M2a Macrophage-Secreted CHI3L1 Promotes Extracellular Matrix Metabolic Imbalances via Activation of IL-13Rα2/MAPK Pathway in Rat Intervertebral Disc Degeneration.

The accumulation of macrophages in degenerated discs is a common phenomenon. However, the roles and mechanisms of M2a macrophages in intervertebral disc degeneration (IDD) have not been illuminated. This study investigated the expression of the M2a macrophage marker (CD206) in human and rat intervertebral disc tissues by immunohistochemistry. To explore the roles of M2a macrophages in IDD, nucleus pulposus (NP) cells were co-cultured with M2a macrophages in vitro. To clarify whether the CHI3L1 protein mediates the effect of M2a macrophages on NP cells, siRNA was used to knock down CHI3L1 transcription. To elucidate the underlying mechanisms, NP cells were incubated with recombinant CHI3L1 proteins, then subjected to western blotting analysis of the IL-13Rα2 receptor and MAPK pathway. CD206-positive cells were detected in degenerated human and rat intervertebral disc tissues. Notably, M2a macrophages promoted the expression of catabolism genes (MMP-3 and MMP-9) and suppressed the expression of anabolism genes (aggrecan and collagen II) in NP cells. These effects were abrogated by CHI3L1 knockdown in M2a macrophages. Exposure to recombinant CHI3L1 promoted an extracellular matrix metabolic imbalance in NP cells via the IL-13Rα2 receptor, along with activation of the ERK and JNK MAPK signaling pathways. This study elucidated the roles of M2a macrophages in IDD and identified potential mechanisms for these effects.

Revealing the Immune Infiltration Landscape and Identifying Diagnostic Biomarkers for Lumbar Disc Herniation.

Intervertebral disc (IVD) degeneration and its inflammatory microenvironment ultimately led to discogenic pain, which is thought to originate in the nucleus pulposus (NP). In this study, key genes involved in NP tissue immune infiltration in lumbar disc herniation (LDH) were identified by bioinformatic analysis. Gene expression profiles were downloaded from the Gene Expression Omnibus (GEO) database. The CIBERSORT algorithm was used to analyze the immune infiltration into NP tissue between the LDH and control groups. Hub genes were identified by the WGCNA R package in Bioconductor and single-cell sequencing data was analyzed using R packages. Gene expression levels were evaluated by quantitative real-time polymerase chain reaction. The immune infiltration profiles varied significantly between the LDH and control groups. Compared with control tissue, LDH tissue contained a higher proportion of regulatory T cells and macrophages, which are associated with the macrophage polarization process. The most significant module contained three hub genes and four subclusters of NP cells. Functional analysis of these genes was performed, the hub gene expression pattern was confirmed by PCR, and clinical features of the patients were investigated. Finally, we identified TGF-ß and MAPK signaling pathways as crucial in this process and these pathways may provide diagnostic markers for LDH. We hypothesize that the hub genes expressed in the specific NP subclusters, along with the infiltrating macrophages play important roles in the pathogenesis of IVD degeneration and ultimately, disc herniation.

Substance P Mediates Estrogen Modulation Proinflammatory Cytokines Release in Intervertebral Disc.

Intervertebral disc degeneration (IDD) is a main contributor to low back pain. A close relationship exists between inflammation and pain. Estrogen can affect inflammation and may play a crucial role in IDD and pain. Substance P (SP) can also regulate the expression of pro-inflammatory cytokines in intervertebral disc (IVD). This study aimed to investigate the potential role of SP in estrogen regulation of IDD. Nine-week-old C57BL/6 female mice were divided into four groups as follows: sham surgery (sham), ovariectomy (OVX), ovariectomy plus estrogen replacement therapy (ERT) group (OVX+E2), and ovariectomy, ERT plus neurokinin 1 receptor (NK1R) agonist (OVX+E2+G). Serum E2, body, and uterus weight were recorded. Immunohistochemistry study and quantitative real-time PCR were used for SP, NK1R, IL-1ß, IL-6, and TNF-α examination and comparison in IVD at protein and gene levels. After OVX, the gene and protein expression of TNF-α, IL-1ß, IL-6, SP, and NK1R in NP cells significantly increased compared with the sham group. ERT can reverse these impacts. ERT plays anti-inflammatory and anti-hyperalgesic roles in IDD of OVX mice. The estrogen-induced changes of the pro-inflammatory cytokines, TNF-α, IL-1ß, and IL-6, are significantly inhibited by NK1R agonists. SP may be a mediator of estrogen regulating pro-inflammatory factors in IDD. Estrogen may affect IVD inflammation through two ways: one is to directly affect the level of pro-inflammatory cytokines and the other is by means of modulation of SP.

