Inflammation In Nucleus Pulposus Cells Research Articles

Mechanism of KMT2D-mediated epigenetic modification in IL-1β-induced nucleus pulposus cell degeneration.

Intervertebral disc (IVD) degeneration (IVDD) is characterized by structural destruction accompanied by accelerated signs of aging. This study aimed to investigate the mechanism of lysine methyltransferase 2D (KMT2D) in the proliferation, apoptosis, and inflammation of nucleus pulposus cells (NPCs) in IVDD. Mouse-derived NPCs were cultured and induced with interleukin-1 beta (IL-1β) to establish cell models. KMT2D expression was detected by western blot and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). KMT2D expression was interfered with, and the contents of IL-18, IL-6, and tumor necrosis factor (TNF) were detected by enzyme-linked immunosorbent assay. Cell proliferation, apoptosis, and the expression of miR-133a-5p and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2) were measured. The enrichment of KMT2D and Histone 3 Lysine 4 monomethylation/dimethylation (H3K4me1/2) on the miR-133a-5p promoter was analyzed by chromatin immunoprecipitation and qPCR. The binding of miR-133a-5p and PFKFB2 was analyzed by a dual-luciferase assay. IL-1β treatment promoted KMT2D expression in NPCs. KMT2D knockdown reduced inflammation and apoptosis and promoted the proliferation of IL-1β-induced NPCs. Mechanistically, KMT2D upregulated miR-133a-5p expression by increasing the level of H3K4me2 at the miR-133a-5p promoter, thereby promoting the binding between miR-133a-5p and PFKFB2 and downregulating the transcription of PFKFB2. miR-133a-5p overexpression or PFKFB2 knockdown alleviated the protective effect of KMT2D knockdown on IL-1β-induced NPCs. KMT2D promoted miR-133a-5p expression through H3K4me2 methylation, thereby promoting the binding of miR-133a-5p to PFKFB2, reducing the mRNA level of PFKFB2, promoting inflammation and apoptosis of IL-1β-induced NPCs, and inhibiting NPC proliferation.

Immune-defensive microspheres promote regeneration of the nucleus pulposus by targeted entrapment of the inflammatory cascade during intervertebral disc degeneration

Sustained and intense inflammation is the pathological basis for intervertebral disc degeneration (IVDD). Effective antagonism or reduction of local inflammatory factors may help regulate the IVDD microenvironment and reshape the extracellular matrix of the disc. This study reports an immunomodulatory hydrogel microsphere system combining cell membrane-coated mimic technology and surface chemical modification methods by grafting neutrophil membrane-coated polylactic-glycolic acid copolymer nanoparticles loaded with transforming growth factor-beta 1 (TGF-β1) (T-NNPs) onto the surface of methacrylic acid gelatin anhydride microspheres (GM) via amide bonds. The nanoparticle-microsphere complex (GM@T-NNPs) sustained the long-term release of T-NNPs with excellent cell-like functions, effectively bound to pro-inflammatory cytokines, and improved the release kinetics of TGF-β1, maintaining a 36 day-acting release. GM@T-NNPs significantly inhibited lipopolysaccharide-induced inflammation in nucleus pulposus cells in vitro, downregulated the expression of inflammatory factors and matrix metalloproteinase, and upregulated the expression of collagen-II and aggrecan. GM@T-NNPs effectively restored intervertebral disc height and significantly improved the structure and biomechanical function of the nucleus pulposus in a rat IVDD model. The integration of biomimetic technology and nano-drug delivery systems expands the application of biomimetic cell membrane-coated materials and provides a new treatment strategy for IVDD.

The long non-coding RNA maternally expressed 3-micorRNA-15a-5p axis is modulated by melatonin and prevents nucleus pulposus cell inflammation and apoptosis.

