Aspirin-Mediated Attenuation of Intervertebral Disc Degeneration by Ameliorating Reactive Oxygen Species In Vivo and In Vitro.
ABSTRACT: Intervertebral disc (IVD) degeneration (IDD) is a major cause of low back pain. The pathogenesis of IDD is associated with the disturbance of reactive oxygen species (ROS) equilibrium, inflammation, and matrix loss. Aspirin is a nonsteroidal anti-inflammatory drug that effectively inhibits inflammation and oxidative stress and has been widely used for the treatment of back pain. Therefore, we hypothesize that aspirin reverses the IDD process via antioxidative and anti-inflammatory effects on the AMPK signaling pathway. In vitro, aspirin diminished cellular oxygen free radicals (ROS, nitric oxide (NO)) and inflammatory cytokines (interleukin- (IL-) 1? and IL-6 and tumor necrosis factor alpha (TNF-?)) induced by lipopolysaccharides (LPS) in nucleus pulposus cells (NPCs). We found that aspirin preserved the extracellular matrix (ECM) content of collagen type II (COL2) and aggrecan while inhibiting the expression of matrix-degenerating enzymes, including matrix metalloproteinase 3 and 13 (MMP-3 and MMP-13) and A disintegrin and metalloproteinase with thrombospondin motifs 4 and 5 (ADAMTS-4, ADAMTS-5). Aspirin significantly promoted the ratios of p-AMPK to AMPK and p-ACC to ACC expression in NPCs. Furthermore, pretreatment with the AMPK inhibitor compound C abrogated the antioxidant effects of aspirin. In vivo, an IDD model was established in Sprague-Dawley rats via percutaneous disc puncture with the 20-gauge needle on levels 8-9 and 9-10 of the coccygeal vertebrae. Imaging assessment showed that after aspirin treatment, improvements in disc height index (DHI) ranged from 1.22-fold to 1.54-fold and nucleus pulposus signal strength improved from 1.26-fold to 1.33-fold. Histological analysis showed that aspirin treatment prevented the loss of COL2 and decreased MMP-3 and MMP-13, inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), IL-1?, and TNF-? expression in the IVD tissues. These results suggest that treatment with aspirin could reverse the IDD process via the AMPK signaling pathway, which provides new insights into the potential clinical applications of aspirin, particularly for IDD treatment.
Project description:Cordycepin is a component of the extract obtained from Cordyceps militaris and has many biological activities, including anti-cancer, anti-metastatic and anti-inflammatory effects. Intervertebral disc degeneration (IDD) is a degenerative disease that is closely related to the inflammation of nucleus pulposus (NP) cells. The effect of cordycepin on NP cells in relation to inflammation and degeneration has not yet been studied. In our study, we used a rat NP cell culture and an intervertebral disc (IVD) organ culture model to examine the inhibitory effects of cordycepin on lipopolysaccharide (LPS)-induced gene expression and the production of matrix degradation enzymes (MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5) and oxidative stress-associated factors (nitric oxide and PGE2). We found a protective effect of cordycepin on NP cells and IVDs against LPS-induced matrix degradation and macrophage infiltration. In addition, western blot and luciferase assay results demonstrated that pretreatment with cordycepin significantly suppressed the LPS-induced activation of the NF-κB pathway. Taken together, the results of our research suggest that cordycepin could exert anti-inflammatory and anti-degenerative effects on NP cells and IVDs by inhibiting the activation of the NF-κB pathway. Therefore, cordycepin may be a potential treatment for IDD in the future.