Interleukin-17A Promotes Human Disc Degeneration by Inhibiting Autophagy Through the Activation of the Phosphatidylinositol 3-Kinase/Akt/Bcl2 Signaling Pathway.

BACKGROUND: Previous studies have suggested that interleukin (IL)-17A is a key factor that contributes to intervertebral disc degeneration (IDD), whereas autophagy has been shown to be a protective mechanism in IDD. However, the relationship between IL-17A and autophagy in IDD remains to be fully elucidated. This study sought to evaluate the association between IL-17 and autophagy and the potential mechanism through which IL-17A affects autophagy in IDD. METHODS: Intervertebral disc specimens were collected from 10 patients with lumbar disc herniation. Human degenerated nucleus pulposus (NP) cells were cultured in the presence or absence of IL-17A treatment. Western blot and monodansylcadaverine staining were used to measure autophagy levels in human degenerated NP cells. Subsequently, phosphatidylinositol 3-kinase (PI3K)/Akt/Bcl-2 pathway inhibitors were used to reveal the potential mechanism. RESULTS: IL-17A treatment inhibited the autophagic activity in human NP cells in a time- and dose-dependent manner. Moreover, monodansylcadaverine staining showed that cells treated with IL-17A had significantly fewer changes in their autophagic vacuoles compared with control-treated cells. After IL-17A treatment, expression levels of PI3K, p-Akt, and Bcl-2 in NP cells were significantly increased. Further assays with PI3K/Akt/Bcl-2 inhibitors revealed that IL-17A suppressed autophagy in NP cells by activating the PI3K/Akt/Bcl-2 signaling pathway. CONCLUSIONS: These data suggest that IL-17A promotes IDD by inhibiting autophagy through activation of the PI3K/Akt/Bcl-2 signaling pathway and may offer new insights for targeted therapy of this disease.

Higenamine inhibits IL-1ß-induced inflammation in human nucleus pulposus cells.

Intervertebral disc degeneration (IDD) is a natural progression of the aging process associated with inflammation. Higenamine, a plant-based alkaloid, has been identified to possess various pharmacological properties, including anti-inflammatory activity. In the present study, we aimed to evaluate the role of higenamine in interleukin (IL)-1ß-induced inflammation in human nucleus pulposus cells (NPCs). The results showed that higenamine improved cell viability in IL-1ß-induced NPCs. The IL-1ß-dependent up-regulation of inflammatory molecules including inducible nitric oxide synthase (iNOS), nitric oxide (NO), prostaglandin E2 (PGE2), cyclooxygenase-2 (COX-2), tumor necrosis factor alpha (TNF-α), and IL-6 was attenuated by higenamine in NPCs. The increased productions of matrix metalloproteinases (MMP-3 and MMP-13), as well as a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS-4 and ADAMTS-5) were significantly mitigated by higenamine treatment. Furthermore, we also found that higenamine suppressed the IL-1ß-induced activation of NF-κB signaling pathway in NPCs. In conclusion, the present study proved that higenamine exhibited anti-inflammatory activity against IL-1ß-induced inflammation in NPCs via inhibiting NF-κB signaling pathway. These results suggested that higenamine might be a therapeutic agent for the treatment of IDD.

Isofraxidin attenuates IL-1ß-induced inflammatory response in human nucleus pulposus cells.

