Response Of Nucleus Pulposus Cells Research Articles

Andrographolide influences IDD cell autophagy and oxidative stress under mechanical pressure via miR-9/FoxO3/PINK1/Parkin molecular axis.

Intervertebral disc degeneration (IDD) is characterized by the decreased function and number of nucleus pulposus cells (NPCs) caused by excessive intervertebral disc (IVD) pressure. This research aims to provide novel insights into IDD prevention and treatment by clarifying the effect of andrographolide (ANDR) on IDD cell autophagy and oxidative stress under mechanical stress. Human primary NPCs were extracted from the nucleus pulposus tissue of non-IDD trauma patients. An IDD cell model was established by posing mechanical traction on NPCs. Through the construction of an IDD rat model, the influence of ANDR on IDD pathological changes was explored in vivo. The proliferation and autophagy of NPCs were decreased while the apoptosis rate and oxidative stress reaction were increased by mechanical traction. ANDR intervention obviously alleviated this situation. MiR-9 showed upregulated expression in IDD cell model, while FoxO3 and PINK1/Parkin were downregulated. Decreased proliferation and autophagy as well as enhanced apoptosis and oxidative stress response of NPCs were observed following miR-9 mimics and H89 intervention, while the opposite trend was observed after FoxO3 overexpression. FoxO3 is a direct target downstream miR-9. The in vivo experiments revealed that after ANDR intervention, the number of apoptotic cells in rat IVD tissue decreased and the autophagy increased. In conclusion, ANDR improves NPC proliferation, and autophagy, inhibits apoptosis and oxidative stress, and alleviates the pathological changes of IDD via the miR-9/FoxO3/PINK1/Parkin axis, which may be a new and effective treatment for IDD in the future.

Laquinimod attenuates oxidative stress-induced mitochondrial injury and alleviates intervertebral disc degeneration by inhibiting the NF-κB signaling pathway

BackgroundLow back pain (LBP) caused by intervertebral disc degeneration (IVDD) is a significant global health concern. It is necessary to investigate the underlying pathological mechanisms leading to IVDD and develop precise treatment strategies for this condition. Considering the well-established anti-inflammatory properties and ability to reduce oxidative stress in various diseases, for the first time we aim to explore the potential of Laquinimod in alleviating IVDD. MethodsWe used hydrogen peroxide (H2O2) to simulate the oxidative stress microenvironment in IVDD, and Laquinimod for intervention purposes. Western blot analysis, quantitative real-time polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence assay were used to measure the expression levels of inflammatory cytokines, catabolic enzymes, and markers of extracellular matrix (ECM) synthesis in nucleus pulposus (NP) cells. In addition, dichlorofluorescin-diacetate (DCFH-DA) and JC-1 fluorescent probes, flow cytometry analysis, and qRT-PCR were used to measure mitochondrial function and apoptosis in NP cells under conditions of oxidative stress. An acupuncture-induced rat model of IVDD was established to further evaluate the efficacy of Laquinimod in alleviating IVDD in vivo. ResultsOur findings showed that Laquinimod significantly reduced the oxidative stress-induced inflammatory response in NP cells, downregulated the expression of catabolic enzymes, and markedly enhanced ECM degradation by inhibiting the NF-κB signaling pathway. The administration of Laquinimod concurrently improved the mitochondrial functional state and reduced apoptosis in NP cells. Additionally, in vivo experiments in rats showed that Laquinimod significantly alleviated acupuncture-induced IVDD. ConclusionsCollectively, the findings of this study provide new insights into the therapeutic potential of Laquinimod as a treatment for oxidative stress-induced IVDD.

Esculetin Alleviates IL-1β-Evoked Nucleus Pulposus Cell Death, Extracellular Matrix Remodeling, and Inflammation by Activating Nrf2/HO-1/NF-kb.