Project description:Intervertebral disc (IVD) degeneration is a major cause of low back pain. The transcription factor c-Fos/Activator Protein-1 (AP-1) controls the expression of inflammatory cytokines and matrix metalloproteinases (MMPs) that contribute to the pathogenesis IVD degeneration. We investigated the effects of inhibition of c-Fos/AP-1 on IVD degeneration and associated pain. A selective inhibitor, T-5224, significantly suppressed the interleukin-1?-induced up-regulation of Mmp-3, Mmp-13 and Adamts-5 transcription in human nucleus pulposus cells and in a mouse explant culture model of IVD degeneration. We used a tail disc percutaneous needle puncture method to further assess the effects of oral administration of T-5224 on IVD degeneration. Analysis of disc height, T2-magnetic resonance imaging (MRI) findings, and histology revealed that IVD degeneration was significantly mitigated by T-5224. Further, oral administration of T-5224 ameliorated pain as indicated by the extended tail-flick latency in response to heat stimulation of rats with needle-puncture-induced IVD degeneration. These findings suggest that the inhibition of c-Fos/AP-1 prevents disc degeneration and its associated pain and that T-5224 may serve as a drug for the prevention of IVD degeneration.
Project description:Surgery is the final choice for most patients with intervertebral disc degeneration (IDD). Operation-caused trauma will cause inflammation in the intervertebral disc. Serious inflammation will cause tissue defects and induce tissue degeneration, IDD recurrence and the occurrence of other diseases. Therefore, we proposed a scheme to treat recurrence after discectomy by inhibiting inflammation with an aspirin (ASP)-loaded hydrogel to restore the mechanical stability of the spine and relieve local inflammation. ASP-liposomes (ASP-Lips) were incorporated into a photocrosslinkable gelatin-methacryloyl (GelMA) via mixing. This material can effectively alleviate inflammation by inhibiting the release of high mobility group box 1 (HMGB1) from the nucleus to the cytoplasm. We further assessed the expression of inflammatory cytokines, such as interleukin 6 (IL-6) and tumor necrosis factor-? (TNF-?), and degeneration-related factors, such as type II collagen (COL-2), Aggrecan, matrix metallopeptidases-3 (MMP-3), MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4) and ADAMTS-5 in rat nucleus pulpous cells. The level of IDD was analyzed through H&E, safranin-O staining and immunohistochemistry in rabbit samples. In vitro, we found that ASP-Lip@GelMA treatment significantly decreased inflammatory cytokines, MMP-3 and -13, and ADAMTS-4 and -5 and up-regulated COL-2 and Aggrecan via the inhibited release of HMGB-1 from the nucleus. In vivo, ASP-Lip@GelMA can effectively inhibit inflammation of local tissue after disc surgery and fill local tissue defects. This composite hydrogel system is a promising way to treat the recurrence of IDD after surgery without persistent complications.
Project description:Because miR-146a expression in articular chondrocytes is associated with osteoarthritis (OA), we assessed whether miR-146a is linked to cartilage degeneration in the spine. Monolayer cultures of nucleus pulposus (NP) cells from the intervertebral discs (IVD) of bovine tails were transfected with a miR-146a mimic. To provoke inflammatory responses and catabolic extracellular matrix (ECM) degradation, cells were co-treated with interleukin-1 (IL-1). Transfection of miR-146a decreases IL-1 induced mRNA levels of inflammatory genes and catabolic proteases in NP cells based on quantitative real-time reverse transcriptase PCR (qRT-PCR) analysis. Similarly, miR146a suppresses IL-1 induced protein levels of matrix metalloproteinases and aggrecanases as revealed by immunoblotting. Disc segments from wild type (WT) and miR-146a knockout (KO) mice were cultured ex vivo in the presence or absence of IL-1 for 3days. Histological and immuno-histochemical (IHC) analyses of disc organ cultures revealed that IL-1 mediates changes in proteoglycan (PG) content and in-situ levels of catabolic proteins (MMP-13 and ADAMTS-5) in the nucleus pulposus of the disc. However, these IL-1 effects are more pronounced in miR-146a KO discs compared to WT discs. For example, absence of miR-146a increases the percentage of MMP-13 and ADAMTS-5 positive cells after treatment with IL-1. Thus, miR-146a appears to protect against IL-1 induced IVD degeneration and inflammation. Stimulation of endogenous miR-146a expression or exogenous delivery of miRNA-146a are viable therapeutic strategies that may decelerate disc degeneration and regain a normal homeostatic balance in extracellular matrix production and turn-over.
