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Nucleus pulposus (NP) cell apoptosis is a significant indication of accelerated intervertebral disc degeneration; however, the precise mechanism is unelucidated as of yet. Ephrin B2 (EFNB2), the only gene down-regulated in the three degraded intervertebral disc tissue microarray groups (GSE70362, GSE147383 and GSE56081), was screened for examination in this study. Subsequently, EFNB2 was verified to be down-regulated in degraded NP tissue samples. Interleukin-1 (IL-1β) treatment of NP cells to simulate the IDD environment indicated that IL-1β treatment decreased EFNB2 expression. In degenerative NP cells stimulated by IL-1β, EFNB2 knockdown significantly increased the rate of apoptosis as well as the apoptosis-related molecules cleaved-caspase-3 and the Bax to Bcl-2 ratio. EFNB2 was found to promote AKT, PI3K, and mTOR phosphorylation; the PI3K/AKT signaling role was investigated using the PI3K inhibitor LY294002. EFNB2 overexpression significantly increased PI3K/AKT pathway activity in IL-1β-stimulated NP cells than the normal control. Moreover, EFNB2 partially alleviated NP cell apoptosis induced by IL-1β, reduced the cleaved-cas3 level, and decreased the Bax/Bcl-2 ratio after the addition of the inhibitor LY294002. Additionally, EFNB2 overexpression inhibited the ERK1/2 phosphorylation; the effects of EFNB2 overexpression on ERK1/2 phosphorylation, degenerative NP cell viability, and cell apoptosis were partially reversed by ERK signaling activator Ceramide C6. EFNB2 comprehensively inhibited the apoptosis of NP cells by activating the PI3K/AKT signaling and inhibiting the ERK signaling, obviating the exacerbation of IDD. EFNB2 could be a potential target to protect against degenerative disc changes., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Nucleus pulposus cells (NPCs) apoptosis and inflammation are the extremely critical factors of intervertebral disc degeneration (IVDD). Nevertheless, the underlying procedure remains mysterious. Macrophage migration inhibitory factor (MIF) is a cytokine that promotes inflammation and has been demonstrated to have a significant impact on apoptosis and inflammation. For this research, we employed a model of NPCs degeneration stimulated by lipopolysaccharides (LPS) and a rat acupuncture IVDD model to examine the role of MIF in vitro and in vivo, respectively. Initially, we verified that there was a significant rise of MIF expression in the NP tissues of individuals with IVDD, as well as in rat models of IVDD. Furthermore, this augmented expression of MIF was similarly evident in degenerated NPCs. Afterwards, it was discovered that ISO-1, a MIF inhibitor, effectively decreased the quantity of cells undergoing apoptosis and inhibited the release of inflammatory molecules (TNF-α, IL-1β, IL-6). Furthermore, it has been shown that the PI3K/Akt pathway plays a vital part in the regulation of NPCs degeneration by MIF. Ultimately, we showcased that the IVDD process was impacted by the MIF inhibitor in the rat model. In summary, our experimental results substantiate the significant involvement of MIF in the degeneration of NPCs, and inhibiting MIF activity can effectively mitigate IVDD., Competing Interests: Declaration of competing interest All authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier Inc.)

Nucleus pulposus cell (NPC) senescence is a major cause of intervertebral disc degeneration (IVDD). Oxidative stress and reactive oxygen species (ROS) play critical roles in regulating cell senescence. Selenophosphate synthetase 1 (SEPHS1) was reported to play an important role in mitigating oxidative stress in an osteoarthritis (OA) model by reducing the production of ROS, thereby, delaying the occurrence and development of osteoarthritis. In this study, we explored the, hitherto unknown, role of SEPHS1 in IVDD in vitro and in vivo using an interleukin-1β (IL-1β)-induced NPC senescence model and a rat needle puncture IVDD model, respectively. SEPHS1 delayed NPC senescence in vitro by reducing ROS production. Age-related dysfunction was also ameliorated by the overexpression of SEPHS1 and inhibition of the Hippo-Yap/Taz signaling pathway. In vivo experiments revealed that the overexpression of SEPHS1 and inhibition of Hippo-Yap/Taz alleviated IVDD in rats. Moreover, a selenium (Se)-deficient diet and lack of SEPHS1 synergistically aggravated IVDD progression. Taken together, our results demonstrate that SEPHS1 plays a significant role in NPC senescence. Overexpression of SEPHS1 and inhibition of Hippo-Yap/Taz can delay NPC senescence, restore the balance of extracellular matrix metabolism, and attenuate IVDD. SEPHS1 could be a promising therapeutic target for IVDD. NEW & NOTEWORTHY Selenophosphate synthetase 1 (SEPHS1) deficiency leads to an increase in reactive oxygen species levels and in the subsequent activation of the Hippo-Yap/Taz signaling pathway. In the rat model of intervertebral disc degeneration (IVDD), overexpression of SEPHS1 and inhibition of Hippo-YAP/Taz mitigated the progression of disc degeneration indicating the involvement of SEPHS1 in IVDD. SEPHS1 is a promising therapeutic target for IVDD.