Inflammation, extracellular matrix metabolic dysfunction, and oxidative stress are key pathogenic characteristics of intervertebral disk degeneration (IVDD), a major pathogenic cause of low back pain. Esculetin possesses anti-injury, anti-inflammation, and antinociceptive properties. This study aimed to explore its role in IVDD. In this research, esculetin exhibited little cytotoxicity to human nucleus pulposus cells (NPCs). Moreover, esculetin increased cell viability under IL-1β stimulation but attenuated IL-1β-induced cell apoptosis and caspase-3 activity. Furthermore, IL-1β-evoked increases in intracellular reactive oxygen species and malondialdehyde (MDA) levels, and decreases in superoxide dismutase (SOD) activity were reversed after esculetin treatment, indicating the antioxidative stress efficacy of esculetin. Esculetin alleviated the inhibitory effects of IL-1β on the transcription and protein expression of anabolic biomarkers (collagen II and aggrecan), accompanied by decreases in expression and release of catabolic biomarkers MMP-3 and MMP-13 from NPCs. Moreover, IL-1β exposure enhanced the expression levels of the inflammatory mediator nitric oxide and inflammatory cytokine IL-6 and TNF-α, which were overturned after esculetin treatment. Additionally, esculetin activated the nuclear factor-erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) to inhibit the activation of nuclear factor κB (NF-κB) signaling in NPCs. Importantly, suppression of Nrf2 signaling reversed the protective efficacy of esculetin against IL-1β-mediated oxidative injury, matrix metabolism disruption, and inflammatory response in NPCs. Together, esculetin may alleviate IL-1β-induced dysfunction in NPCs by regulating the Nrf2/HO-1/NF-kb signaling, indicating its potential as a promising therapeutic agent against IVDD.

Systemic pharmacology reveal the mechanism by which the Qiangjin Zhuanggu Qufeng mixture inhibits LPS-induced pyroptosis of rat nucleus pulposus cells

ObjectiveLow back pain (LBP) is a worldwide health issue primarily attributed to intervertebral disc degeneration (IVDD). Qiangjin Zhuang Qufeng mixture (QJZG), an approved hospital-based formula with years of clinical application, has demonstrated notable therapeutic effects in the treatment of LBP. Nevertheless, the underlying mechanism by which it alleviates LBP remains uncertain. MethodsThe bioactive constituents of QJZG were initially identified using ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS). Subsequently, network pharmacology was employed to explore the core components and targets. In vivo and in vitro experiments were then conducted to validate the specific mechanism of action of QJZG based on the identified targets and pathways. Following that, ultra-high-performance liquid chromatography/mass spectrometry combined with 16S rRNA gene sequencing of blood and faecal samples was utilized to assess the impact of gut microbiota on faecal and serum metabolites subsequent to QJZG administration in intervertebral disc degeneration (IVDD) rats. ResultsThe principal constituents of QJZG were identified using UPLC-Q-TOF-MS/MS, revealing a substantial enrichment of flavonoids and triterpenes. Network pharmacology analysis indicated the potential inhibitory effects of QJZG on the NLRP3 inflammasome and downstream inflammatory factors. Furthermore, investigations demonstrated that intervertebral disc degeneration may be attributed to pyroptotic cell death within the nucleus pulposus. In vitro experiments were performed utilizing LPS to induce the inflammatory response in nucleus pulposus cells (NPC), and it was observed that QJZG-containing serum significantly suppressed key pyroptosis-related genes and downstream inflammatory factors. Additionally, in vivo experiments substantiated the capacity of QJZG to preserve disc height and ameliorate the progression of disc degeneration. Concurrently, oral pharmacotherapy in animal studies prominently involved the effects of Enterobacteriaceae and Clostridium, closely intertwined with lipid metabolism. ConclusionsQJZG exhibited a delaying effect on IVDD by preserving the equilibrium between extracellular matrix (ECM) synthesis and degradation in NPCs. This effect was achieved through the suppression of NLRP3 inflammasome expression and the prevention of pyroptosis in NPCs.

The effect of microRNA-663b in the inhibition of interleukin-1-induced nucleus pulposus cell apoptosis and inflammatory response.

