imaging PBS; phosphate-buffered salin PBS; phosphate-buffered salin PVDF; polyvinylidene fluoride TBST; Tris-buffered saline Tween ECL; enhanced chemiluminescenceRIP; RNA immunoprecipitation

TNF-α-stimulated nucleus pulposus cells induce cell apoptosis through the release of exosomal miR-16 targeting IGF-1 and IGF-1R in rats.

BackgroundExosomes may contain excess cellular components released by cells in response to harmful external stimuli to maintain cellular homeostasis. Inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), can induce cell apoptosis, alter cellular component expression levels, and stimulate exosome release. In this study, we examined whether exosomes released from nucleus pulposus cells (NPCs) under inflammatory conditions could induce normal NP cell apoptosis in rats and its underlining mechanism.MethodsExosomes were isolated from TNF-α‐treated NPCs and used to treat normal NPCs. The effects were assessed by flow cytometry and western blot analysis. Anti-apoptotic insulin-like growth factor-1 (IGF-1) expression in NPCs was assessed by western blot analysis. Given the exosomal miRNAs might be the key factors of exosomes, bioinformatics approaches and quantitative real-time polymerase chain reaction (qRT-PCR) were used to identify IGF-1-regulating micro RNAs (miRNAs), including miR-16. Luciferase reporter assay assessed miR-16 regulation of IGF-1 and IGF-1 receptor (IGF-1R). NPCs were transfected with miR-16 mimic, and exosomes were applied to normal NPCs. NPCs were pretreated with 10 ng/mL TNF-α, transfected with miR-16 inhibitors, and the exosomes were isolated. Cell and exosome miR-16 levels were detected by qRT-PCR. Western blot analysis determined IGF-1, IGF-1R, and apoptotic marker levels in exosome-treated NPCs.ResultsExosomes from TNF-α-treated NPCs induced apoptosis in normal NPCs and repressed IGF-1 expression. Exosomal miR-16 regulated IGF-1 and induced NPC apoptosis. The dual-luciferase reporter assay revealed that miR-16 binds the 3' untranslated regions (3'-UTRs) of IGF-1 and IGF-1R. Exosomal miR-16 repressed IGF-1 and the IGF-1R/phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway which therefore induced NPC apoptosis. Rescue experiments using miR-16 inhibitors further validated these findings.ConclusionsThe inflammatory factor TNF-α stimulated exosome release from NPCs, which induced the apoptosis of normal NPCs through the actions of exosomal miR-16. Exosomal miR-16 directly repressed the anti-apoptotic IGF-1/IGF-1R pathway, increasing the apoptosis of NPCs.

LncRNA GAS5 as miR-26a-5p Sponge Regulates the PTEN/PI3K/Akt Axis and Affects Extracellular Matrix Synthesis in Degenerative Nucleus Pulposus Cells in vitro.

The role of lncRNA growth arrest specific 5 (GAS5) in degenerative nucleus pulposus cell (NPC) apoptosis has been reported, but the mechanism of GAS5 in extracellular matrix (ECM) synthesis in intervertebral disc degeneration (IDD) remains unknown. We aimed to investigate the mechanism of GAS5 in ECM synthesis in degenerative NPCs. GAS5 expression was measured in degenerative NPCs (CP-H170) and normal NPCs (CP-H097). siRNA-mediated GAS5 knockdown was transfected to NPCs to detect cell viability and the expression of ECM-related genes (Collagen II, aggrecan, Collagen I, and MMP-3). Subcellular localization of GAS5 was analyzed. The downstream gene and pathway of GAS5 in degenerative NPCs were explored. As our results indicated, lncRNA GAS5 was upregulated in degenerative NPCs. Silencing GAS5 improved the viability of degenerative NPCs and increased ECM synthesis. GAS5 was mainly located in the cytoplasm of NPCs. LncRNA GAS5 sponged miR-26a-5p to regulate PTEN. Overexpression of miR-26a-5p promoted ECM synthesis in degenerative NPCs. Akt inhibitor LY294002 reversed the promotion of silencing GAS5 on ECM synthesis of degenerative NPCs. In conclusion, lncRNA GAS5 sponged miR-26a-5p to upregulate PTEN and inhibit the PI3K/Akt pathway, thus inhibiting ECM synthesis of degenerative NPCs.

LncRNA H19 Aggravates Intervertebral Disc Degeneration by Promoting the Autophagy and Apoptosis of Nucleus Pulposus Cells Through the miR-139/CXCR4/NF-κB Axis.