Inflammation has been demonstrated to be the key factor for intervertebral disc degeneration (IVD), which remains a major public health problem. Isofraxidin is a coumarin compound that possesses strong anti-inflammatory activity. However, the role of isofraxidin in IVD remains unclear. The aim of this study was to evaluate the effects of isofraxidin on inflammatory response in human nucleus pulposus cells (NPCs) exposed to interleukin-1ß (IL-1ß). The results proved that isofraxidin attenuated the IL-1ß-induced significant increases in inflammatory mediators and cytokines including nitric oxide (NO), inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-α), and IL-6. Besides, isofraxidin also inhibited the induction effect of IL-1ß on matrix metalloproteinases (MMP)-3 and MMP-13. Moreover, the NF-κB activation caused by IL-1ß was significantly inhibited by isofraxidin treatment. These findings suggested that isofraxidin alleviates IL-1ß-induced inflammation in NPCs. Our work provided an idea that isofraxidin might act as a novel preventive role in IVD.

Propionibacterium acnes induces discogenic low back pain via stimulating nucleus pulposus cells to secrete pro-algesic factor of IL-8/CINC-1 through TLR2-NF-κB p65 pathway.

Latent infection of Propionibacterium acnes was considered as a new pathogeny for low back pain (LBP); however, there is no credible animal evidence or mechanism hypothesis. This study proved that P. acnes is a causative pathogen of bacteria-induced LBP and investigated its underlying mechanism. For this, P. acnes was firstly identified in patients' degenerated intervertebral disc (IVDs) samples. The results of patients' Japanese Orthopaedic Association Back Pain Evaluation Questionnaire (JOABPEQ), Japanese Orthopaedic Association (JOA), and Oswestry Disability Index (ODI) scores indicated that P. acnes-positive patients showed more severe LBP and physical disability. Then, a P. acnes-inoculated lumbar IVDs model was established in rats. The results of paw/foot withdrawal threshold and qRT-PCR indicated that P. acnes-inoculated rats had obvious LBP in behavioral evaluation and over-expression of substance P (SP) and calcitonin gene-related peptide (CGRP) in IVDs. Subsequently, enzyme-linked immunosorbent assay (ELISA) results demonstrated that increased expression of IL-8 or CINC-1 (the homolog of IL-8 in rats) in the P. acnes-positive IVDs of human and rats. The CINC-1 injected animal model proved that the cytokines were able to induce LBP. Finally, the co-culture experiments showed that nucleus pulposus cells (NPCs) were able to respond to P. acnes and secreted IL-8/CINC-1 via TLR-2/NF-κB p65 pathway. In conclusion, P. acnes had strong association with LBP by stimulating NPCs to secrete pro-algesic factor of IL-8/CINC-1 via TLR2/NF-κBp65 pathway. The finding may provide a promising alternative therapy strategy for LBP in clinical. KEY MESSAGES: Patients with P. acnes-positive IVDs tended to have more severe LBP, physical disability, and increased IL-8 expressions. P. acnes can induce LBP via IL-8/CINC-1 in IVDs. P. acnes stimulate the NPCs to secrete pro-algesic factor of IL-8/CINC-1 via TLR2/NF-κBp65 pathway.

Nuclear factor-kappa B-dependent X-box binding protein 1 signalling promotes the proliferation of nucleus pulposus cells under tumour necrosis factor alpha stimulation.

OBJECTIVES: Tumour necrosis factor alpha (TNF-α) expressed by nucleus pulposus cells (NPCs) plays a critical role in intervertebral disc (IVD) degeneration. A key unfolded protein response (UPR) component, X-box binding protein 1 (XBP1) and nuclear factor-kappa B (NF-κB) are essential for cell survival and proliferation. The aim of our study was to elucidate the roles of XBP1 and NF-κB in IVD degeneration (IDD). MATERIALS AND METHODS: Rat NPCs were cultured with TNF-α in the presence or absence of XBP1 and NF-κB-p65 small interfering RNA. The associated genes and proteins were evaluated through quantitative real-time PCR, Western blot analyses and immunofluorescence staining to monitor UPR and NF-κB signalling and identify the regulatory mechanism of p65 by XBP1. Cell counting kit-8 assay, cell cycle analysis and related gene and protein expression were performed to examine the proliferation of NPCs. RESULTS: The acute exposure of TNF-α accelerated the proliferation of rat NPCs by activating the UPR/XBP1 pathway. XBP1 signalling favoured the phosphorylation and nuclear translocation of p65 subunit of NF-κB. The activation of NF-κB in the later phase also enhanced NPC proliferation. CONCLUSIONS: Unfolded protein response reinforces the survival and proliferation of NPCs under TNF-α stimulation by activating the XBP1 pathway, and NF-κB serves as a vital mediator in these events. The XBP1 signalling of UPR can be a novel therapeutic target in IDD.