Nucleus pulposus (NP) cell apoptosis is regarded as a critical risk factor for intervertebral disc degeneration (IVDD). Melatonin exerts a protective role on NP cells. The study concentrates on the role and mechanism of lncRNA MEG3 in melatonin-mediated effects on NP cells. An in vitro IVDD model was constructed using IL-1β on human NP cells. qRT-PCR investigated MEG3, miR-15a-5p and PGC-1α mRNA levels in tissues and NP cells. IL-1β-treated NP cells subsequent to transfection, followed by melatonin treatment. NP cell proliferation, viability, apoptosis and inflammatory reactions were assayed. Western blot checked the profiles of PGC-1α, SIRT1 and NF-κB p65. Student's t-test or one-way analysis of variance (ANOVA) followed by Tukey's test was used for statistical tests. As indicated by the data, melatonin weakened NP cell inflammation and apoptosis and enhanced MEG3 expression. MEG3 expression was attenuated in IVDD tissues. MEG3 knockdown impaired the function of melatonin, which was, however, strengthened by miR-15a-5p knockdown. MEG3 targeted miR-15a-5p, which targeted PGC-1α and repressed the PGC-1α/SIRT1 pathway. Collectively, this study has disclosed that the MEG3-miR-15a-5p-PGC-1α/SIRT1 pathway modulated by melatonin can hamper NP cell apoptosis and inflammation elicited by IL-1β.

LncRNA HCG18 promotes inflammation and apoptosis in intervertebral disc degeneration via the miR-495-3p/FSTL1 axis.

Intervertebral disc degeneration (IDD) causes pain in the back and neck. This study investigated the role of long non-coding RNA HLA complex group 18 (HCG18) in a cell model of IDD. An IDD model was established by stimulating nucleus pulposus (NP) cells with interleukin (IL)-1β. MTT assay was performed to evaluate NP cell viability. The apoptosis was detected by flow cytometry. The expressions of HCG18, microRNA (miR)-495-3p, and follistatin-like protein-1 (FSTL1) were measured by RT-qPCR. The interactions of miR-495-3p with HCG18 and FSTL1 were analyzed by luciferase reporter assay. IL-1β stimulation upregulated HCG18 and FSTL1, but downregulated miR-495-3p in NP cells. Silencing of HCG18 or FSTL1, as well as miR-495-3p overexpression in NP cells alleviated IL-1β-induced apoptosis and inflammation of NP cells. Both HCG18 and FSTL1 had binding sites for miR-495-3p. Overexpression of FSTL1 abolished the effects of HCG18 silencing on IL-1β-induced apoptosis and inflammation. The HCG18/miR-495-3p/FSTL1 axis is essential for IDD development. Therapeutic strategies targeting this axis may be used for IDD treatment.

Emodin protects against apoptosis and inflammation by regulating reactive oxygen species-mediated NF-κB signaling in interleukin-1β-stimulated human nucleus pulposus cells.

Intervertebral disc degeneration (IDD) is a complex degradative disorder associated with inflammation. Emodin, an anthraquinone derivative, possesses strong anti-inflammatory activity. This study focused on the in vitro therapeutic action of emodin in a cellular model of IDD. Human nucleus pulposus cells (NPCs) were stimulated with interleukin-1β (IL-1β) to induce inflammation. Cell Counting Kit-8 and terminal deoxynucleotidyl transferase dUTP nick end labeling staining assays were performed to evaluate the viability and apoptosis of NPCs, respectively. Caspase-3 activity was measured to indirectly assess cell apoptosis. Western blot analysis was performed to detect protein expression levels. Reverse transcription-polymerase chain reaction was performed for the detection of relative mRNA levels of tumor necrosis factor-α (TNF-α) and IL-6. Enzyme-linked immunosorbent assay was performed to analyze TNF-α and IL-6 secretion. Our results showed that emodin treatment mitigated IL-1β-induced reduction of cell viability in NPCs. Moreover, the increase in reactive oxygen species (ROS) production, apoptotic rate, and caspase-3 activity in IL-1β-stimulated NPCs was reduced by emodin treatment. Treatment with emodin also abolished IL-1β-induced inflammation in NPCs, as indicated by reduced secretion of IL-6 and TNF-α. Besides, the increase in expression levels of phosphorylated p65 and nuclear p65 in IL-1β-stimulated NPCs was suppressed by emodin treatment. Furthermore, inhibition of nuclear factor kappa B (NF-κB) activation with pyrrolidine dithiocarbamate aggravated the protective effects of emodin. These results suggested that emodin protected NPCs against IL-1β-induced apoptosis and inflammation via inhibiting ROS-mediated activation of NF-κB.

Regulatory roles of noncoding RNAs in intervertebral disc degeneration as potential therapeutic targets (Review).