The etiology of lumbocrural pain is closely related to intervertebral disc degeneration (IDD). Long noncoding RNAs (lncRNAs) serve crucial roles in IDD progression. This study investigated the effect of lncRNA H19 on autophagy and apoptosis of nucleus pulposus cells (NPCs) in IDD. The rat model of IDD was established. Normal NPCs and degenerative NPCs (DNPCs) were cultured in vitro. H19 expression in IDD rat was detected. DNPCs were treated with si-H19 to evaluate autophagy and apoptosis of DNPCs. The binding relationships between H19 and miR-139-3p, and miR-139-3p and CXCR4 were verified. DNPCs were co-transfected si-H19 and miR-139-3p inhibitor. The phosphorylation of NF-κB pathway-related p65 in DNPCs was detected. LncRNA H19 was upregulated in IDD rats. Downregulation of H19 inhibited autophagy and apoptosis of DNPCs. LncRNA H19 sponged miR-139-3p to inhibit CXCR4 expression. si-H19 and miR-139-3p inhibitor co-treatment induced autophagy and apoptosis, and enhanced CXCR4 expression. si-H19 decreased p-p65 phosphorylation, while si-H19 and miR-139-3p inhibitor co-treatment partially elevated p-p65 phosphorylation. In conclusion, lncRNA H19 facilitated the autophagy and apoptosis of DNPCs by the miR-139-3p/CXCR4/NF-κB axis, thereby aggravating IDD. This study may offer new insights for the management of IDD.

Propionibacterium acnes Accelerates Intervertebral Disc Degeneration by Inducing Pyroptosis of Nucleus Pulposus Cells via the ROS-NLRP3 Pathway.

Our previous study verified the occurrence of Propionibacterium acnes (P. acnes), a low-virulence anaerobic bacterium, latently residing in degenerated intervertebral discs (IVDs), and the infection had a strong association with IVD degeneration. We explored whether P. acnes induces nucleus pulposus cell (NPC) pyroptosis, a more dangerous cell death process than apoptosis, and accelerates IVD degeneration via the pyroptotic products interleukin- (IL-) 1β and IL-18. After coculturing with P. acnes, human NPCs showed significant upregulation of NOD-like receptor 3 (NLRP3), cleaved IL-1β, cleaved caspase-1, and cleaved gasdermin D in response to the overexpression of IL-1β and IL-18 in a time- and dose-dependent manner. In addition, the gene expression of inflammatory factors and catabolic enzymes significantly increased in normal NPCs when cocultured with pyroptotic NPCs in a transwell system, and the adverse effects were inhibited when NPC pyroptosis was suppressed. Furthermore, inoculation of P. acnes into the IVDs of rats caused significant pyroptosis of NPCs and remarkable IVD degeneration. Finally, coculture of NPCs with P. acnes induced the overexpression of reactive oxygen species (ROS) and NLRP3, while inhibition of both factors reduced NPC pyroptosis. Therefore, P. acnes induces NPC pyroptosis via the ROS-NLRP3 signaling pathway, and the pyroptotic NPCs cause an IVD degeneration cascade. Targeting the P. acnes-induced pyroptosis of NPCs may become an alternative treatment strategy for IVD degeneration in the future.

Autophagy-activated nucleus pulposus cells deliver exosomal miR-27a to prevent extracellular matrix degradation by targeting MMP-13.

Although autophagy may be beneficial for maintaining the metabolic balance of the extracellular matrix (ECM) in the nucleus pulposus (NP) and its vitality under inflammation, the underlying mechanism still remains unclear. A previous study found that autophagy activation stimulated the release of exosomes in normal chondrocytes, which are located in a similar avascular environment and share many common features with those of nucleus pulposus cells (NPCs). This study explored the protective effect on matrix degradation in the NP by exosomes derived from autophagy-activated NPCs and exosomal microRNAs. NPCs-derived exosomes (NPCs-Exos) were isolated from culture medium of either normal NPCs or rapamycin-treated NPCs and quantified by nanoparticle tracking analysis. The effect of rapamycin-treated NPC-derived exosomes on NPCs were assessed by coculture with interleukin 1β (IL-1β)-stimulated NPCs. After examination of six major proteinases of the ECM, matrix metalloproteinase 13 (MMP-13) was chosen for further study. miR-27a, which targets MMP-13, was investigated through previous studies and bioinformatics tool. The levels of miR-27a were upregulated in both rapamycin-treated NPCs and their exosomes, compared to the control. When exosomal miR-27a was transferred into NPCs, it alleviated IL-1β-induced degradation of the NPC ECM by targeting MMP-13. Autophagy activation may promote the release of NPCs-derived exosomes and thereby prevent the NPC matrix from degradation. Autophagy activation also alleviates intervertebral disc degeneration (IDD), at least partly via exosomal miR-27a, which restrains MMP-13 expression under IL-1β stimulation. Our work elucidates a new mechanism for how autophagy may participate in preventing IDD, which may be a promising therapeutic strategy.