Duhuo Jisheng Decoction inhibits SDF-1-induced inflammation and matrix degradation in human degenerative nucleus pulposus cells in vitro through the CXCR4/NF-κB pathway.

Lower back pain (LBP) is the most common disease in orthopedic clinics world-wide. A classic Fangji of traditional Chinese medicine, Duhuo Jisheng Decoction (DHJSD), has been proven clinically effective for LBP but its therapeutic mechanisms remain unclear. We hypothesized that DHJSD might relieve LBP through inhibiting the exaggerated proinflammatory cytokines and extracellular matrix (ECM) degradation. Thus, we studied the effects of DHJSD on stromal cell-derived factor-1 (SDF-1)-induced inflammation and ECM degradation in human nucleus pulposus cells (hNPCs). The primary hNPCs were isolated from either degenerated human intervertebral disc (HID) of LBP patients or normal HID of lumbar vertebral fracture patients, and cultured in vitro. The cells were treated with SDF-1 (10 ng/mL) and subsequently with different concentrations (100-500 µg/mL) of DHJSD for 24 h, respectively. We found that application of DHJSD significantly antagonized the SDF-1-induced production of proinflammatory cytokines and reduction of aggrecan and type II collagen in the hNPCs. DHJSD also markedly reduced the SDF-1-induced increase of CXCR4 and p-p65 and inhibited the nuclear translocation of p65 in the hNPCs. DHJSD, CXCR4-siRNA, and NF-κB inhibitor (BAY11-7082) caused the same inhibition of exaggerated proinflammatory cytokines in the SDF-1-treated hNPCs. These results provided compelling evidence that DHJSD may inhibit the generation of proinflammatory mediators and ECM degradation of HID through an orchestrated targeting at multiple molecules in the SDF-1/CXCR4/NF-κB pathway, thus offered novel mechanistic insights into the clinical effectiveness of DHJSD on LBP.

Moracin M inhibits lipopolysaccharide-induced inflammatory responses in nucleus pulposus cells via regulating PI3K/Akt/mTOR phosphorylation.

Moracin M, a phenolic component obtained from Mori Cortex, has been reported to have anti-inflammatory activities. The present study was designed to investigate the effects and mechanisms of Moracin M on lipopolysaccharide (LPS)-treated nucleus pulposus cells (NPCs) in intervertebral disc. NPCs were treated with moracin M at different concentrations for 1 h and then stimulated with LPS (0.5 µg/mL) for 24 h. The result demonstrated that moracin M could significantly inhibit LPS-induced inflammation. The elevated levels of IL-1ß, TNF-α and IL-6 induced by LPS could be reversed by moracin M in NPCs. Moreover, moracin M increased the expressions of autophagy-related proteins and up-regulated the phosphorylation of PI3K/Akt/mTOR in LPS-treated NPCs. In conclusion, our data demonstrated that moracin M might inhibit LPS-induced PI3K and Akt phosphorylation, which leading to promote the autophagy and inhibit the inflammatory mediator production in NPCs.

Phototherapy suppresses inflammation in human nucleus pulposus cells for intervertebral disc degeneration.