The aim of this study was to explore the role and pathological mechanism of microRNA-663b in interleukin-1beta (IL-1β)-induced inflammation and apoptosis of nucleus pulposus cells. First, the best concentration and time to construct the nucleus pulposus cell inflammation model was screen out. Overexpression or inhibition of miR-663b expression was performed by adding microRNA-663b mimic or microRNA-663b inhibitor. 293T cells were transfected according to experimental requirements. The luciferase activity of each group was detected to analyze the targeted regulation of microRNA-663b on interleukin-1 receptor (IL1R1). Compared with the mimic negative control (NC) group, the expression of inflammatory factors in the microRNA-663b overexpression group was inhibited (P<0.05), and the expression of type 2 collagen and polysaccharide protein increased (P<0.05), and the apoptosis of nucleus pulposus cells was inhibited (P<0.01), and the number of TUNEL-positive cells decreased significantly (P<0.01), and the microRNA and protein expression of IL1R1, the ratio of P-P65/P65 and phospho-nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (P-IκBα)/nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) protein expression were significantly decreased (P<0.05). The expression of inflammatory factors in the miR-663b inhibitor group was significantly higher than that in the inhibitor NC group (P<0.01), and the expression of type 2 collagen and polysaccharide protein was significantly decreased (P<0.01), and the number of apoptosis cells and TUNEL staining positive cells increased (p<0.01). The expression of IL1R1 gene and protein was significantly increased (P<0.01). The ratio of P-P65/P65 and P-IκBα/IκBα protein expression increased (P<0.05). IL1R1 is a downstream target gene of microRNA-663b. MicroRNA-663b may down-regulate the expression of IL1R1 at the transcriptional level by targeting IL1R1, inhibit the inflammatory response of nucleus pulposus cells, and slow down the degeneration of nucleus pulposus cells.

The effect of microRNA-663b in the inhibition of interleukin-1-induced nucleus pulposus cell apoptosis and inflammatory response.

The aim of this study was to explore the role and pathological mechanism of microRNA-663b in interleukin-1beta (IL-1β)-induced inflammation and apoptosis of nucleus pulposus cells. First, the best concentration and time to construct the nucleus pulposus cell inflammation model was screen out. Overexpression or inhibition of miR-663b expression was performed by adding microRNA-663b mimic or microRNA-663b inhibitor. 293T cells were transfected according to experimental requirements. The luciferase activity of each group was detected to analyze the targeted regulation of microRNA-663b on interleukin-1 receptor (IL1R1). Compared with the mimic negative control (NC) group, the expression of inflammatory factors in the microRNA-663b overexpression group was inhibited (P<0.05), and the expression of type 2 collagen and polysaccharide protein increased (P<0.05), and the apoptosis of nucleus pulposus cells was inhibited (P<0.01), and the number of TUNEL-positive cells decreased significantly (P<0.01), and the microRNA and protein expression of IL1R1, the ratio of P-P65/P65 and phospho-nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (P-IκBα)/nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) protein expression were significantly decreased (P<0.05). The expression of inflammatory factors in the miR-663b inhibitor group was significantly higher than that in the inhibitor NC group (P<0.01), and the expression of type 2 collagen and polysaccharide protein was significantly decreased (P<0.01), and the number of apoptosis cells and TUNEL staining positive cells increased (p<0.01). The expression of IL1R1 gene and protein was significantly increased (P<0.01). The ratio of P-P65/P65 and P-IκBα/IκBα protein expression increased (P<0.05). IL1R1 is a downstream target gene of microRNA-663b. MicroRNA-663b may down-regulate the expression of IL1R1 at the transcriptional level by targeting IL1R1, inhibit the inflammatory response of nucleus pulposus cells, and slow down the degeneration of nucleus pulposus cells.

Pericellular heparan sulfate proteoglycans: Role in regulating the biosynthetic response of nucleus pulposus cells to osmotic loading.