Project description:Intervertebral disc (IVD) degeneration is associated with local inflammation and increased expression of neurotrophins. Acidic microenvironment is believed to cause the progression of IVD degeneration. However, there is a paucity of information regarding the relationship between acidic microenvironment and the inflammation and expression of neurotrophins in IVD. G-protein-coupled receptor 4 (GPR4) is a pH-sensing receptor, which can activate the inflammation and increase the expression levels of nerve growth factor in acidic microenvironment. In this study, culture media with pH?7.2 (representing the normal IVD-like acidic condition) and pH?6.5 (degenerated IVD-like acidic condition) were prepared. The gene and protein expression levels of GPR4 in SD rat nucleus pulposus cells were determined under the acidic conditions. And cyclic AMP (cAMP), the second messenger of GPR4, was assayed. Furthermore, the expression levels of receptor activator of nuclear factor ? B (RANK), RANKL ligand (RANKL), osteoprotegerin (OPG), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) were also determined. To clarify the involvement of GPR4 in the upregulation of the expression of RANK/RANKL/OPG system and neurotrophins, gene knockdown and forced expression of GPR4 and inhibiting its downstream cAMP accumulation and Ca2+ mobilization were performed. The alternation of the expression levels of matrix metalloproteinase-3 (MMP-3), MMP-13, and aggrecanase-2 (ADAMTS-5) were evaluated by RT-PCR and western blot. The results showed that GPR4 was expressed in rat nucleus pulposus cells, and the expression was upregulated under the degenerated IVD-like acidic microenvironment. cAMP accumulation levels were increased under the degenerated IVD-like acidic culture conditions. The expression levels of RANK, RANKL, OPG, NGF, and BNDF were significantly upregulated under the degenerated IVD-like acidic microenvironment. GPR4 knockdown and reduction of cAMP by the inhibitor SQ22536 abolished the upregulation of the expression of RANK, RANKL, OPG, NGF, and BNDF under the degenerated IVD-like acidic microenvironment. On the opposite, acidosis-induced cAMP accumulation and upregulation of RANK, RANKL, OPG, NGF, and BNDF were further promoted by GPR4 overexpression. The expression levels of MMP-3, MMP-13, and ADAMTS-5 were upregulated under the degenerated IVD-like acidic condition, which can be promoted or attenuated by GPR4 overexpression or knockdown, respectively. We concluded that GPR4-mediated cAMP accumulation was involved in the increased expression of RANK/RANKL/OPG system and neurotrophins by nucleus pulposus cells under the degenerated IVD-like acidic microenvironment.
Project description:P38MAPK mediates cytokine induced inflammation in nucleus pulposus (NP) cells and involves in multiple cellular processes which are related to intervertebral disc degeneration (IDD). The aim of this study was to investigate the expression, activation and function of p38 MAPK isoforms (?,?, ? and ?) in degenerative NP and the effect of p38 activation in NP cells on macrophage polarization. P38 ?, ? and ? isoforms are preferential expressed, whereas the p38? isoform is absent in human NP tissue. LV-sh-p38?, sh-p38? transfection in NP cells significantly decreased the ADAMTS-4,-5, MMP-13,CCL3 expression and restored collagen-II and aggrecan expression upon IL-1? stimulation. As compared with p38? and p38?, p38? exhibited an opposite effect on ADAMTS-4,-5, MMP-13 and aggrecan expression in NP cells. Furthermore, the production of GM-CSF and IFN? which were trigged by p38? or p38? in NP cells induced macrophage polarization into M1 phenotype. Our finding indicates that p38 MAPK ?, ? and ? isoform are predominantly expressed and activated in IDD. P38 positive NP cells modulate macrophage polarization through the production of GM-CSF and IFN?. Hence, Our study suggests that selectively targeting p38 isoforms could ameliorate the inflammation in IDD and regard IDD progression.