The accumulation of metabolites in the intervertebral disc is considered an important cause of intervertebral disc degeneration (IVDD). Lactic acid, which is a metabolite that is produced by cellular anaerobic glycolysis, has been proven to be closely associated with IVDD. However, little is known about the role of lactic acid in nucleus pulposus cells (NPCs) senescence and oxidative stress. The aim of this study was to investigate the effect of lactic acid on NPCs senescence and oxidative stress as well as the underlying mechanism. A puncture-induced disc degeneration (PIDD) model was established in rats. Metabolomics analysis revealed that lactic acid levels were significantly increased in degenerated intervertebral discs. Elimination of excessive lactic acid using a lactate oxidase (LOx)-overexpressing lentivirus alleviated the progression of IVDD. In vitro experiments showed that high concentrations of lactic acid could induce senescence and oxidative stress in NPCs. High-throughput RNA sequencing results and bioinformatic analysis demonstrated that the induction of NPCs senescence and oxidative stress by lactic acid may be related to the PI3K/Akt signaling pathway. Further study verified that high concentrations of lactic acid could induce NPCs senescence and oxidative stress by interacting with Akt and regulating its downstream Akt/p21/p27/cyclin D1 and Akt/Nrf2/HO-1 pathways. Utilizing molecular docking, site-directed mutation and microscale thermophoresis assays, we found that lactic acid could regulate Akt kinase activity by binding to the Lys39 and Leu52 residues in the PH domain of Akt. These results highlight the involvement of lactic acid in NPCs senescence and oxidative stress, and lactic acid may become a novel potential therapeutic target for the treatment of IVDD., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Objective: This study aimed to explore the specific function of M2 macrophages in intervertebral disc degeneration (IDD)., Methods: Intervertebral disc (IVD) samples from normal (n = 4) and IDD (n = 6) patients were collected, and the expression of M2-polarized macrophage marker, CD206, was investigated using immunohistochemical staining. Nucleus pulposus cells (NPCs) in a TNF-α environment were obtained, and a mouse caudal IVD puncture model was established. Mice with Rheb deletions, specifically in the myeloid lineage, were generated and subjected to surgery-induced IDD. IDD-induced damage and cell apoptosis were measured using histological scoring, X-ray imaging, immunohistochemical staining, and TdT-mediated dUTP nick end labeling (TUNEL) assay. Finally, mice and NPCs were treated with R-spondin-2 (Rspo2) or anti-Rspo2 to investigate the role of Rspo2 in IDD., Results: Accumulation of CD206 in human and mouse IDD tissues was detected. Rheb deletion in the myeloid lineage (RheBcKO) increased the number of CD206+ M2-like macrophages (mean difference 18.6% [15.7-21.6%], P < 0.001), decreased cell apoptosis (mean difference -15.6% [-8.9 to 22.2%], P = 0.001) and attenuated the IDD process in the mouse IDD model. NPCs treated with Rspo2 displayed increased extracellular matrix catabolism and apoptosis; co-culture with a conditioned medium derived from RheBcKO mice inhibited these changes. Anti-Rspo2 treatment in the mouse caudal IVD puncture model exerted protective effects against IDD., Conclusions: Promoting CD206+ M2-like macrophages could reduce Rspo2 secretion, thereby alleviating experimental IDD. Rheb deletion may help M2-polarized macrophages accumulate and attenuate experimental IDD partially by inhibiting Rspo2 production. Hence, M2-polarized macrophages and Rspo2 may serve as therapeutic targets for IDD., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2023 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Pyroptosis, a newly discovered pro-inflammatory programmed necrosis of cells, serves as an initiating and promoting event that leads to intervertebral disc (IVD) degeneration (IDD). Endoplasmic reticulum stress (ERS) and autophagy are vital regulatory mechanisms of cellular homeostasis, which is also closely related to IDD. However, the role and relationship of ERS and autophagy in the pyroptosis of nucleus pulposus cell (NPC) are not well understood. In this research, we aimed to elucidate the role and mechanism of ERS-C/EBP homologous protein (CHOP) in lipopolysaccharide (LPS)-induced cell pyroptosis and determine its interaction with autophagy. ERS and autophagy inducers or inhibitors were used or not in the preconditioning of rat NPCs. Cell viability, pyroptosis-related protein expression, caspase-1 activity assay, and enzyme-linked immunosorbent assay were performed to observe rat NPC pyroptosis after the treatment of LPS. Activation of the ERS pathway and autophagy were assessed by quantitative real-time PCR, western blot analyses, and immunofluorescence staining assay to classify the molecular mechanisms. Our results showed that LPS stimulation induced NPC pyroptosis with concomitant activation of the ERS-CHOP pathway and initiated autophagy. Activation of the ERS-CHOP pathway exacerbated rat NPC pyroptosis, whereas autophagy inhibited cell pyroptosis. LPS-induced cell pyroptosis and CHOP upregulation were negatively regulated by autophagy. LPS-induced autophagy was depressed by the ERS inhibitor but aggravated by the ERS inducer. Taken together, our findings suggested that LPS induced NPC pyroptosis by activating ERS-CHOP signaling and ERS mediated LPS-induced autophagy, which in turn alleviated NPC pyroptosis by inhibiting CHOP signaling., (© 2023 Wiley Periodicals LLC.)