Upregulated lnc‑HRK‑2:1 prompts nucleus pulposus cell senescence in intervertebral disc degeneration.

Intervertebral disc (IVD) degeneration is a complicated physiological change involving cellular senescence, inflammation and the degradation of the extracellular matrix. Long non-coding RNAs (lncRNAs) have been identified as new players in IVD degeneration. The present study aimed to identify lncRNAs implicated in IVD degeneration via the regulation of cellular senescence. In the present study, nucleus pulposus (NP) cells isolated from moderately degenerated IVD tissues exhibited a senescent phenotype with increased senescence rates, detected by senescence-associated β-galactosidase (SA-β-gal) staining, and reduced growth and migratory abilities. Microarray and target prediction analyses identified 353 differentially expressed lncRNAs, and 251 cis- and 2,170 trans-acting targets in degenerated NP cells. Bioinformatic analyses revealed that these predicted targets were enriched in the regulation of response to DNA damage stimulus, positive regulation of cell cycle processes and interferon-β production. In addition, a network of the top 10 upregulated and top 10 downregulated lncRNA targets was constructed, and two trans-acting targets, C-C motif chemokine ligand 5 (CCL5) and polyribonucleotide nucleotidyltransferase 1 (PNPT1) involved in aging or senescence, and their corresponding lncRNAs, lnc-ST8SIA5-1:2 and lnc-HRK-2:1, were identified. Reverse transcription-quantitative PCR validation demonstrated that the two targets and two candidate lncRNAs were significantly upregulated in degenerated NP cells. Overexpression of lnc-HRK-2:1, with validated higher expression levels, in normal NP cells induced a senescent phenotype, with enhanced rates of senescence detected by SA-β-gal staining in cells, decreased growth and migratory abilities and improved expression levels of CCL5 and PNPT1. Collectively, these results suggested that upregulation of lnc-HRK-2:1 prompted NP cell senescence in IVD degeneration, which may be associated with increased expression levels of CCL5 and PNPT1.

MicroRNA‑25‑3p regulates human nucleus pulposus cell proliferation and apoptosis in intervertebral disc degeneration by targeting Bim

Intervertebral disc degeneration (IDD) is a degenerative disease of the spine originating from the intervertebral disc. MicroRNAs (miRNAs or miRs) are a group of endogenous small non-coding RNAs that act on target genes and play a critical role in numerous biological processes. However, the underlying mechanism of miR-25-3p in IDD remains unclear. Therefore, the present study aimed to explore the role of miR-25-3p in the pathogenesis of IDD. The results demonstrated that miR-25-3p was downregulated in rat degenerative nucleus pulposus (NP) cells and that Bcl-2 interacting mediator of cell death (Bim) was a direct target of miR-25-3p. Next, to investigate the effect of miR-25-3p on normal NP cell proliferation and apoptosis, NP cells were transfected with an miR-25-3p inhibitor, a negative control of miR-25-3p inhibitor, miR-25-3p inhibitor + control-small interference RNA (siRNA) or miR-25-3p inhibitor + Bim-siRNA for 48 h and cell proliferation and apoptosis were then analyzed. The results demonstrated that the miR-25-3p inhibitor could decrease NP cell proliferation and induce cell apoptosis, and these effects were reversed by Bim-siRNA. In addition, an in vitro cell model of IDD was established by subjecting NP cells to 10 ng/ml interleukin (IL)-1β for 24 h. Further experiments suggested that IL-1β treatment induced a reduction in NP cell proliferation and an increase in cell apoptosis, which were prevented by the miR-25-3p mimic. All the effects of miR-25-3p mimic on IL-1β-treated NP cells were significantly reversed by Bim upregulation. These findings suggested that miR-25-3p may be a novel therapeutic target for IDD prevention.