BackgroundDaily physiologic loading causes fluctuations in hydration of the intervertebral disc (IVD); thus, the embedded cells experience cyclic alterations to their osmotic environment. These osmotic fluctuations have been described as a mechanism linking mechanics and biology, and have previously been shown to promote biosynthesis in chondrocytes. However, this phenomenon has yet to be fully interrogated in the IVD. Additionally, the specialized extracellular matrix surrounding the cells, the pericellular matrix (PCM), transduces the biophysical signals that cells ultimately experience. While it is known that the PCM is altered in disc degeneration, whether it disrupts normal osmotic mechanotransduction has yet to be determined. Thus, our objectives were to assess: (1) whether dynamic osmotic conditions stimulate biosynthesis in nucleus pulposus cells, and (2) whether pericellular heparan sulfate proteoglycans (HSPGs) modulate the biosynthetic response to osmotic loading.MethodsBovine nucleus pulposus cells isolated with retained PCM were encapsulated in 1.5% alginate beads and treated with or without heparinase III, an enzyme that degrades the pericellular HSPGs. Beads were subjected to 1 h of daily iso‐osmotic, hyper‐osmotic, or hypo‐osmotic loading for 1, 2, or 4 weeks. At each timepoint the total amount of extracellular and pericellular sGAG/DNA were quantified. Additionally, whether osmotic loading triggered alterations to HSPG sulfation was assessed via immunohistochemistry for the heparan sulfate 6‐O‐sulfertransferase 1 (HS6ST1) enzyme.ResultsOsmotic loading significantly influenced sGAG/DNA accumulation with a hyper‐osmotic change promoting the greatest sGAG/DNA accumulation in the pericellular region compared with iso‐osmotic conditions. Heparanase‐III treatment significantly reduced extracellular sGAG/DNA but pericellular sGAG was not affected. HS6ST1 expression was not affected by osmotic loading.ConclusionResults suggest that hyper‐osmotic loading promotes matrix synthesis and that modifications to HSPGs directly influence the metabolic responses of cells to osmotic fluctuations. Collectively, results suggest degeneration‐associated modifications to pericellular HSPGs may contribute to the altered mechanobiology observed in disease.

Polystyrene nanoparticles carrying miR-375 alleviate inflammatory response of intervertebral disc nucleus pulposus cells via NF-κB signaling pathway

The aim of this study was to investigate the protective role of miR-375-loaded polystyrene nanoparticles (PSNPs) in inflammatory response within nucleus pulposus cells (NPC) in intervertebral disc. RT-qPCR was used to determine the miR-375 expression in the intervertebral disc NPCs and tissues from patients with inflammatory reaction. After transfection of PS-NPs carrying miR-375, MTT and flow cytometry experiment were conducted to assess proliferation, apoptosis and cell cycle of inflammatory cells in the intervertebral disc. The relationship between miR-375-loaded PS-NPs and NF-κB pathway was analyzed using a luciferase reporter assay. miR-375 expression was decreased in inflammatory cells and intervertebral disc NPCs. Administration of PS-NPs carrying miR-375 resulted in decreased proliferation rate of NPCs when promoting cell apoptosis. PS-NP inhibitor in return suppressed apoptosis of NPCs in the intervertebral disc. The expression of NF-κB pathway was directly regulated by miR-375 PS-NPs and participated in the inflammatory response of intervertebral disc NPCs. Down-regulation of miR-375 induced continuous proliferation of inflammatory cells in the nucleus pulposus of intervertebral disc and hindered cell apoptosis. miR-375 PS-NPs can inhibit the inflammatory response of NPCs on intervertebral disc through NF-κB pathway, which provides a new direction for nano-therapeutics for inflammatory responses of intervertebral disc NPCs.

Long intergenic non-coding RNA -00917 regulates the proliferation, inflammation, and pyroptosis of nucleus pulposus cells via targeting miR-149-5p/NOD-like receptor protein 1 axis

ABSTRACT Intervertebral disc degeneration (IDD) has caused great trouble in people’s lives. Dysregulated long noncoding RNAs (lncRNAs) are closely linked to IDD progression. Our study aims to analyze the role of LINC00917 in the progression of IDD. Forty nucleus pulposus (NP) IDD tissues and 40 NP tissues of intervertebral discs without apparent degeneration were collected. TBHP was used to induce IDD. Cell proliferation was measured using the MTT and EdU assays. Pyroptosis was detected using flow cytometry. RT-qPCR and Western blot assays were performed to determine mRNA, miRNA, and protein expression. Dual-luciferase reporter and RNA pull-down assays were performed to verify the relationship between LINC00917 or NLRP1 and miR-149-5p. LINC00917 expression was enhanced in TBHP-treated nucleus pulposus cells (NPCs). The knockdown of LINC00917 promoted proliferation and inhibited cytotoxicity, inflammatory response, and pyroptosis of NPCs. LINC00917 functions as a sponge for miR-149-5p. Having silenced miR-149-5p, the effects of LINC00917 knockdown on NPC proliferation and inflammation-induced pyroptosis were alleviated. NLRP1 overexpression induced cellular dysfunction and pyroptosis of NPCs. LINC00917 knockdown restored NPC cellular functions and inhibited IDD progression by modulating the miR-149-5p/NLRP1 axis.

