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The effect of a cyclooxygenase 2 inhibitor on early degenerated human nucleus pulposus explants.

ABSTRACT: Study Design?Preclinical in vitro culture of human degenerated nucleus pulposus (NP) tissue. Objective?Cyclooxygenase 2 inhibitors (e.g., celecoxib) inhibit prostaglandin E2 (PGE2) production, and they have been shown to upregulate regeneration of articular cartilage. In this study, we developed an explant culture system for use with human tissue and tested the potential of celecoxib. Methods?NP explants were cultured with or without 1 ?M of celecoxib and were analyzed at days 0 and 7 for biochemical content (water, sulfated glycosaminoglycans, hydroxyproline, and DNA), gene expression (for disk matrix anabolic and catabolic markers), and PGE2 content. Results?Water and biochemical contents as well as gene expression remained close to native values after 1?week of culture. PGE2 levels were not increased in freshly harvested human NP tissue and thus were not reduced in treated tissues. Although no anabolic effects were observed at the dosage and culture duration used, no detrimental effects were observed and some specimens did respond by lowering PGE2. Conclusions?Human degenerated NP explants were successfully cultured in a close to in vivo environment for 1 week. Further research, especially dosage-response studies, is needed to understand the role of PGE2 in low back pain and the potential of celecoxib to treat painful disks.

SUBMITTER: van Dijk B

PROVIDER: S-EPMC3908972 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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The effect of a cyclooxygenase 2 inhibitor on early degenerated human nucleus pulposus explants.

van Dijk Bart B Potier Esther E Licht Ruud R Creemers Laura L Ito Keita K

Global spine journal 20131023 1

Study Design Preclinical in vitro culture of human degenerated nucleus pulposus (NP) tissue. Objective Cyclooxygenase 2 inhibitors (e.g., celecoxib) inhibit prostaglandin E2 (PGE2) production, and they have been shown to upregulate regeneration of articular cartilage. In this study, we developed an explant culture system for use with human tissue and tested the potential of celecoxib. Methods NP explants were cultured with or without 1 μM of celecoxib and were analyzed at days 0 and 7 for bioche ...[more]

PMID: 24494179

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Project description:Study designIsolated human nucleus pulposus (hNP) cells from degenerated intervertebral disc were incubated under hydrostatic pressure and evaluated for regenerative potential.ObjectivesTo characterize metabolic turnover in hNP cells isolated from degenerated intervertebral discs classified by Pfirrmann grade under physiologically relevant hydrostatic pressure at high osmolality in vitro.Summary of background dataWe demonstrated that bovine caudal nucleus pulposus cells isolated from healthy cows produced more extracellular matrix under cyclic hydrostatic pressure followed by constant pressure (mimicking physiological intradiscal pressure in humans) than under no pressure in vitro. We assessed the effects of pressure on human degenerated cells isolated under the same regimen of pressure used for bovine cells.MethodshNP cells isolated from discarded tissue classified as Pfirrmann Grade 2-3 (n=13: age, 46.7±14.0) and Grade 4 (n=13: age, 53.0±11.5) were incubated under cyclic hydrostatic pressure at 0.2-0.7 MPa, 0.5 Hz for 2 days followed by constant pressure at 0.3 MPa for 1 day, repeated twice over 6 days. The gene expression and immunohistology of matrix molecules, catabolic and anti-catabolic proteins were evaluated.ResultsAggrecan and collagen type II expression was significantly more upregulated under HP in Grade 2-3 than in Grade 4 tissues (both, P<0.01). Linear regression analysis showed a positive correlation between matrix metalloproteinase 13 and tissue inhibitor for metalloproteinase 2 expression in Grade 2-3, while a negative correlation was found in Grade 4 (P<0.05). Immunohistological staining revealed the activation of a mechanoreceptor, transient receptor potential vanilloid 4, under HP.ConclusionsResident cells in mild-moderate degenerated disc classified as Pfirrmann grade 2-3 have the potential to promote extracellular matrix production and maintain adequate cell viability under physiological spinal loading.RelevanceThis study explored the potential of degenerated remnant NP cells under physiological environment, possibly leading to establishing strategies for IVD regeneration.

Project description:Intervertebral disc (IVD) degeneration is a pathological process, which may lead to lower back pain. The present study aimed to investigate the pathogenesis of IVD degeneration. GSE42611 was downloaded from Gene Expression Omnibus, including 4 nucleus pulposus samples isolated from degenerated IVDs and 4 nucleus pulposus samples separated from normal IVDs. The differentially expressed genes (DEGs) between the degenerated and normal samples were screened using the limma package in R. Functional and pathway enrichment analyses were conducted separately for the upregulated and downregulated genes, using Database for Annotation, Visualization and Integrated Discovery software. In addition, protein‑protein interaction (PPI) networks were constructed using the Search Tool for the Retrieval of Interacting Genes database and Cytoscape software. Finally, module analyses were conducted for the PPI networks using the MCODE plug‑in in Cytoscape. A total of 558 DEGs were identified in the degenerated nucleus pulposus cells: 253 upregulated and 305 downregulated. Pathway enrichment analysis revealed that downregulated thrombospondin 1 (THBS1) was enriched in extracellular matrix‑receptor interaction. Interleukin (IL)‑6 in the PPI network for the upregulated genes and vascular endothelial growth factor A (VEGFA) in the PPI network for the downregulated genes had higher degrees. Additionally, four modules (µM1, µM2, µM3 and µM4) were identified from the PPI network for the upregulated genes. Four modules (dM1, dM2, dM3 and dM4) were identified from the PPI network for the downregulated genes. In the dM2 module, collagen genes and integrin subunit α4 (ITGA4) may interact with each other. Additionally, functional enrichment indicated that collagen genes were enriched in extracellular matrix organization. In conclusion, IL‑6, VEGFA, THBS1, ITGA4 and collagen genes may contribute to the progression of IVD degeneration. These results suggested that the manipulation of these genes and their products may have potential as a novel therapeutic strategy for the treatment of patients with IVD.