Intervertebral disc degeneration (IDD) is the leading cause of lower back pain, which is one of the primary factors that lead to disability and pose a serious economic burden. The key pathological processes involved are extracellular matrix degradation, autophagy, apoptosis, and inflammation of nucleus pulposus cells. Non-coding RNAs (ncRNAs), including microRNAs, long ncRNAs and circular RNAs, are key regulators of the aforementioned processes. ncRNAs are differentially expressed in tissues of the intervertebral disc between healthy individuals and patients and participate in the pathological progression of IDD via a complex pattern of gene regulation. However, the regulatory mechanisms of ncRNAs in IDD remain unclear. The present review summarizes the latest insights into the regulatory role of ncRNAs in IDD and sheds light on potentially novel therapeutic strategies for IDD that may be implemented in the future.

BMSC-Derived Exosomes Alleviate Intervertebral Disc Degeneration by Modulating AKT/mTOR-Mediated Autophagy of Nucleus Pulposus Cells.

The pathogenesis of intervertebral disc degeneration (IDD) is still unclear. It has been shown that the pathological process of IDD is most closely related to inflammation of nucleus pulposus cells (NPCs), in which inflammatory factors play an important role. Exosomes are the main paracrine mediators and are microvesicles with biological functions similar to those of the cells from which they are derived. Studies have shown that bone mesenchymal stem cells (BMSCs) can inhibit apoptosis of NPCs by sending exosomes as anti-inflammatory and antioxidant, which has been proved to be effective on IDD. However, the specific mechanism of inhibiting apoptosis of NPCs is still unclear. In our study, BMSC-derived exosomes (BMSC-Exo) were isolated from the BMSC culture medium, and their antiapoptotic effects were evaluated in cells and rat models to explore the possible mechanisms. We observed that BMSC-Exo promotes autophagy in NPCs and inhibits the release of inflammatory factors such as IL-1β and TNF-α in LPS-treated NPCs and inhibits apoptosis in NPCs. Further studies showed that BMSC-Exo inhibited the Akt-mTOR pathway. Intramuscular injection of BMSC-Exo alleviates disc degeneration in rat IDD models. In conclusion, our results suggest that BMSC-Exo can reduce NPC apoptosis and alleviate IDD by promoting autophagy by inhibiting the Akt-mTOR pathway. Our study confers a promising therapeutic strategy for IDD.

Kukoamine A attenuates lipopolysaccharide-induced apoptosis, extracellular matrix degradation, and inflammation in nucleus pulposus cells by activating the P13K/Akt pathway

ABSTRACT Intervertebral disc degeneration (IDD) is the leading cause of back, neck, and radicular pain. This study aims to look at the roles of Kukoamine A (KuA) in nucleus pulposus cells (NPCs) of IDD and its related potential mechanisms. Cell viability of NPCs in the control, lipopolysaccharide (LPS) and LPS+KuA groups was firstly detected by cell counting kit (CCK)-8. Meanwhile, the protein expression of collagen II in LPS-induced NPCs was measured by western blot. Then, the experiments following the treatment of KuA in LPS-induced NPCs included cell proliferation assessment by 5-ethynyl-2’-deoxyuridine (EdU) kit, cell apoptosis and extracellular matrix degradation (ECM) analysis by Terminal dUTP nick-end labeling (TUNEL) and western blot, the detection of inflammatory cytokines by western blot and enzyme-linked immunosorbent assay (ELISA), P13K/Akt pathway-related protein levels analysis by western blot. Finally, after the addition of P13K/Akt pathway inhibitor LY294002, cell apoptosis, ECM and inflammation in KuA-treated NPCs induced by LPS were again examined by the same methods. Results indicated that KuA prevented loss of cell viability and attenuated the apoptosis, ECM, and inflammation in LPS-induced NPCs. Furthermore, western blot experiment verified the activation of KuA on P13K/Akt pathway in LPS-induced NPCs. However, inhibition of P13K/Akt pathway reversed the roles of KuA in LPS-induced NPCs. Thus, KuA attenuates LPS-induced apoptosis, ECM and inflammation in LPS-induced NPCs by activating the P13K/Akt pathway.