SIRT1 alleviates high-magnitude compression-induced senescence in nucleus pulposus cells via PINK1-dependent mitophagy.

Mechanical overloading-induced nucleus pulposus (NP) cells senescence plays an important role in the pathogenesis of intervertebral disc degeneration (IVDD). The silent mating type information regulator 2 homolog-1 (SIRT1)-mediated pathway preserves the normal NP cell phenotype and mitochondrial homeostasis under multiple stresses. We aimed to investigate the role of SIRT1 in IVDD by assessing the effects of SIRT1 overexpression on high-magnitude compression-induced senescence in NP cells. High-magnitude compression induced cellular senescence and mitochondrial dysfunction in human NP cells. Moreover, SIRT1 overexpression tended to alleviate NP cell senescence and mitochondrial dysfunction under compressive stress. Given the mitophagy-inducing property of SIRT1, activity of mitophagy was evaluated in NP cells to further demonstrate the underlying mechanism. The results showed that SIRT1-overexpression attenuated senescence and mitochondrial injury in NP cells subjected to high-magnitude compression. However, depletion of PINK1, a key mitophagic regulator, impaired mitophagy and blocked the protective role of SIRT1 against compression induced senescence in NP cells. In summary, these results suggest that SIRT1 plays a protective role in alleviating NP cell senescence and mitochondrial dysfunction under high-magnitude compression, the mechanism of which is associated with the regulation of PINK1-dependent mitophagy. Our findings may provide a potential therapeutic approach for IVDD treatment.

Effects of the NF‑κB/p53 signaling pathway on intervertebral disc nucleus pulposus degeneration.

The incidence of intervertebral disc degeneration (IDD) is increasing, especially among elderly individuals. The present study aimed to investigate the effects of the NF-κB/p53 signaling pathway on IDD and its regulatory effect on associated cytokines. In the present study, human nucleus pulposus cells were isolated from patients with thoracic-lumbar fractures and patients with IDD to observe cellular morphology and detect phosphorylated (p)-p65/p53 expression levels. The locality and expression levels of p65 in interleukin (IL)-1β-stimulated nucleus pulposus cells, with or without the addition of ammonium pyrrolidinedithiocarbamate (PDTC; a NF-κB signaling pathway-specific blocker), were measured. Furthermore, the effects of IL-1β stimulation on the protein and gene expression levels of IDD-related cytokines were determined following p53 knockdown and inhibition of the NF-κB signaling pathway. The results suggested that p-p65 and p53 expression was significantly increased in IDD cells compared with normal nucleus pulposus cells. Moreover, nucleus pulposus cells isolated from patients with IDD contained less cytoplasm compared with normal nucleus pulposus cells, and p65 expression levels were higher in the cytoplasm than the nucleus of IL-1β-stimulated PDTC-treated healthy nucleus pulposus cells. Moreover, the p53 expression levels were significantly decreased following transfection with sip53. PDTC treatment and p53 knockdown significantly decreased matrix metallopeptidase (MMP)-3, MMP-13, metallopeptidases with thrombospondin type 1 motif (ADAMTS)-4 and ADAMTS-5 expression levels, and increased aggrecan and collagen type II expression levels in IL-1β-stimulated cells. The present study indicated that activation of the NF-κB/p53 signaling pathway might be related to the occurrence of IDD; therefore, the NF-κB/p53 signaling pathway may serve as a therapeutic target for IDD.

Effect of miR-202-5p-mediated ATG7 on autophagy and apoptosis of degenerative nucleus pulposus cells.

This study aimed to research the effect of miR-202-5p-mediated ATG7 on autophagy and apoptosis of degenerative nucleus pulposus cells. The intervertebral disc nucleus pulposus (NP) tissue of patients with intervertebral disc degenerative disease and normal intervertebral disc nucleus pulposus (NP) tissue of patients with spinal fractures was collected as the research object. Normal NP cells and degenerative NP cells were isolated. Low expression of miR-202-5p and overexpression of ATG7 were carried out in degenerative NP cells. The expression of miR-202-5p and ATG7 mRNA was detected by RT-PCR. The expression of ATG7, LC3-II, Bax, and Bcl-2 proteins was detected by Western blot. The autophagy of cells was detected by MDC staining. The apoptosis of NP cells was detected by flow cytometry. The targeting relationship between miR-202-5p and ATG7 was detected by Dual-Luciferase reporter. In the degenerative NP tissues, miR-202-5p was highly expressed and ATG7 was low expressed. The inhibition of miR-202-5p expression can effectively promote autophagy of NP cells, increase the expression of ATG7 and LC3-II, inhibit the apoptosis of NP cells, inhibit the expression of pro-apoptotic proteins Bax, and promote the expression of pro-apoptotic proteins Bcl-2 proteins. The upregulation of ATG7 expression in degenerative NP cells alone had the same effect as the downregulation of miR-202-5p. The assay of the Dual-Luciferase reporter confirmed the targeting relationship between miR-202-5p and ATG7. MiR-202-5p can affect the autophagy and apoptosis of degenerative nucleus pulposus cells through targeted adjustment of ATG7, which may be a new therapeutic target for intervertebral disc degenerative diseases.

