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TLR4/NF-?B axis signaling pathway-dependent up-regulation of miR-625-5p contributes to human intervertebral disc degeneration by targeting COL1A1.

ABSTRACT: The activation of the toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-?B) signaling pathway has been found to play a critical role in many inflammatory diseases by controlling the expression of many cytokines. However, this pathway's role in the pathological process of intervertebral disc degeneration (IDD) has not been reported to date. In the present study, we found universal activation of the TLR4/NF-?B signaling pathway and elevated levels of pro-inflammatory cytokines in IDD patients. The in vitro analyses in human nucleus pulposus cells (hNPC) and annulus fibrosus cells (hAFC) also indicated that Lipopolysaccharide (LPS) treatment could activate TLR4/NF-?B signaling and induce pro-inflammatory cytokine levels. By comparing the results of two microRNA (miRNA)-based microarrays, we identified 15 miRNAs that were dysregulated in both IDD tissues and LPS-treated cells. Of these miRNAs, the most prominently up-regulated was miR-625-5p, which was predicted to bind to the three prime untranslated region (3'-UTR) of collagen type I alpha 1 (COL1A1). In vitro overexpression or down-regulation of miR-625-5p was able to repress or induce the expression of COL1A1, respectively. The in vitro analyses showed that treatment with LPS, recombinant IL-6 or TNF-? could induce miR-625-5p levels but decrease COL1A1 expression. In contrast, the treatments with their corresponding inhibitors, CLI095, siltuximab and D2E7, respectively, resulted in the exact opposite effects. Taken together, our results suggest that activation of the TLR4/NF-?B signaling pathway induces pro-inflammatory cytokines, which further up-regulates the expression of miR-625-5p, resulting in the down-regulation of COL1A1 and eventually contributing to the pathological process of IDD.

SUBMITTER: Shen L

PROVIDER: S-EPMC6456508 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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TLR4/NF-κB axis signaling pathway-dependent up-regulation of miR-625-5p contributes to human intervertebral disc degeneration by targeting <i>COL1A1</i>.

Shen Liqi L Xiao Yun Y Wu Qishun Q Liu Ling L Zhang Caiguo C Pan Xuekun X

American journal of translational research 20190315 3

The activation of the toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) signaling pathway has been found to play a critical role in many inflammatory diseases by controlling the expression of many cytokines. However, this pathway's role in the pathological process of intervertebral disc degeneration (IDD) has not been reported to date. In the present study, we found universal activation of the TLR4/NF-κB signaling pathway and elevated levels of pro-inflammatory cytokines in IDD patients ...[more]

PMID: 30972168

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Project description:Background contextNonphysiological mechanical loading and inflammation are both critically involved in intervertebral disc (IVD) degeneration, which is characterized by an increase in cytokines and matrix metalloproteases (MMPs) in the nucleus pulposus (NP). This process is known to be mediated by the NF-κB pathway.Clinical significanceCurrent clinical treatments for IVD degeneration focus on the alleviation of symptoms rather than targeting the underlying mechanism. Injection of an NF-κB inhibitor may attenuate the progression of IVD degeneration.PurposeTo investigate the ability of the NF-κB inhibitor, NEMO binding domain peptide (NBD), to alter IVD degeneration processes by reducing IL-1β- and mechanically-induced cytokine and MMP levels in human nucleus pulposus cells in vitro, and by attenuating IVD degeneration in an in vivo rat model for disc degeneration.Study designExperimental in vitro and animal model.Patient sampleDiscarded specimens of lumbar disc from 21 patients, and 12 Sprague Dawley rats.Outcome measuresGene and protein expression, cell viability, µMRI and histology.MethodsIL-1β-prestimulated human nucleus pulposus cells embedded into fibrin constructs were loaded in the Flexcell FX-5000 compression system at 5 kPa and 1 Hz for 48 hours in the presence and absence of NBD. Unloaded hNPC/fibrin constructs served as controls. Cell viability in loaded and unloaded constructs was quantified, and gene and protein expression levels determined. For in vivo testing, a rat needle disc puncture model was employed. Experimental groups included injured discs with and without NBD injection and uninjured controls. Levels of disc degeneration were determined via µMRI, qPCR and histology. Funding sources include $48,874 NASS Young Investigator Research Grant and $119,174 NIH 5K01AR071512-02. There were no applicable financial relationships or conflicts of interest.ResultsMechanical compression of hNPC/fibrin constructs resulted in upregulation of MMP-3 and IL-8. Supplementation of media with 10 μM NBD during loading increased cell viability, and decreased MMP-3 gene and protein levels. IVD injury in rat resulted in an increase in MMP-3, IL-1β and IL-6 gene expression. Injections of 250 µg of NBD during disc injury resulted in decreased IL-6 gene expression. µMRI analysis demonstrated a reduction of disc hydration in response to disc needle injury, which was attenuated in NBD-treated IVDs. Histological evaluation showed NP and AF lesion in injured discs, which was attenuated by NBD injection.ConclusionsThe results of this study show NBD peptide's capacity to reduce IL-1β- and loading-induced MMP-3 levels in hNPC/fibrin constructs while increasing the cells' viability, and to attenuate IVD degeneration in rat, involving downregulation of IL-6. Therefore, NBD may be a potential therapeutic agent to treat IVD degeneration.

Project description:BackgroundIntervertebral disc degeneration (IVDD) is a leading cause of low back pain (LBP). The pathological process of IVDD is associated with inflammatory reactions and extracellular matrix (ECM) disorders. Digoxin is widely used for treating heart failure, and it has been reported to have anti-inflammatory effects.ObjectiveThis study is to investigate the role of digoxin in the pathogenesis of intervertebral disc degeneration as well as the involved molecular mechanism, particularly the potential target protein.MethodsWe exploited a rat needle model to investigate digoxin's role in intervertebral disc degeneration in vivo. Safranin O staining was used to measure cartilaginous tissue in the intervertebral disc. The morphological changes of intervertebral discs in animal models were determined by Hematoxylin-Eosin (H&E) staining and the pathological score. Primary nucleus pulposus cells (NP cells) from intervertebral discs of patients and murine were used in the present study. Western-Blotting assay, Real-time PCR assay, immunofluorescence staining, and immunochemistry were used to detect the role of digoxin in anti-TNF-α-induced inflammatory effects in vitro. Transfection of siRNA was used to regulate low-density lipoprotein receptor-related protein 4 (LRP4) expression in NP cells to investigate the potential protein target of digoxin.ResultsDigoxin protected against intervertebral disc degeneration in rat needle models. Digoxin was found to exert its disc-protective effects through at least three different pathways by a) suppressing TNF-α-induced inflammation, b) attenuating ECM destruction, c) significantly promoting ECM anabolism. Additionally, LRP4 was found to be the downstream molecule of digoxin in NP cells for anti-inflammation and regulation of ECM metabolism. The knockdown of LRP4 downregulated the protective effect of digoxin in NP cells.ConclusionThese findings suggest that digoxin may be a potential therapeutic agent for intervertebral disc degeneration through anti-catabolism and pro-anabolism. Digoxin might also work as an alternative for other inflammation-related diseases.

Dataset Information

TLR4/NF-?B axis signaling pathway-dependent up-regulation of miR-625-5p contributes to human intervertebral disc degeneration by targeting COL1A1.

Publications

TLR4/NF-κB axis signaling pathway-dependent up-regulation of miR-625-5p contributes to human intervertebral disc degeneration by targeting <i>COL1A1</i>.

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