Circ_0004354 might compete with circ_0040039 to induce NPCs death and inflammatory response by targeting miR-345-3p-FAF1/TP73 axis in intervertebral disc degeneration.

The abnormal function of nucleus pulposus cells (NPCs) plays a crucial role in the pathogenesis of intervertebral disc degeneration (IVDD). Recent studies have demonstrated that circular RNAs (circRNAs) are involved in the pathological process of IVDD by regulating NPCs' function. Nevertheless, the investigation on circRNA-circRNA interaction has not yet been reported. Here, we identified the top upregulated circ_0040039 and circ_0004354 in IVDD, derived from the syntrophin beta 2 gene but had different degrees of biological functions. Accumulating studies have reported PANoptosis is composed of apoptosis, pyroptosis, and necroptosis. Based on this, we think there should be a new pro-inflammatory cell death PAoptosis in the form of apoptosis and pyroptosis. Circ_0004354 might compete with circ_0040039 to induce the development of IVDD by modulating miR-345-3p-FAF1/TP73 axis-mediated PAoptosis, inflammatory response, growth inhibition, and ECM degradation of NPCs. Thus, these findings offer a novel insight into the circRNAs-mediated posttranscriptional regulatory network in IVDD, contributing to further clarification of the pathological mechanism of IVDD to develop a promising therapeutic target for IVDD diseases.

Microfluidic Chip with Low Constant-Current Stimulation (LCCS) Platform: Human Nucleus Pulposus Degeneration In Vitro Model for Symptomatic Intervertebral Disc

Intervertebral disc (IVD) degeneration is a major cause of low back pain (LBP) in the lumbar spine. This phenomenon is caused by several processes, including matrix degradation in IVD tissues, which is mediated by matrix metalloproteinases (MMPs) and inflammatory responses, which can be mediated by interactions among immune cells, such as macrophages and IVD cells. In particular, interleukin (IL)-1 beta (β), which is a master regulator secreted by macrophages, mediates the inflammatory response in nucleus pulposus cells (NP) and plays a significant role in the development or progression of diseases. In this study, we developed a custom electrical stimulation (ES) platform that can apply low-constant-current stimulation (LCCS) signals to microfluidic chips. Using this platform, we examined the effects of LCCS on IL-1β-mediated inflammatory NP cells, administered at various currents (5, 10, 20, 50, and 100 μA at 200 Hz). Our results showed that the inflammatory response, induced by IL-1β in human NP cells, was successfully established. Furthermore, 5, 10, 20, and 100 μA LCCS positively modulated inflamed human NP cells’ morphological phenotype and kinetic properties. LCCS could affect the treatment of degenerative diseases, revealing the applicability of the LCCS platform for basic research of electroceuticals.

RETRACTED: Role of microRNA-15a-5p/Sox9/NF-κB axis in inflammatory factors and apoptosis of murine nucleus pulposus cells in intervertebral disc degeneration

MicroRNAs are well-established players in post-transcriptional gene modulation. We aim to explore the role of microRNA-15a-5p (miR-15a-5p)/sex determining region Y-box 9 (Sox9)/nuclear factor-κB (NF-κB) axis in inflammation and apoptosis of murine nucleus pulposus cells (NPCs) in intervertebral disc degeneration (IVDD). Expression levels of miR-15a-5p and Sox9 in disc tissues from IVDD patients were determined. The IVDD mouse models were established by disc puncture, and the modeled mice were accordingly injected with miR-15a-5p antagomir and/or overexpressed Sox9 plasmid, or their negative controls. Then, the expression of miR-15a-5p, Sox9 and p-p65, pathological changes and the apoptosis of NPCs in IVDD mouse intervertebral disc tissues were measured. The NPCs were isolated and cultured, which were then transfected with miR-15a-5p inhibitor, overexpressed or silenced Sox9 plasmids, or the NCs. Next, the expression of miR-15a-5p and Sox9, apoptosis, proliferation and cell cycle distribution of NPCs, and the contents of inflammatory factors in the NPCs were evaluated. MiR-15a-5p expression was increased while Sox9 expression was reduced in intervertebral disc tissues from IVDD patients and mice. Mouse NPCs were successfully isolated. The down-regulated miR-15a-5p could elevate Sox9 to activate p-p65 expression, suppress NPC apoptosis and inflammatory factor contents, promote proliferation of NPCs, and arrest the NPCs at S and G2/M phases. However, these effects could be reversed by silencing Sox9. Reduction of miR-15a-5p elevated Sox9 to inhibit the inflammatory response and apoptosis of NPCs in IVDD mice through the NF-κB pathway. This study may be helpful for IVDD treatment.