Biological activity and regulatory effect of extracellular matrix expression of bone marrow mesenchymal stem cellsconditioned medium on the degenerated nucleus pulposus cells

Objective To observe the biological activities and expression of extracellular matrix of degenerative nucleus pulposus cells which were cultured in the bone marrow mesenchymal stem cells conditioned medium. Methods Bone marrow mesenchymal stem cells were extracted from the femur and tibia of adult SD rats. After subculture, the conditioned medium of bone marrow mesenchymal stem cells was collected. We destroy the annulus fibrosus of the adult rats, and the intervertebral disc was obtained 2 weeks later. Degenerative rat model was used to extract rat nucleus pulposus cells. The nucleus pulposus cells were cultured in the bone marrow mesenchymal stem cells conditioned medium for 24 hours. Results Compared with normal nucleus pulposus cells, the activity of degenerative nucleus pulposus cells [(59.43±12.33)%, t=11.647, P 0.05) and type Ⅱ collagen in cells (1.52±0.18, t=0.299, P>0.05, 1.21±0.31, t=1.543, P>0.05), proteoglycan (1.81±0.32, t=0.486, P>0.05, 0.99±0.16, t=0.094, P>0.05) and matrix metalloproteinase 3 (0.24±0.14, t=0.486, P>0.05, 0.55±0.11, t=0.593, P>0.05) Protein and mRNA expression levels were not significantly affected. Conclusion The bone marrow mesenchymal stem cell conditioned medium promoted the proliferation of extramedullary nucleus cells and the expression of extracellular matrix proteins. Key words: Intervertebral disc herniation; Nucleus pulposus cells; Extracellular matrix protein; Bone marrow mesenchymal stem cell conditioned medium; Type Ⅱ collagen; Aggrecan; Matrix metalloproteinase 3

Upregulation of suppressor of cytokine signaling 3 ameliorates spinal degenerative disease in adolescents by mediating leptin and tumor necrosis factor-α levels.

Spinal degenerative changes may occur following the rapid growth observed in adolescents, causing a reduced quality of life. The suppressor of cytokine signaling (SOCS) is involved in various degenerative diseases. The current study recruited adolescents with spinal degenerative disease (SDD) to identify the effect of SOCS-3 on leptin and tumor necrosis factor-α (TNF-α) levels in this disorder. From January 2010 to January 2016, 120 adolescents (aged 14 to 25) were enrolled in the current study, with 68 diagnosed with SDD and the remaining 52 treated as controls. Nucleus pulposus cells (NPCs) were extracted and cultured in vitro. TNF-α levels in NPCs were determined using flow cytometry. Degenerative NPCs were then transfected with pCR3.1-SOCS-3 and ELISA was performed to determined TNF-α and leptin levels. RT-qPCR was performed to measure the mRNA level of SOCS-3 and leptin in NPCs and western blotting was utilized to detect the protein level of leptin and the extent of leptin receptor phosphorylation. The results revealed that TNF-α levels in degenerative NPCs were higher than those in normal NPCs. The overexpression of SOCS-3 reduced levels of TNF-α and leptin in degenerative NPCs. In addition, the upregulation of leptin increased SOCS-3 levels in a concentration-dependent manner. Furthermore, the expression of the leptin receptor and phosphorylated leptin receptor gradually decreased with increasing leptin concentrations and the level of phosphorylated leptin receptor negatively correlated with SOCS-3 expression. The inductive effect of leptin on the level of SOCS-3 and the inhibitory effect of SOCS-3 on the activity of leptin were identified. The current study demonstrated that SOCS-3 reduces leptin and TNF-α levels in degenerative NPCs from adolescents, indicating its potential role in the development of novel SDD therapies.