The mitochondrial antioxidant SS-31 attenuated lipopolysaccharide-induced apoptosis and pyroptosis of nucleus pulposus cells via scavenging mitochondrial ROS and maintaining the stability of mitochondrial dynamics

Evidence has shown that effects from inflammation and mitochondrial dysfunction lead to pyroptosis and apoptosis of nucleus pulposus (NP) cells. Damaged mitochondria release dangerous molecules such as reactive oxygen species (ROS), activating the NLRP3 inflammasome. SS-31 is a mitochondria-targeting peptide that has been used in the treatment of many diseases by scavenging ROS and ameliorating mitochondrial function. This study found that SS-31 ameliorated lipopolysaccharide (LPS)-induced loss of cell viability, ROS production, and apoptosis in NP cells. Moreover, mitochondrial dynamics and ATP synthesis were restored on pretreatment with SS-31 compared with the LPS group. For the molecular mechanism research, SS-31 stabilized mitochondrial morphology and inhibited the activation of the NF-κB pathway and the activation of the NLRP3 inflammasome. To evaluate whether the inhibition of NLRP3 inflammasome activation by SS-31 is dependent on the clearance of mitochondrial ROS, we comparatively analyzed the activation of NLRP3 inflammasome in NP cells pretreated with SS-31 and the ROS scavenger N-acetyl-L-cysteine (NAC). The results indicate that SS-31 could inhibit NLRP3 inflammasome activation by limiting the production of mitochondrial ROS. To sum up, our results revealed that SS-31 inhibits LPS-induced apoptosis, pyroptosis, and inflammation in NP cells via scavenging ROS and maintaining the stability of mitochondrial dynamics, which could be considered a promising therapeutic intervention for disk degeneration.

Dehydrocostus Lactone Attenuates the Senescence of Nucleus Pulposus Cells and Ameliorates Intervertebral Disc Degeneration via Inhibition of STING-TBK1/NF-κB and MAPK Signaling.

The progression of intervertebral disc degeneration (IDD) is multifactorial with the senescence of nucleus pulposus (NP) cells and closely related to inflammation in NP cells. Dehydrocostus lactone (DHE) is a natural sesquiterpene lactone isolated from medicinal plants that has anti-inflammatory properties. Thus, DHE may have a therapeutic effect on the progression of IDD. In this study, NP cells were used to determine the appropriate concentration of DHE in vitro. The role of DHE in tumor necrosis factor-α (TNF-α)–induced activation of inflammatory signaling pathways and cellular senescence, together with anabolism and catabolism of extracellular matrix (ECM) in NP cells, was examined in vitro. The therapeutic effect of DHE in vivo was determined using a spinal instability model of IDD in mice. The TNF-α–induced ECM degradation and the senescence of NP cells were partially attenuated by DHE. Mechanistically, DHE inhibited the activation of NF-κB and MAPK inflammatory signaling pathways and ameliorated the senescence of NP cells caused by the activation of STING-TBK1/NF-κB signaling induced by TNF-α. Furthermore, a spinal instability model in mice demonstrated that DHE treatment could ameliorate progression of IDD. Together, our findings indicate that DHE can alleviate IDD changes and has a potential therapeutic function for the treatment of IDD.

A20 regulates inflammation through autophagy mediated by NF-κB pathway in human nucleus pulposus cells and ameliorates disc degeneration in vivo

Intervertebral disc degeneration (IDD) is closely related to loss of the extracellular matrix (ECM), apoptosis and inflammation in nucleus pulposus cells (NPCs). It has been reported that Zinc finger protein A20/TNFAIP3 (A20) can inhibit the activity of the NF-κB pathway and promote autophagy. Therefore, we speculated that A20 can regulate inflammation and ameliorate IDD through autophagy mediated by NF-κB in human NPCs. Our results indicated that the expression of A20 and inflammatory factors in IDD tissues was increased. A20 is an essential negative regulator in the NF-κB pathway. Constructed adenoviral shRNA and overexpression vectors for A20 could effectively regulate the inflammation, autophagy, and activity of NF-κB, which in turn affected the progression of IDD. Inhibition of NF-κB on the basis of knocking down A20 results in increased autophagy, suggesting that A20-regulated autophagy was mediated by NF-κB. In vivo, A20 overexpression could ameliorate the progression of IDD and promote autophagy at the same time, while deletion of A20 leads to low levels of autophagy and severe degeneration. In summary, A20 plays an important role in inhibiting inflammation through autophagy mediated by NF-κB in NPCs and ameliorating IDD.

Long non-coding RNA MT1DP interacts with miR-365 and induces apoptosis of nucleus pulposus cells by repressing NRF-2-induced anti-oxidation in lumbar disc herniation.