The etiology of intervertebral disc (IVD) degeneration accompanied by low back pain (LBP) is largely unknown, and there are no curative therapies. Painful IVD degeneration is associated with infiltrated macrophage-mediated inflammatory response of human nucleus pulposus (NP) cells. The present study aimed to address the hypothesis that pro-inflammatory cytokines derived from macrophages lead to the altered molecular phenotype of human NP cells and to investigate the effects of phototherapy (630, 525, 465 nm with 16, 32, 64 J/cm2) on pain-related cytokine interleukin (IL)-6 and chemokine IL-8 under inflammatory conditions in human NP cells. Human NP cells were treated with soluble factors derived from macrophages in an inflammatory microenvironment, similar to that found in degenerative IVD. Human NP cells were also treated with phototherapy (630, 525, 465 nm with 16, 32, 64 J/cm2), and their cytokine and chemokine levels were detected. The soluble factors caused modulated expression of IL-6, IL-8, and matrix metalloproteinases (MMPs) at the gene and protein levels, causing a shift toward matrix catabolism through the expression of MMPs and increased pain-related factors via preferential activation of the nuclear factor-kappa B (NF-κB) p50 protein. Importantly, phototherapy attenuated the protein and gene expression of pain-related factor IL-6 at all doses and wavelengths. Interestingly, phototherapy also modulated the protein and gene expression of IL-8, which is responsible for the anabolic response, at a wavelength of 465 nm at all doses, in human NP cells. These findings suggested that phototherapy, at an optimal dose and wavelength, might be a useful therapeutic tool to treat IVD degeneration.

Modic type 1 change is an autoimmune response that requires a proinflammatory milieu provided by the 'Modic disc'.

BACKGROUND CONTEXT: Modic changes (MCs) are magnetic resonance imaging (MRI) evidence of inflammatory and fibrotic vertebral bone marrow lesions that associate with adjacent disc degeneration and end plate damage. Although MC etiology is uncertain, historical data suggest a linkage to an autoimmune response of bone marrow triggered by the nucleus pulposus (NP). PURPOSE: The aim of this study was to test whether bone marrow has an autoimmune response to NP cells that is amplified by an inflammatory milieu and ultimately leads to MC development in vivo. We hypothesized that an inflammatory co-stimulus is required for bone marrow/NP crosstalk to stimulate MC. STUDY DESIGN: This is an in-vitro cell co-culture study plus in-vivo experiments in rat caudal vertebrae. METHODS: In in-vitro study, bone marrow mononuclear cells (BMNCs) and NP cells (NPCs) from rats were co-cultured with and without interleukin (IL)-1α stimulation. Cell viability (n=3) of BMNCs and NPCs and gene expression (n=7) were analyzed. In in-vivo study, proinflammatory lipopolysaccharide (LPS) and control disc nucleus surrogates (NP micromass pellets) were generated in vitro from rat NPCs and implanted into rat tail vertebrae, and the response was compared with sham surgery (n=12 each). Tissue changes were investigated with T1w and T2w MRI (7T), histology, and immunohistochemistry (tumor necrosis factor, CD3) 1 (n=6) and 2 weeks (n=6) after implantation. RESULTS: BMNC/NPC co-culture significantly increased lymphocyte viability (42%-69%, p<.05) and reduced NPC viability (96%-88%, p<.001), indicating immunogenicity of NPC. However, IL-1α was required to cause significant transcriptional upregulation of IL-1, IL-6, IL-10, and tropomyosin receptor kinase A. Therefore, an inflammatory activation is required to amplify the immune response. Immunogenicity of the NP was corroborated in vivo by CD3 cell accumulation around LPS and control disc surrogates at Day 7. However, only the LPS disc surrogate group demonstrated infiltration of CD3 cells at Day 14. Furthermore, end plate defects (p<.05, LPS: n=4/6, Ctrl: n=0/6, sham: n=0/6) and MC1-like MRI changes (T2w hyperintensity, p<.05) were only seen with LPS disc surrogates. CONCLUSIONS: NPCs are immunogenic but cannot trigger MC without an additional proinflammatory stimulus. Our data suggest that MC requires end plate defects that allow marrow/NPC co-mingling plus an adjacent inflammatory "MC disc" that can amplify the immune response.

Mast Cell-Intervertebral disc cell interactions regulate inflammation, catabolism and angiogenesis in Discogenic Back Pain.