Project description:Intervertebral disc degeneration (IDD) is a form of chronic inflammation and is one of the most common disorders reported to be involved in low back pain (LBP). The pathophysiology of degeneration is not completely understood, but the consensus is that the degradation of extracellular matrix (ECM) proteins in the disc is the leading factor contributing to IDD. High temperature requirement A1 (HtrA1) is serine protease that has been shown to be increased in degenerated intervertebral discs as a result of an increase in the expression of matrix metalloproteinases (MMPs), but no study has focused on the effect of HtrA1 on a disintegrin-like and metalloproteinase with thrombospondin motifs (ADAMTSs). In the present study, we successfully isolated human nucleus pulposus cells (HNPCs) from IDD patients who were our research subjects to elaborate on the potential role of HtrA1 in the pathogenesis of IDD. We confirmed that HtrA1 has the potential to induce the expression of ADAMTS-5 in a dose-dependent manner. Consistently, this was mediated by the ERK, NF-κB and JNK pathways. By using inhibitors of these pathways, the increase in ADAMTS-5 could be reduced. Our findings indicated that HtrA1 can induce the expression of ADAMTS-5 in HNPCs via the ERK/NF-κB/JNK signaling pathway, and our study also elucidated the involved induction mechanisms in HNPCs, which may provide new insights for the treatment of IDD.
Project description:Basic science, biologic study.To determine the potential benefits of using resveratrol (RSV) for intervertebral disc (IVD) matrix repair and regeneration.The phytoestrogen RSV is a natural compound found in various plants including grapes and red wines. RSV has been reported to provide a protective effect on articular cartilage in rabbit models for arthritis, but its effect on spine cartilage is unknown. METHODS.: We studied the effect of RSV on bovine IVD cartilage homeostasis by assessing MMP-13 (potent catabolic factor) production, proteoglycan (PG) accumulation and synthesis, and the interaction between RSV and known catabolic factors such as bFGF or IL-1. To understand the molecular mechanisms by which RSV modulates MMP-13 and PG production, we also investigated its downstream target regulatory molecules.Stimulation of bovine disc cells cultured in monolayer with bFGF or IL-1 augmented the production of MMP-13 and ADAMTS-4 at the transcriptional level and this augmentation was blocked by RSV. Incubation of nucleus pulposus cells with RSV for 21 days significantly increased PG accumulation per cell in a dose-dependent manner, increased PG synthesis, rescued PG losses induced by catabolic reagents bFGF and IL-1, and promoted cell survival to levels seen after incubation with the anabolic protein BMP7 100 ng/mL. Protein-DNA interaction array results suggest that RSV effectively suppresses downstream target molecules of bFGF and IL-1 responsible for oxidative stress, proliferation, and apoptosis.Resveratrol is a potent anabolic mediator of bovine IVD cartilage homeostasis, revealing its potential as a unique biologic treatment to slow the progression of IVD degeneration. These data suggests RSV may have considerable promise in the treatment of disc degeneration.