Background: Low back pain (LBP) has become the second leading cause of disability worldwide, which has brought great economic burden to people. It is generally believed that intervertebral disc degeneration (IDD) is the main cause of LBP. This study aimed to explore the role of circ-STC2 in the pathogenesis of IDD., Methods: Nucleus pulposus cells (NPCs) were treated with T-Butyl Hydrogen Peroxide (TBHP) to establish IDD model in vitro. RT-qPCR was performed to detect mRNA expressions. The cell viability was detected with CCK-8 assay. The levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), Fe 2+ and glutathione (GSH) of NPCs were measured by corresponding kits. The protein expressions were determined by western blot. Dual-luciferase reporter and RNA pull-down assays were conducted to verify the relationship between circ-STC2 or transferrin recepto 2 (TFR2) and miR-486-3p., Results: Circ-STC2 and TFR2 expressions were up-regulated in IDD tissues, and miR-486-3p expression was down-regulated. Knockdown of circ-STC2 promoted the cell viability and inhibited the ferroptosis of the NPCs. The GSH levels, and glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) protein expressions were increased, the LDH, MDA and Fe 2+ levels and achaete-scute complexlike 4 (ASCL4) protein expressions were decreased after circ-STC2 knockdown. Knockdown of miR-486-3p abrogated the si-circ-STC2 effects and overexpression of TFR2 reversed the miR-486-3p mimic effects., Conclusions: Circ-STC2 inhibits the cell viability, induced the ferroptosis of the TBHP treated NPCs via targeting miR-486-3p/TFR2 axis., (© 2023. The Author(s).)

Background: Accumulating evidence indicates that intervertebral disc degeneration (IDD) is associated with diabetes mellitus (DM), while the underlying mechanisms still remain elusive. Herein, the current study sought to explore the potential molecular mechanism of IDD in diabetic rats based on transcriptome sequencing data., Methods: Streptozotocin (STZ)-induced diabetes mellitus type 1 (T1DM) rats were used to obtain the nucleus pulposus tissues for transcriptome sequencing. Next, differentially expressed genes (DEGs) in transcriptome sequencing data and GSE34000 microarray dataset were obtained and intersected to acquire the candidate genes. Moreover, GO and KEGG enrichment analyses were performed to analyze the cellular functions and molecular signaling pathways primarily regulated by candidate DEGs., Results: A total of 35 key genes involved in IDD of T1DM rats were mainly enriched in the extracellular matrix (ECM) and cytokine adhesion binding-related pathways. NLRP3 inflammasome activation promoted the pyroptosis of nucleus pulposus cells (NPCs). Besides, BMP7 could affect the IDD of T1DM rats by regulating the inflammatory responses. Additionally, NPCs were isolated from STZ-induced T1DM rats to illustrate the effects of BMP7 on IDD of T1DM rats using the ectopic expression method. Both in vitro and in vivo experiments validated that BMP7 alleviated IDD of T1DM rats by inhibiting NLRP3 inflammasome activation and pyroptosis of NPCs., Conclusion: Collectively, our findings provided novel mechanistic insights for understanding of the role of BMP7 in IDD of T1DM, and further highlighted BMP7 as a potential therapeutic target for preventing IDD in T1DM., (© 2023. The Author(s).)