BackgroundThis study investigated the effects of the long non-coding (lnc) RNA MT1DP on the apoptosis of nucleus pulposus (NP) cells. The interactions between MT1DP and the microRNA miR-365, and its effects on the anti-oxidant activity of nuclear factor erythroid 2-related factor 2 (NRF-2) were investigated in lumbar disc herniation (LDH).MethodsHuman degenerative intervertebral disc NP tissues were obtained from 10 patients with LDH who underwent lumbar spine surgery. Normal intervertebral disc NP tissues were obtained from 10 patients with lumbar vertebrae fractures and used as negative controls (NCs).ResultsThe gene expressions of MT1DP and miR-365 in human degenerative disc NP tissues and nucleus pulposus cells (NPCs) were significantly increased, while the level of NRF-2 was significantly decreased. Overexpression of MT1DP and miR-365 (MT1DP + miR-365) and inhibition of NRF-2 suppressed NP cell viability and induced apoptosis. MT1DP + miR-365 caused inflammation in NP cells by damaging the mitochondrial membrane. The combination of lnc-MT1DP and miR-365 reduced cell mitochondrial function and led to a decrease in the ability of cells to elimination reactive oxygen species (ROS).ConclusionsThe combination of lnc-MT1DP and miR-365 damaged the cell mitochondrial membrane, reduced mitochondrial function and the ability to eliminate ROS, increased cell apoptosis, and caused LDH.

Lumican silencing alleviates tumor necrosis factor-α-induced nucleus pulposus cell inflammation and senescence by inhibiting apoptosis signal regulating kinase 1/p38 signaling pathway via inactivating Fas ligand expression

ABSTRACT A recent study has reported that lumican (LUM) is expressed at a high level in the nucleus pulposus specimens from herniated lumbar disc, without description of the specific mechanism. This study was designed to investigate the function and mechanism of LUM in intervertebral disc degeneration (IDD). In this study, human nucleus pulposus cells (hNPCs) cells were challenged with tumor necrosis factor (TNF)-α to establish the IDD in vitro model. After LUM silencing, cell viability was detected using CCK-8 kit, and the expression of inflammatory factors was evaluated using RT-qPCR and ELISA. Flow cytometry and β-galactosidase staining were used to determine cell cycle and cell senescence. The expression of cycle and senescence-related proteins was evaluated with western blotting. Then, Fas ligand (FasL) was overexpressed and proteins in apoptosis signal regulating kinase 1 (ASK1)/p38 signaling were tested. Finally, GS-4997, an inhibitor of ASK1, was used to explore the regulatory effects of LUM on ASK1/p38 signaling in TNF-α-induced hNPCs. Results indicated that LUM expression was upregulated in TNF-α-challenged hNPCs. LUM gene interference mitigated TNF-α-induced inflammatory response, cell cycle arrest, and senescence of hNPCs. It was then found that LUM silencing could inhibit ASK1/p38 signaling in TNF-α-treated hNPCs, which was reversed by FasL overexpression. Additionally, ASK1/p38 participated in the mediation by LUM of TNF-α-induced inflammation, cell cycle arrest, and senescence of hNPCs. To conclude, interference with LUM effectively mitigated TNF-α-induced inflammatory response, cell cycle arrest, and cell senescence. Further experiments showed the involvement of ASK1/p38 pathway in LUM-mediated NP cell phenotypes through FasL.

Fibronectin Fragments and Inflammation During Canine Intervertebral Disc Disease.