MicroRNA-16 inhibits the lipopolysaccharide-induced inflammatory response in nucleus pulposus cells of the intervertebral disc by targeting TAB3.

IntroductionA variety of inflammatory mediators are produced by the degenerative human intervertebral disc (IVD) tissues spontaneously, suggesting their role in the development of intervertebral disc degeneration (IDD). Our present study was designed to investigate the regulatory effect of microRNA-16 (miR-16) on the lipopolysaccharide (LPS)-induced inflammatory response in nucleus pulposus (NP) cells of the IVD.Material and methodsNP cells were treated with LPS to induce inflammatory responses. The expression of miRNA and genes was determined by qRT-PCR. Western blot and an ELISA kit were used to detect the proteins and protein expression, respectively. A dual luciferase reporter assay was applied to identify the correlation between a miRNA and a gene, and to test nuclear factor-κB (NF-κB) activity.ResultsThe results suggested that miR-16 positively regulated the mRNA and protein expression of extracellular matrix (ECM) genes (including aggrecan and collagen II) in NP cells, while it was negatively correlated with ECM degrading enzymes (including MMP3, MMP13, ADAMTS4, ADAMTS5) and related genes of nitric oxide (NO) reaction. Further studies revealed that miR-16 could oppositely regulate NF-κB and MAPK pathways by directly mediating their upstream gene TAB3.ConclusionsOur study suggested that, in NP cells of the IVD, miR-16 could exert inhibitory effects on the LPS-induced inflammatory response through NF-κB and MAPK pathways by directly mediating TAB3. In this way, miR-16 would play a protective role against LPS-induced IDD and inflammation. Therefore, miR-16 may be a novel therapeutic target for the inhibit of the ECM in the IVD.

Dimethyl fumarate protects nucleus pulposus cells from inflammation and oxidative stress and delays the intervertebral disc degeneration.

Lower back pain is a common problem in middle-aged and elderly people, and intervertebral disc degeneration (IVDD) is often the main cause. The present study aimed to explore the effects of dimethyl fumarate (DMF) on inflammation and oxidative stress in the intervertebral disc. C57/BL6 mice were used to construct an IVDD model by tail suspension and daily intraperitoneal injections of 10 mg/kg DMF were administered to analyze the effects of DMF on IVDD. In addition, human nucleus pulposus (NP) cells were cultured and stimulated cells with recombinant human IL-1β and DMF to examine the effects of DMF on inflammation and oxidative stress in NP cells. DMF significantly increased the intervertebral disc height index of mice and inhibited the degradation of the extracellular matrix of mouse NP tissue. In addition, DMF also decreased the expression of inflammatory factors [including IL-6, IL-8, matrix metalloproteinase (MMP)3 and MMP13] in NP cells. In terms of oxidative stress, DMF significantly increased the antioxidative stress response in NP cells and reduced endoplasmic reticulum stress. DMF also increased the activity of the nuclear factor erythroid 2-related factor (Nrf) 2/heme oxygenase (HO)-1 signaling pathway in NP cells and increased the phosphorylation of Akt. DMF also increased the anti-inflammatory and antioxidative ability of NP cells by promoting the activity of the Nrf2/HO-1 and PI3K/Akt signaling pathways, thus delaying IVDD.

Regenerative Response of Degenerate Human Nucleus Pulposus Cells to GDF6 Stimulation.

Growth differentiation factor (GDF) family members have been implicated in the development and maintenance of healthy nucleus pulposus (NP) tissue, making them promising therapeutic candidates for treatment of intervertebral disc (IVD) degeneration and associated back pain. GDF6 has been shown to promote discogenic differentiation of mesenchymal stem cells, but its effect on NP cells remains largely unknown. Our aim was to investigate GDF6 signalling in adult human NP cells derived from degenerate tissue and determine the signal transduction pathways critical for GDF6-mediated phenotypic changes and tissue homeostatic mechanisms. This study demonstrates maintained expression of GDF6 receptors in human NP and annulus fibrosus (AF) cells across a range of degeneration grades at gene and protein level. We observed an anabolic response in NP cells treated with recombinant GDF6 (increased expression of matrix and NP-phenotypic markers; increased glycosaminoglycan production; no change in catabolic enzyme expression), and identified the signalling pathways involved in these responses (SMAD1/5/8 and ERK1/2 phosphorylation, validated by blocking studies). These findings suggest that GDF6 promotes a healthy disc tissue phenotype in degenerate NP cells through SMAD-dependent and -independent (ERK1/2) mechanisms, which is important for development of GDF6 therapeutic strategies for treatment of degenerate discs.