Project description:STUDY DESIGN:Development of an in vitro model for assessing the anti-inflammatory efficacies of naringin (Nar) and naringenin (NG). PURPOSE:To evaluate the efficacy of natural flavonoids as therapeutic drugs against anti-inflammatory processes in the nucleus pulposus (NP) cells using in-vitro and in-silico methods. OVERVIEW OF LITERATURE:Intervertebral disc (IVD) disease is a common cause of low back pain. Chronic inflammation and degeneration play a significant role in its etiopathology. Thus, a better understanding of anti-inflammatory agents and their role in IVD degeneration and pro-inflammatory cytokines expression is necessary for pain management and regeneration in IVD. METHODS:We performed primary cell culture of NP cells; immunocytochemistry; gene expression studies of cytokines, metalloproteases, extracellular proteins, and apoptotic markers using quantitative polymerase chain reaction and reverse transcription-polymerase chain reaction (RT-PCR); cytotoxicity assay (MTT); and molecular docking studies using AutoDock 4.2 software (Molecular Graphics Laboratory, La Jolla, CA, USA) to confirm the binding mode of proteins and synthesized complexes. We calculated the mean±standard deviation values and performed analysis of variance and t-test using SPSS ver. 17.0 (SPSS, Inc., Chicago, IL, USA). RESULTS:Molecular docking showed that both Nar and NG bind to the selected genes of interest. Semi-quantitative RT-PCR analysis reveals differential gene expression of collagen (COL)9A1, COL9A2, COL9A3, COL11A2, COMT (catechol-O-methyltransferase), and THBS2 (thrombospondin 2); up regulation of ACAN (aggrecan), COL1A1, COL11A1, interleukin (IL)6, IL10, IL18R1, IL18RAP, metalloprotease (MMP)2, MMP3, MMP9, ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs 5), IGF1R (insulin-like growth factor type 1 receptor), SPARC (secreted protein acidic and cysteine rich), PARK2 (parkin), VDR (vitamin D receptor), and BCL2 (B-cell lymphoma 2); down regulation of IL1A, CASP3 (caspase 3), and nine genes with predetermined concentrations of Nar and NG. CONCLUSIONS:The present study evaluated the anti-inflammatory and regenerative efficiencies of Nar and NG in degenerated human NP cells. Altered gene expressions of cytokines, metalloproteases, extracellular proteins, apoptotic genes were dose responsive. The molecular docking (in silico) studies showed effective binding of these native ligands (Nar and NG) with genes identified as potent inhibitors of inflammation. Thus, these natural flavonoids could serve as anti-inflammatory agents in the treatment of low back pain and sciatica.

Project description:BackgroundIntervertebral disc degeneration (IVDD) is a major contributor to spinal disorders. Previous studies have indicated that the infiltration of immunocytes, specifically macrophages, plays a crucial role in the advancement of IVDD. Exosomes (exo) are believed to play a significant role in intercellular communication. This study aims to investigate the role of exosomes derived from degenerated nucleus pulposus (dNPc) in the process of macrophages M1 polarization.MethodsNucleus pulposus (NP) tissue and nucleus pulposus cells (NPc) were collected from patients with intervertebral disc degeneration (IVDD) and idiopathic scoliosis. Immunohistochemistry analysis was performed to determine the number of M1 macrophages in NP tissue. Subsequently, exosomes derived from degenerated NP cells (dNPc-exo) and non-degenerated NP cells (nNPc-exo) were collected and co-cultured with M0 macrophages, which were induced from THP-1 cells. The M1 phenotype was assessed using western blot, flow cytometry, immunofluorescence staining, and qRT-PCR. RNA-sequencing analysis was conducted to examine the expression levels of microRNAs in the dNPc-exo and nNPc-exo groups, and qRT-PCR was performed to investigate the effect pf different microRNA to induce macrophage polarization. Furthermore, western blot and qRT-PCR were employed to demonstrate the regulatory effect of microRNAs carried by dNPc-exo on downstream target signaling pathways in macrophages. Finally, an animal model of IVDD was utilized to investigate the impact of dNPc-exo on inducing M1 polarization of macrophages and its role in the IVDD process.ResultsIn this study, we observed an increase in the number of M1 macrophages as the intervertebral disc (IVD) degraded. Additionally, we discovered that dNPc releases exosomes (dNPc-exo) could promote the polarization of macrophages towards the M1 phenotype. Notably, through RNA-sequencing analysis of dNPc-exo and nNPc-exo groups, we identified miR-27a-3p as a highly expressed miRNA in the dNPc-exo group, which significantly influences the induction of M1 polarization of macrophages. And then, we discovered that dNPc-exo has the ability to transport miR-27a-3p and target the PPARγ/NFκB/PI3K/AKT signaling pathway, thereby influencing the M1 polarization of macrophages. We conducted experiments using rat model of IVDD and observed that the exosomes carrying miR-27a-3p actually induced the M1 polarization of macrophages and exacerbated the degradation of IVD.ConclusionIn conclusion, our findings highlight the significant role of dNPc-exo in IVDD process and provide a basis for further investigation into the mechanism of IVDD and the potential of exosome-based therapy.

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The effect of a cyclooxygenase 2 inhibitor on early degenerated human nucleus pulposus explants.

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The effect of a cyclooxygenase 2 inhibitor on early degenerated human nucleus pulposus explants.

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