The availability of an appropriate and reliable research model is helpful for researchers to understand the occurrence and development of diseases. Historically, animal models have been beneficial in the study of intervertebral disc degenerative diseases, but intervertebral disc degeneration (IDD) is a precise and complex process that needs to appear and occur in a specific tissue microenvironment, and animal degeneration models cannot fully simulate these parameters. These challenges must be overcome, especially when animal models cannot fully generalise the complex pathology of humans. In the past few years, the research on the cell disease model has made important progress, and the construction of the nucleus pulposus cell (NPC) degeneration model has become an indispensable step in studying the occurrence and development of IDD. Here, several different methods of constructing NPC degeneration models and indicators for testing the effect of modelling are introduced. The practical applications of cell models constructed by different methods are enumerated to screen and evaluate effective methods of establishing degenerative cell models and explore the mechanism of IDD.

Background: In recent years, intervertebral disc (IVD) degeneration (IDD) has increased in age. There is still a lack of effective treatment in clinics, which cannot improve the condition of IDD at the level of etiology., Objective: To explore IDD pathogenesis at the cellular and gene levels and investigate lactotransferrin (LTF) expression in IDD patients and its possible mechanism., Methods: We downloaded the IDD data set from the Gene Expression Omnibus (GEO) database, screened the differentially expressed genes (DEGs) and hub genes and performed Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis to construct a protein-protein interaction (PPI) network. Subsequently, we verified LTF's regulatory mechanism through cell experiments. IL-1β was used to intervene in nucleus pulposus cells (NPCs) to construct the IDD cell model, and LTF and Fas expression was detected by qRT-PCR. LTF inhibitor, Fas inhibitor, LTF mimic, and Fas mimic were used to intervene in each group. Western blotting was used to detect Fas, Caspase-3, Bax, and Bcl-2 expression., Results: A total of 131 DEGs and 10 hub genes were screened. LTF mRNA in the IDD model was significantly higher than that in the control group, while Fas' mRNA was significantly lower. When LTF was upregulated or downregulated in NPCs, apoptosis marker expression showed the opposite trend. The rescue test showed that LTF and Fas' overexpression greatly enhanced NPC apoptosis., Conclusion: LTF promotes IDD progression by regulating Fas in NPCs, and it may be an effective gene therapy target.

The study aimed to explore the effects of normal CESC-derived exosomes (N-CESC-exo) on autophagy, apoptosis and extracellular matrix (ECM) metabolism of nucleus pulposus cells (NPCs) and their underlying molecular mechanisms in vivo and in vitro. Tert-buty l hydroperoxide (TBHP) was used to induce CESCs and NPCs degeneration models in vitro. Flow cytometry and TUNEL staining were used to assess apoptosis. Proteins expression were detected by Western blotting. qRT-PCR was applied to detect miR-125-5p and SUV39H1 expression. The miRNA differences were analyzed by bioinformatics. Dual-luciferase reporter assay was applied to detect the target relationship. The degeneration of intervertebral disc tissue was observed by hematoxylin-eosin (H&E) staining. The disc damage was assessed with Safranin-O and Fast Green staining. LC3B expression was detected by immunofluorescence. We observed that NPCs could ingest N-CESC-exo. N-CESC-exo reduced degenerated nucleus pulposus cells (TBHP-NPC) apoptosis, bax, MMP13, and p62 expression, while increased bcl2, ACAN, LC3-II/I expression, and the fluorescence intensity of GFP-LC3. Bioinformatics analysis confirmed that miR-125-5p was low expression, while SUV39H1 was overexpressed in IDD. Further, the dual-luciferase reporter assays confirmed the targeting relationship between miR-125-5p and SUV39H1. CESC-exo miR-125-5p inhibited TBHP-NPCs apoptosis by inhibiting SUV39H1, bax, MMP13, and p62 expression, while increased bcl2, ACAN, LC3-II/I expression, and the fluorescence intensity of GFP-LC3, thereby alleviating rat IDD., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Intervertebral disc (IVD) diseases are common spinal disorders that cause neck or back pain in the presence or absence of an underlying neurological disorder. IVD diseases develop on the basis of degeneration, and there are no established treatments for degeneration. IVD diseases may therefore represent a candidate for the application of regenerative medicine, potentially employing normal human dermal fibroblasts (NHDFs) induced to differentiate into nucleus pulposus (NP) cells. Here, we used a three-dimensional culture system to demonstrate that ectopic expression of MYC , KLF4 , NOTO , SOX5 , SOX6 , and SOX9 in NHDFs generated NP-like cells, detected using Safranin-O staining. Quantitative PCR, microarray analysis, and fluorescence-activated cell sorting revealed that the induced NP cells exhibited a fully differentiated phenotype. These findings may significantly contribute to the development of effective strategies for treating IVD diseases.