Mesenchymal stem cells regulate inflammatory milieu within degenerative nucleus pulposus cells via p38 MAPK pathway.

It has been established that excessive apoptosis of nucleus pulposus cells (NPCs) are responsible for pathogenesis of human intervertebral disc degeneration (IDD). The present study aimed to shed light on the molecular mechanisms underlying the protective effects of mesenchymal stem cells (MSCs) on NPCs in an inflammatory environment. NPCs were treated with TNF-α to induce inflammation and then co-cultured with Wharton's Jelly-derived MSCs (WJ-MSCs)without direct interaction. The levels of inflammation markers (IL-1β, IL-6 and IL-8) in NPCs were detected by performing enzyme-linked immunosorbent assay (ELISA), and expression of metalloproteases and aggrecan, as well as the activity of p38 MAPK pathway were determined through immunoblotting. SB-203580 was used to inhibit p38 signaling, prior to evaluation of the effects of Wharton's Jelly-derived MSCs (WJ-MSCs) on inflammatory response within the co-cultured NPCs. After TNF-α treatment, the levels of inflammatory cytokines, MMP-3, and MMP-13 in NPCs were increased whereas aggrecan was decreased, which was then dramatically reversed by WJ-MSCs co-culture. Likewise, WJ-MSCs suppressed TNF-α-induced phosphorylation of p38 MAPK signaling components including p38, ASK-1, MKK-3 and MKK-6. Blocking p38 MAPK pathway enhanced the anti-inflammatory impact of WJ-MSCs, and there was no significant difference between NPCs co-cultured with WJ-MSCs or the cells cultured alone. WJ-MSCs co-culture mitigate TNF-α-induced inflammatory response and ECM degeneration in NPCs, the major pathological events are implicated in IDD development, probably by suppressing the p38 MAPK signaling cascade.

The Anti-Inflammatory and Cytoprotective Efficiency of Curvularin, a Fungal Macrolactone against Lipopolysaccharide-Induced Inflammatory Response in Nucleus Pulposus Cells: An &lt;i&gt;In Vitro&lt;/i&gt; Study

The Anti-Inflammatory and Cytoprotective Efficiency of Curvularin, a Fungal Macrolactone against Lipopolysaccharide-Induced Inflammatory Response in Nucleus Pulposus Cells: An &lt;i&gt;In Vitro&lt;/i&gt; Study

The Anti-Inflammatory and Cytoprotective Efficiency of Curvularin, a Fungal Macrolactone against Lipopolysaccharide-Induced Inflammatory Response in Nucleus Pulposus Cells: An In Vitro Study.

Study DesignDeveloping an in vitro model for assessing the anti-inflammatory properties of curvularin.PurposeTo evaluate the efficacy of natural fungal macrolactone as a therapeutic drug against lipopolysaccharide (LPS)-induced inflammation in primary human nucleus pulposus cells (NPCs) in vitro.Overview of LiteratureLumbar disk disease is a common cause of lower back pain (LBP) and sciatica. It is an established fact that inflammation, rather than mechanical compression on the nerve root, plays a role in the cause of LBP and sciatica. Current treatment options for reducing inflammation are either nonsteroidal anti-inflammatory drugs or steroids, prolonged use of which can potentially lead to adverse effects such as gastrointestinal disturbances and renal and cardiac issues. Hence, there is a need for better antiinflammatory drugs with no or minimal complications for treating inflammation-induced LBP and sciatica. Curvularin (Cur), a fungal macrolactone, is known for its anti-inflammatory activity, but nothing is known about its impact on inflammation due to disk pathologies.MethodsPrimary NPCs were cultured and characterized by flow cytometry and immunocytochemistry using the CD24 antibody and treated with 10 μg/mL LPS for 36 hours and then treated with Cur, betamethasone, and dexamethasone (10 μg/mL) for 48 hours, after which cell cycle analysis, cell viability assay, and gene expression studies (quantitative polymerase chain reaction [PCR] and quantitative real-time-PCR) were conducted. The NPCs treated with Cur downregulated the expression of pro-inflammatory cytokines (tumor necrosis factor-α, interleukin [IL]-1β, and IL-6); matrix metalloproteinases (MMPs; MMP-2 and MMP-3), ADAMTS; and apoptotic marker (cytochrome c).ResultsIn our study, Cur-treated cells showed enhanced expression of collagen 9A1 and insulin-like growth factor receptor 1, indicating the recovery of NPCs from inflammatory assault.ConclusionsBased on observations, the anti-inflammatory properties of Cur render it an excellent drug molecule for treating disk degeneration nonsurgically, by direct injection into spinal disks when treating LBP and sciatica.