Background: Canine intervertebral disc disease (IVDD) represents a significant clinical problem in veterinary medicine, with similarities to the human pathology. Host-derived damage-associated molecular patterns like fibronectin fragments (FnF) that develop during tissue dysfunction may be of specific relevance to IVD pathologies by inducing an inflammatory response in resident cells.Aim: This project aimed to determine the presence and pathobiological role of FnF during IVD herniation in dogs, with a focus on inflammation.Methods: Herniated nucleus pulposus (NP) material from five dogs as well as non-herniated adjacent NP material from three dogs was collected during spinal surgery required due to acute IVD herniation. The presence of different types of FnF were determined by Western blot analysis. NP cells isolated from six herniated canine IVDs were then exposed to 30 kDa FnF. NP cell inflammation and catabolism was examined by investigating the expression of IL-1β, IL-6, IL-8, and COX-2, as well as MMP-1 and MMP-3 by qPCR (all targets) and ELISA (IL-6, PGE2).Results: Amongst multiple sized FnF (30, 35, 45, and >170kDa), N-terminal fragments at a size of ~30 kDa were most consistently expressed in all five herniated IVDs. Importantly, these fragments were exclusively present in herniated, but not in non-herniated IVDs. Exposure of canine NP cells to 500 nM 30 kDa FnF caused a significant upregulation of IL-6 (62.5 ± 79.9, p = 0.032) and IL-8 (53.0 ± 75.7, p = 0.031) on the gene level, whereas IL-6 protein analysis was inconclusive. Donor-donor variation was observed in response to FnF treatment, whereby this phenomenon was most evident for COX-2, with three donors demonstrating a significant downregulation (0.67 ± 0.03, p = 0.003) and three donors showing upregulation (6.9 ± 5.5, p = 0.21). Co-treatment with Sparstolonin B, a TRL-2/TRL-4 antagonist, showed no statistical difference to FnF treatment alone in all tested target genes.Conclusion: Given the presence of the 30 kDa FnF in canine herniated IVDs and the proinflammatory effect of 30 kDa FnF on NP cells, we concluded that the accumulation of FnF may be involved in the pathogenesis of canine IVDD. These results correspond to the findings in humans with IVDD.

Dexmedetomidine suppresses oxidative stress and inflammation of nucleus pulposus cells by activating the PI3K/Akt signaling pathway.

Objective: Intervertebral disc degeneration (IVDD) is very common in the elderly, so it is particularly important to find appropriate prevention or treatment. The aim of this study was to explore the effect of dexmedetomidine (DEX) on the degeneration of nucleus pulposus (NP) cells and its mechanism. Methods: We established a mouse model of IVDD and cultured mouse NP cells and treated them with IL-1β and DEX. The effect of DEX on NP cells was determined by detecting the extracellular matrix of NP cells, changes in ROS levels and inflammatory mediators. LY294002, a PI3K inhibitor, is used to inhibit the activity of the PI3K/Akt signaling pathway. The effect of DEX on the PI3K/Akt signaling pathway was determined by studying the effects of DEX on PI3K/ Akt signaling pathway-related molecules and the effect of LY294002 on NP cells degeneration. DEX significantly increased the disc height index and attenuated IVDD in mice. Results: DEX significantly inhibited the expression of MMP3/9 in NP cells, effectively inhibiting the degradation of extracellular matrix. In addition, oxidative stress levels and inflammatory levels in NP cells are also attenuated by DEX. The expression of PI3K, Akt and p-Akt was significantly increased in DEX-stimulated NP cells, indicating that DEX increased the activity of the PI3K/Akt signaling pathway. DEX promotes PI3K/Akt signaling pathway, inhibits oxidative stress and inflammatory of NP cells, thereby slowing the degeneration of NP cells. Conclusion: DEX promotes PI3K/Akt signaling pathway, inhibits oxidative stress and inflammatory of NP cells, thereby slowing the degeneration of NP cells.

Piezo1 activates the NLRP3 inflammasome in nucleus pulposus cell-mediated by Ca2+/NF-κB pathway

Studying and understanding the mechanism of inflammation in nucleus pulposus is the key to understand and prevent intervertebral disc degeneration. We propose a model of mechanical sensitive ion channel Piezo1 mediated inflammation of nucleus pulposus cells. Piezo1 can up-regulate the level of interleukin-1β (IL-1β) in nucleus pulposus cells once it is activated. It is suggested that Piezo1 may mediate inflammation by activating Nod-like receptor protein 3 (NLRP3) inflammasome to accelerate the production and maturation of IL-1β. The primary objective of this study was to explore whether Piezo1 activates NLRP3 inflammasome in nucleus pulposus cells. Piezo1 sensitization was induced by mechanical stretch following which we analyzed the priming and assembly of NLRP3 inflammasome and also studied if the downstream Ca2+/NF-κB pathway mediated this activation in nucleus pulposus cells. The expression of Piezo1 and NLRP3 inflammasome increased in a time-dependent manner upon mechanical stretch. Piezo1 activation promoted NLRP3 inflammasome assembly, which was demonstrated by the upregulation of caspase-1 activation and IL-1β production. Transfection of Piezo1-siRNA reversed this process. The inhibition of Ca2+/NF-κB pathway reduced Piezo1-dependent activation of NLRP3 inflammasome. Thus, we propose that activation of NLRP3 inflammasome in nucleus pulposus cells mediated by Piezo1 through the Ca2+/NF-κB pathway is a novel pathogenesis for the progress of intervertebral disc degeneration. As per our knowledge this is the first study which has provided evidence linking Piezo1-mediated inflammation in nucleus pulposus cells with the production of NLRP3 inflammasome.