Functionalized self-assembling peptides, which display functional growth-factor bioactivity, can be designed by connecting the C-terminus of a pure self-assembling peptide with a short functional motif. In this study, we designed a novel functionalized peptide (RADA16-SNVI) in which an SNVI motif with hBMP-7 activity was conjugated onto the C-terminus of the RADA16 peptide via solid-phase synthesis. A mix of RADA16-SNVI and RADA16 solutions was used to create a functionalized peptide nanofiber scaffold (SNVI-RADA16). The hydrogels were analyzed by atomic force microscopy, circular dichroism, and scanning electron microscopy. The results showed that the SNVI-RADA16 solution effectively formed hydrogel. Next, we seeded the SNVI-RADA16 scaffold with adipose-derived stem cells (ADSCs) and investigated whether it displayed biological properties of nucleus pulposus tissue. SNVI-RADA16 displayed good biocompatibility with the ADSCs and induced their expression. Cells in SNVI-RADA16 gel had a greater secretion of the extracellular matrix marker collagen type II and aggrecan compared to ADSCs grown in monolayer and control gel (p < 0.05). The ratio of the aggrecan to collagen in cells in SNVI-RADA16 gel is approximately 29:1 after culture for 21 days. ADSCs in SNVI-RADA16 gels expressed the hypoxia-inducible factor 1α(HIF-1α) mRNA by real-time PCR. However, HIF-1 mRNA is absence in control gel and monolayer. The results suggested that the functionalized self-assembled peptide promotes the differentiation of ADSCs into nucleus pulposus-like cells. Thus, the designed SNVI-RADA16 self-assembling peptide hydrogel scaffolds may be suitable for application in nucleus pulposus tissue regeneration., (© 2022. The Author(s).)

Nucleus pulposus cells (NPCs) degeneration is an essential pathological basis of intervertebral disc diseases, and autologous cell transplantation is a means of regeneration of NPCs. This study aimed to evaluate the effects of autologous facet joint chondrocytes (CHs) with Sirtuin 1 (sirt1)-overexpression on NPCs degeneration. We used human NPCs and CHs isolated from the patients' tissue and transduced CHs with the plasmid vector to overexpress the sirt1 gene. Further, NPCs were seeded as monolayers and treated with IL-1β to obtain the degeneration, and the sirt1-overexpressed CHs (sirt1-CHs) in the transwell insert were co-cultured in the same well. The NPCs' degenerated degree was determined by the levels of living cells, proliferation, p16, and collagen I/II, and aggrecan expression at the time point of 1, 3, or 5 days. Besides, the ROS accumulation, antioxidative enzymes, sirt1, and inflammatory factors gene expression were also tested. After IL-1β treatment, when co-cultured with sirt1-CHs, NPCs accumulated more living cells, proliferation, collagen II, aggrecan, but less p16 and collagen I expression than cultured without sirt1-CHs. Additionally, SOD1, CAT, and TIMP4 mRNA were protected, and the production of TNF-α, IL-6, MMP3, and ROS were alleviated with the presence of sirt1-CHs. Thus, co-culture with sirt1-CHs delays NPCs' degeneration via the suppression of ROS accumulation and inflammatory response. Transplanting autologous CHs with sirt1-overexpressed into the NP tissue might be a novel treatment for intervertebral disc degeneration., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Bergenin is a C-glucoside of 4-O-methyl gallic acid with a variety of biological activities, such as antioxidant and anti-inflammatory. Herein, we investigated the involvement of bergenin in the protective effect against H 2 O 2 -induced oxidative stress and apoptosis in human nucleus pulposus cells (HNPCs) and the underlying mechanisms. HNPCs were cotreated with various concentrations of bergenin and 200 μM H 2 O 2 for 24 h. Cell viability was detected by Cell Counting Kit-8 and lactate dehydrogenase release assays. Reactive oxygen species (ROS) was evaluated utilizing 2',7'-dichlorofluorescein-diacetate. Superoxide dismutase (SOD) and catalase (CAT) activities and glutathione (GSH) levels were measured to assess oxidative stress. Apoptosis was evaluated using terminal deoxynucleotidyl transferase dUTP nick end labeling and caspase-3/7 activity assays. Expression of protein was determined by western blotting. Results indicated that treatment with bergenin significantly alleviated H 2 O 2 -induced viability reduction and ROS overproduction in HNPCs in a dose-dependent manner. Bergenin alleviated H 2 O 2 -induced oxidative stress in HNPCs by increased activity of superoxide dismutase and level of glutathione peroxidase. H 2 O 2 -induced apoptosis and activity of caspase-3/7 were also suppressed by bergenin treatment in HNPCs. Western blotting showed that H 2 O 2 -induced decrease in expression of peroxisome proliferator-activated receptor γ (PPAR-γ) and increase in nuclear factor κB (NF-κB) were inhibited by bergenin. However, the inhibitory effect of bergenin on H 2 O 2 -induced viability reduction, oxidative stress and apoptosis were noticeably abrogated in PPAR-γ knockdown HNPCs. In conclusion, our results indicated that bergenin alleviates H 2 O 2 -induced oxidative stress and apoptosis in HNPCs by activating PPAR-γ and suppressing NF-κB pathway., (© 2021 Wiley Periodicals LLC.)