Low back pain (LBP) is a widespread debilitating disorder of significant socio-economic importance and intervertebral disc (IVD) degeneration has been implicated in its pathogenesis. Despite its high prevalence the underlying causes of LBP and IVD degeneration are not well understood. Recent work in musculoskeletal degenerative diseases such as osteoarthritis have revealed a critical role for immune cells, specifically mast cells in their pathophysiology, eluding to a potential role for these cells in the pathogenesis of IVD degeneration. This study sought to characterize the presence and role of mast cells within the IVD, specifically, mast cell-IVD cell interactions using immunohistochemistry and 3D in-vitro cell culture methods. Mast cells were upregulated in painful human IVD tissue and induced an inflammatory, catabolic and pro-angiogenic phenotype in bovine nucleus pulposus and cartilage endplate cells at the gene level. Healthy bovine annulus fibrosus cells, however, demonstrated a protective role against key inflammatory (IL-1ß and TNFα) and pro-angiogenic (VEGFA) genes expressed by mast cells, and mitigated neo-angiogenesis formation in vitro. In conclusion, mast cells can infiltrate and elicit a degenerate phenotype in IVD cells, enhancing key disease processes that characterize the degenerate IVD, making them a potential therapeutic target for LBP.

Cytokine and chemokine profile changes in patients with lower segment lumbar degenerative spondylolisthesis.

BACKGROUND: Lumbar degenerative spondylolisthesis (DS) develops as a result of inflammatory and remodeling processes in facet joints (FJs). Several inflammatory cytokines are involved in the osteoarthritic and remodeling changes that occur and in low-back and/or radicular pain, the most prevalent clinical symptom of disease. This study improves knowledge related to the roles that 27 cytokines, chemokines and growth factors play in the pathophysiology of lumbar DS. MATERIAL AND METHODS: Cytokine levels were examined using capture sandwich immunoassay using the Bio-Plex® 200 System and the Bio-PlexTM Human Cytokine Standard 27-Plex, Group I (Bio-Rad, Hercules, California, USA) separately in intervertebral discs (IVDs) and FJ bone tissue. The samples were obtained during primary spinal surgery from 9 patients suffering from lower segment lumbar DS. The pain intensity was assessed using a visual analog scale. The controls were tissue samples collected from both lower lumbar segment levels of 6 male subjects during a multiorgan procurement procedure. RESULTS: The Bio-Plex® assay revealed significant differences between the patients and controls in cytokines, chemokines and growth factor profiles: i, The elevated interleukin-6 (IL-6), IL-7, IL-13, tumor necrosis factor α (TNF-α), interferon γ and platelet-derived growth factor levels in lumbar DS samples of subchondral FJ bone. These indicated ongoing inflammation, bone formation and increased fibroblasts activity in the FJ bone. ii, The elevated levels of IL-6, IL-8, TNF-α, granulocyte-macrophage colony-stimulating factor and monocyte chemoattractant protein-1 in anulus fibrosus together with increased IL-6, IL-8, TNF-α and eotaxin and decreased IL-1-receptor antagonist in nucleus pulposus confirmed advanced IVD degeneration in the patient samples. CONCLUSION: This study identified, for the first time, protective levels of cytokines, chemokines and growth factors in healthy subjects and supported their significant involvement in the pathogenesis of lumbar DS. The control samples and analytical methods used avoided any false changes in the cytokine levels due to secondary factors (e.g., death of donor and limited cytokine stability).

Sp1 downregulates proinflammatory cytokine­induced catabolic gene expression in nucleus pulposus cells.