Roles of TREM2 in degeneration of human nucleus pulposus cells via NF-κB p65.

Nuclear factor (NF)-κB p65 plays a key role in the development of intervertebral disc degeneration (IDD). Herein, we found that messenger RNA levels of human triggering receptor expressed on myeloid cells-2 (TREM2) and NF-κB p65 were upregulated and strongly positive correlated (r2 = 0.299, P = 0.0126) in nucleus pulposus (NP) tissues of patients with IDD. To investigate the role of TREM2 in the development of IDD and whether NF-κB p65 was the underlying mechanism, whereby TREM2 played its role, we established TREM2-siRNA-transfected human degenerative NP cells and TREM2-overexpression vector-transfected human normal NP cells. Degeneration of human NP cells was assessed by measuring cell apoptosis, cell proliferation, and the secretion of tumor necrosis factor-α, interleukin (IL)-1β, and IL-6. Protein levels of Bcl2, Bax, total NF-κB p65 in whole cell lysates, cytoplasm NF-κB p65, and nuclear NF-κB p65 were determined to evaluate underlying mechanisms. Our data elucidated that TREM2 silencing was a therapy for human degenerative NP cells through inhibiting cell apoptosis, promoting cell proliferation, suppressing production of tumor necrosis factor-α, IL 1β, and IL-6, and the mechanisms included decreasing Bax while enhancing Bcl2, downregulating total NF-κB p65, and retarding NF-κB p65 nuclear translocation. On the contrary, upregulated TREM2 showed the opposite effects, accelerating the degeneration of human normal NP cells. Downregulating TREM2 and total NF-κB p65 and inhibiting NF-κB p65 nucleus translocation were also confirmed in NP tissue samples of four IDD rats. We concluded that TREM2 functioned as a promoter in the degeneration of human NP cells. Downregulating TREM2 may be a novel therapeutic strategy for human IDD.

Circular RNA GRB10 as a competitive endogenous RNA regulating nucleus pulposus cells death in degenerative intervertebral disk

Intervertebral disc degeneration (IDD) is an important factor leading to low back pain, but the underlying mechanisms remain poorly understood. Compared with normal nucleus pulposus (NP) tissues, the expression of circ-GRB10 was downregulated in IDD. Furthermore, overexpression of circ-GRB10 inhibited NP cell apoptosis. circ-GRB10 could sequester miR-328-5p, which could potentially lead to the upregulation of target genes related to cell proliferation via the ErbB pathway. In conclusion, the present study revealed that circ-GRB10/miR-328-5p/ERBB2 signaling pathway is involved in IDD development, suggesting that circ-GRB10 might be a novel therapeutic target for IDD.

Methylation of microRNA-129-5P modulates nucleus pulposus cell autophagy by targeting Beclin-1 in intervertebral disc degeneration.

MicroRNAs play an important role in the etiology and progression of many diseases, including intervertebral disc degeneration (IVDD). The miRNA miR-129-5P regulates autophagy in various cancers, but its role in human nucleus pulposus (NP) cells is unclear. The present study investigated whether miR-129-5p regulates the expression of Beclin-1 which is known to induce autophagy in NP cells by evaluating their levels in normal and degenerative disc tissues and human NP cells transfected with miR-129-5P mimic or inhibitor by quantitative real-time (qRT-)PCR, western blotting, flow cytometry, and immunofluorescence analysis. A bioinformatics analysis was used to predict the relationship between miR-129-5P and Beclin-1, which was confirmed by the dual luciferase assay. DNA methylation status was assessed by methylation-specific PCR, and the effect of demethylation on miR-129-5P level and autophagy was examined by qRT-PCR, western blotting, and flow cytometry. We found that miR-129-5P expression was downregulated while that of Beclin-1 and LC3-II was upregulated in degenerative disc NP cells. Meanwhile, autophagy was reduced in human NP cells transfected with miR-129-5P mimic, whereas the opposite result was observed upon treatment with miR-129-5P inhibitor. Bioinformatics analysis and the luciferase reporter assay revealed that Beclin-1 is a target of and is inhibited by miR-129-5P. We also found that CpG islands in the miR-129-5P promoter region were hypermethylated in degenerative as compared to normal disc tissue. Thus, miR-129-5P blocks NP cell autophagy by directly inhibiting Beclin-1, a process that is dependent on miR-129-5P promoter methylation.