The crosstalk between IGF-1R and ER-α in the proliferation and anti-inflammation of nucleus pulposus cells.

Insulin-like growth factors-1 receptor (IGF-1R) and estrogen receptor (ER) are reported to co-express and engage in crosstalk involving in the synergistic effect of various aspects. It is unknown whether this crosstalk exists in the nucleus pulposus (NP) cells. We aimed to investigate the interaction between IGF-1R and ER-α in regulating NP cell proliferation and inflammation response under IGF-1 stimulation. We analyzed the IGF-1, IGF-1R, and ER-α in different degenerated degree human NP tissues. NP cells were cultured with IGF-1 protein with or without the inhibitor of IGF-1R or ER-α to investigate their effects on the proliferation and inflammation response. In addition, we also upregulated the IFG-1R and ER-α expression by plasmid transfection to investigate the impact on each other. The content of IGF-1, IFG-1R, and ER-α was analyzed by enzyme-linked immunosorbent assay (ELISA). The proliferative cell rate was determined by flow cytometry. Additionally, intracellular collagen-II, p16, PCNA, IL-1β, IL-6, TNF-α, and MMP-13 expression were also detected. We found IGF-1, IFG-1R, and ER-α content were decreased in higher degenerated NP tissues. IGF-1 protein treatment upregulated the IFG-1R and ER-α expression and promoted NP cell proliferation, collagen-II, and PCNA expression. However, the suppression of IGF-1R (or ER-α) weakened the IGF-1 induced collagen-II expression, proliferation, and anti-inflammation effects on NP cells, decreased ER-α (or IGF-1R) expression, and partly reversed the protective effect of NP cells caused by IGF-1 Similarly, the upregulation of one of IGF-1R and ER-α may increase the other as well. There is an interaction between IGF-1R and ER-α acts synergistically to promote the proliferation and suppress inflammation in NP cells.

MicroRNA-149 Suppresses Inflammation in Nucleus Pulposus Cells of Intervertebral Discs by Regulating MyD88.

BackgroundIntervertebral disc degeneration (IDD) is associated with low back and neck pain, but the mechanisms underlying its pathogenesis are unclear. In this study, we explored the function of microRNA-149 (miR-149) in inflammatory response mediated by lipopolysaccharide (LPS) in nucleus pulposus (NP) cells.Material/MethodsQuantitative real-time PCR was used to detect miRNA and mRNA levels, while Western blotting was utilized to determine protein levels. ELISA was used to examine chemokine production. The correlation between miR-149 and MyD88 was assessed by reporter assay. Apoptosis was examined by flow cytometry.ResultsmiR-149 expression was significantly decreased after LPS exposure in NP cells. Overexpression of miR-149 reversed LPS-induced inhibition in aggrecan and collagen II expression and attenuated LPS-mediated promotion in the levels of MMP3, ADAMTS4, and inflammatory cytokines. Moreover, we found that miR-149 exerted its function by targeting MyD88 in NP cells.ConclusionsmiR-149 can inhibit the inflammatory response mediated by LPS in NP cells, and might be a potential target for the treatment of IDD.

Higenamine inhibits IL-1β-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)-1β-induced inflammation in human nucleus pulposus cells (NPCs). The results showed that higenamine improved cell viability in IL-1β-induced NPCs. The IL-1β-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-1β-induced activation of NF-κB signaling pathway in NPCs. In conclusion, the present study proved that higenamine exhibited anti-inflammatory activity against IL-1β-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-1β-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-1β (IL-1β). The results proved that isofraxidin attenuated the IL-1β-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-1β on matrix metalloproteinases (MMP)-3 and MMP-13. Moreover, the NF-κB activation caused by IL-1β was significantly inhibited by isofraxidin treatment. These findings suggested that isofraxidin alleviates IL-1β-induced inflammation in NPCs. Our work provided an idea that isofraxidin might act as a novel preventive role in IVD.