Oxymatrine suppresses IL-1β-induced degradation of the nucleus pulposus cell and extracellular matrix through the TLR4/NF-κB signaling pathway.

Intervertebral disc degeneration is the main cause of low back pain. However, its pathomechanism has not been fully clarified yet. Previous studies have indicated that inflammation may lead to apoptosis of nucleus pulposus cells and break the balance between anabolism and catabolism of the nucleus pulposus extracellular matrix. The purpose of this study is to explore the mitigative effect of oxymatrine on extracellular matrix degradation and apoptosis of nucleus pulposus cells after interleukin-1 beta-induced inflammation, and its possible signaling pathway. We examined the gene and protein levels of collagen II, aggrecan, and MMPs (MMP2/3/9/13) and interleukin 6 in nucleus pulposus cells. The results demonstrated that oxymatrine could reduce extracellular matrix degradation and apoptosis of nucleus pulposus cells; interleukin-1 beta prompted the expression of MMPs and interleukin 6 through TLR4/NF-κB axis, while oxymatrine reduced the expression of MMPs and TNF-α induced by interleukin-1 beta. Moreover, TAK 242, as a small molecule inhibitor of TLR4 signaling, was used to detect the effect of oxymatrine on the TLR4/NF-κB signaling. The final experimental results show that oxymatrine could reduce the inflammatory response of nucleus pulposus cells and degradation of nucleus pulposus tissue. Oxymatrine may be a potential medicine to reduce disc inflammation and relieve intervertebral disc degeneration by inhibiting the TLR4/NF-κB signal pathway. Impact statement Currently, drug therapy is a potential treatment for patients with intervertebral disc degeneration. In the present research, oxymatrine intervenes in intervertebral disc degeneration effectively via regulating inflammation in intervertebral disc degeneration rats. Our research highlights the therapeutic potential of oxymatrine in the treatment of intervertebral disc degeneration.

Impact of NF-κB pathway on the apoptosis-inflammation-autophagy crosstalk in human degenerative nucleus pulposus cells.

The NF-κB pathway has been reported to play a very important role in the process of intervertebral disc degeneration (IVDD). Our results demonstrated that knockdown of NF-κB with P65-siRNA can significantly decrease cell apoptosis and the expression of pro-inflammation factors TNF-α and IL-1β in LPS-induced nucleus pulposus cells (NPCs). However, the molecular mechanism of NF-κB pathway exerting anti-inflammation and anti-apoptosis function remains unclear. Some researchers reported that inhibiting NF-κB pathway can attenuate the catabolic effect by promoting autophagy during inflammatory conditions in rat nucleus pulposus cells. Therefore, we hypothesized that in human NPCs, inhibiting NF-κB pathway may also promote autophagy. Our results indicated that after knockdown of NF-κB, the autophagy was significantly increased and the expression of p-AKT and p-mTOR protein markedly decreased, but the level of autophagy was inhibited after treatment with AKT activator SC79, suggesting the involvement of AKT/mTOR–mediated autophagy was under autophagy activation. However, both LPS-induced NPCs apoptosis and expression of pro-inflammation factors were further increased by pretreatment with the autophagy inhibitor chloroquine (CQ). These suggested that inhibiting NF-κB pathway can promote autophagy and decrease apoptosis and inflammation response in LPS-induced NPCs. Meanwhile, autophagy triggered by NF-κB inhibition plays a protective role against apoptosis and inflammation.