During the pathogenesis of intervertebral disc degeneration, pro­inflammatory cytokines, including tumor necrosis factor­α (TNF­α), stimulate the degradation of the extracellular matrix (ECM) of intervertebral discs via the activity of catabolic enzymes including matrix metalloproteinases (MMPs), disintegrins and metalloproteinases with thrombospondin motifs (ADAMTSs), and cyclooxygenase 2 (Cox2). The transcriptional promoters of the human catabolic enzymes MMPs, ADAMTS, Cox2 and Syndecan 4 contain at least one specificity protein­1 (Sp1) transcription factor­binding site. The present study investigated the role of Sp1 in the regulation of the mRNA and protein expression of the aforementioned catabolic enzyme genes in nucleus pulposus cells, using reverse transcription­quantitative polymerase chain reaction, western blot, transfection and RNA interference. The data demonstrated that Sp1 transcription factor protein expression is induced by TNF­α and interleukin­1ß. Specific inhibitors of Sp1 DNA binding to its GC­rich consensus site, WP631 and mithramycin A, partially suppressed TNF­α­induced catabolic enzyme expression and activity. Genetic inhibition of Sp1 by small interfering RNA­mediated Sp1 knockdown partially inhibited catabolic enzyme induction by TNF­α. In addition, Sp1 transcription factor inhibitors decreased the activity of MMP3, ADAMTS4 and ADAMTS5 promoters. Furthermore, chromatin immunoprecipitation revealed functional Sp1 binding sites at ­577/­567 bp within the ADAMTS4 promoter and ­718/­708 bp within the ADAMTS5 promoter. These results provide pharmacological and genetic evidence of the importance of Sp1 in catabolic enzyme gene regulation during TNF­α stimulation. Thus, Sp1 may represent an effective target in reducing intervertebral disc­associated ECM loss.

Inflammatory cytokines induce caveolin-1/ß-catenin signalling in rat nucleus pulposus cell apoptosis through the p38 MAPK pathway.

OBJECTIVES: Apoptosis of nucleus pulposus (NP) cells is a major cause of intervertebral disc degeneration. To elucidate relationships between caveolin-1 and cytokine-induced apoptosis, we investigated the role of caveolin-1 in cytokine-induced apoptosis in rat NP cells and the related signalling pathway. MATERIALS AND METHODS: Rat NP cells were treated with interleukin (IL)-1ß or tumour necrosis factor alpha (TNF-α), and knockdown of caveolin-1 and ß-catenin was achieved using specific siRNAs. Then, apoptotic level of rat NP cells and expression and activation of caveolin-1/ß-catenin signalling were assessed by flow cytometric analysis, qRT-PCR, western blotting and luciferase assays. The relationship between the mitogen-activated protein kinase (MAPK) pathway and caveolin-1 promoter activity was also determined by luciferase assays. RESULTS: IL-1ß and TNF-α induced apoptosis, upregulated caveolin-1 expression and activated Wnt/ß-catenin signalling in rat NP cells, while the induction effect of cytokines was reversed by caveolin-1 siRNA and ß-catenin siRNA. Promotion of rat NP cell apoptosis and nuclear translocation of ß-catenin induced by caveolin-1 overexpression were abolished by ß-catenin siRNA. Furthermore, pretreatment with a p38 MAPK inhibitor or dominant negative-p38, blocked cytokine-dependent induction of caveolin-1/ß-catenin expression and activity. CONCLUSIONS: The results revealed the role of p38/caveolin-1/ß-catenin in inflammatory cytokine-induced apoptosis in rat NP cells. Thus, controlling p38/caveolin-1/ß-catenin activity seemed to regulate IL-1ß- and TNF-α-induced apoptosis in the NP during intervertebral disc degeneration.

Differential expression of p38 MAPK α, ß, γ, δ isoforms in nucleus pulposus modulates macrophage polarization in intervertebral disc degeneration.

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 (α,ß, γ and δ) in degenerative NP and the effect of p38 activation in NP cells on macrophage polarization. P38 α, ß and δ isoforms are preferential expressed, whereas the p38γ isoform is absent in human NP tissue. LV-sh-p38α, sh-p38ß transfection in NP cells significantly decreased the ADAMTS-4,-5, MMP-13,CCL3 expression and restored collagen-II and aggrecan expression upon IL-1ß stimulation. As compared with p38α and p38ß, p38δ exhibited an opposite effect on ADAMTS-4,-5, MMP-13 and aggrecan expression in NP cells. Furthermore, the production of GM-CSF and IFNγ which were trigged by p38α or p38ß in NP cells induced macrophage polarization into M1 phenotype. Our finding indicates that p38 MAPK α, ß and δ isoform are predominantly expressed and activated in IDD. P38 positive NP cells modulate macrophage polarization through the production of GM-CSF and IFNγ. Hence, Our study suggests that selectively targeting p38 isoforms could ameliorate the inflammation in IDD and regard IDD progression.