Project description:Background/objective:Intervertebral disc degeneration (IDD) remains to be an intractable clinical challenge. Although IDD is characterised by loss of notochordal cells (NCs) and dysfunction of nucleus pulposus (NP) cells, little is known about the origin, heterogeneity, fate and maintenance of NCs and NP cells, which further stunts the therapeutic development. Thus, effective tools to spatially and temporally trace specific cell lineage and clarify cell functions in intervertebral disc (IVD) development and homoeostasis are urgently required. Methods:In this study, NP specimens were obtained from 20 patients with degenerative disc disease or scoliosis. LepR-Cre mice was crossed with R26R-Tdtomato mice to generate LepR-Cre; R26R-Tdtomato mice, which enabled fate-mapping of NPs from embryo stage to late adult. LMNA G609G/G609G mice was used to determine the effect of premature-aging induced IDD on LepR NPs. X-ray imaging was used to measure lumber disc height of mice. Results:Here, we provide the first evidence that the leptin receptor (LepR) is preferentially expressed in NCs at embryonic stages and notochord-derived cells in the postnatal IVD. By using R26R-Tdtomato fluorescent reporter mice, we systematically analysed the specificity of activity and targeting efficiency of leptin receptor-Cre (LepR-Cre) in IVD tissues from the embryonic stage E15.5 to 6-month-old LepR-Cre; Rosa26-Tdtomato (R26R-Tdtomato) mice. Specifically, LepR-Cre targets a distinct subpopulation of notochord-derived cells closely associated with disc homoeostasis. The percentage of LepR-expressing NP cells markedly decreases in the postnatal mouse IVD and, more importantly, in the human IVD with the progression of IDD. Moreover, both spine instability-induced and premature ageing-induced IDD mouse models display the phenotype of IDD with decreased percentage of LepR-expressing NP cells. These findings uncover a potential role of LepR-expressing notochord-derived cells in disc homoeostasis and open the gate for therapeutically targeting the NP cell subpopulation. Conclusion:In conclusion, our data prove LepR-Cre mice useful for mapping the fate of specific subpopulations of IVD cells and uncovering the underlying mechanisms of IDD. The translational potential of this article:The translation potential of article is that we first identified LepR as a candidate marker of subpopulation of nucleus pulposus (NP) cells and provided LepR as a potential target for the treatment of intervertebral disc degeneration (IDD), which have certain profound significance.
Project description:Fibroblast growth factor 2 (FGF2) is a growth factor that is immediately released after cartilage injury and plays a pivotal role in cartilage homeostasis. In human adult articular cartilage, FGF2 mediates anti-anabolic and potentially catabolic effects via the suppression of proteoglycan (PG) production along with the upregulation of matrix-degrading enzyme activity. The aim of the present study was to determine the biological effects of FGF2 in spine disc cells and to elucidate the complex biochemical pathways utilized by FGF2 in bovine intervertebral disc (IVD) cells in an attempt to further understand the pathophysiologic processes involved in disc degeneration.We studied the effect of FGF2 on IVD tissue homeostasis by assessing MMP-13 expression (potent matrix-degrading enzyme), PG accumulation, and PG synthesis in the bovine spine IVD, as well as evaluating whether FGF2 counteracts known anabolic factors such as BMP7. To understand the molecular mechanisms by which FGF2 antagonizes BMP7 activity, we also investigated the signaling pathways utilized by FGF2 in bovine disc tissue.The primary receptor expressed in bovine nucleus pulposus cartilage is FGFR1, and this receptor is upregulated in degenerative human IVD tissue compared with normal IVD tissue. Stimulation of bovine nucleus pulposus cells cultured in monolayer with FGF2 augmented the production of MMP-13 at the transcriptional and translational level in a dose-dependent manner. Stimulation of bovine nucleus pulposus cells cultured in alginate beads for 21 days with FGF2 resulted in a dose-dependent decrease in PG accumulation, due at least in part to the inhibition of PG synthesis. Further studies demonstrate that FGF2 (10 ng/ml) antagonizes BMP7-mediated acceleration of PG production in bovine nucleus pulposus cells via the upregulation of noggin, an inhibitor of the transforming growth factor beta/bone morphogenetic protein signaling pathway. Chemical inhibitor studies showed that FGF2 utilizes the mitogen-activated protein kinase and NF-kappaB pathways to upregulate noggin, serving as one potential mechanism for its anti-anabolic effects.FGF2 is anti-anabolic in bovine spine disc cells, revealing the potential of FGF2 antagonists as unique biologic treatments for both prevention and reversal of IVD degeneration.