Background: Nucleus pulposus cell (NPC) degeneration is widely accepted as one of the major causes of intervertebral disc (IVD) degeneration (IVDD). The pathogenesis of IVDD is complex and consists of inflammation, oxidative stress, and the loss of extracellular matrix (ECM). Cannabinoid type 2 receptor (CB2) has been shown to be involved in the pathological mechanism of a variety of diseases due to its anti-inflammatory effects and antioxidative stress capacity., Method: In Vitro, H 2 O 2 was used to induce degeneration of nucleus pulposus cells, mRNA and protein expression level was determined by RT-PCR and Western Blot, and Immunocytochemical staining were used to detect expression of collagen II, aggrecan, MMP3/13, superoxide dismutase 2 (SOD2) and inducible nitric oxide synthase (iNOS). In vivo, the potential therapeutic effect of CB2 was detected in the rat acupuncture model., Result: In vitro, we found that the CB2 agonist (JWH133) treatment reduced the oxidative stress level in NPCs induced by hydrogen peroxide (H 2 O 2 ) treatment. Furthermore, the expression of inflammatory cytokines was also decreased by JWH133 treatment. We found that collagen II and aggrecan expression was preserved, whereas matrix metalloproteinase levels were reduced. In vivo, we established a rat model by needle puncture. Imaging assessment revealed that the disc height index (DHI) and morphology of IVD were significantly improved, and the disc degeneration process was delayed by treatment of JWH133. Furthermore, immunohistochemical (IHC) staining revealed that JWH133 could inhibit the degradation of collagen II and decrease the expression of MMP3., Conclusions: The experiment indicates the oxidative stress and inflammatory response of rat NPCs induced by H 2 O 2 could be inhibited by activating CB2. This study reveals that CB2 activation can effectively delay the development of IVDD, providing an effective therapeutic target for IVDD., (© 2021. The Author(s).)

Decorin (Dcn) is a member of the class I small leucine-rich proteoglycans, whose expression in the nucleus pulposus (NP) of intervertebral discs (IVDs) has been shown to increase with aging in humans and sheep. Dcn induces autophagy in endothelial cells; however, its precise role in NP and IVD degeneration during aging is not well understood. We addressed this question in the present study by treating rat nucleus pulposus cells (NPCs) with different concentrations of Dcn. The Western blot analysis and terminal deoxynucleotidyl transferase dUTP nick end labeling assay results showed that Dcn treatment induced autophagy and decreased apoptosis caused by interleukin (IL)-1β application. This effect was dependent on the protein kinase B/mechanistic target of rapamycin (mTOR)/p70 S6 kinase signaling. Dcn treatment also decreased the expression of matrix metalloproteinase-3 and -13 and decreased the IL-1β-induced attenuation of collagen type II and aggrecan levels. The role of Dcn in stimulating autophagy was further supported by the fact that the observed effects were abrogated by knocking down autophagy-related protein 7 with Atg7 small interfering RNA. Thus, Dcn protects NPCs in IVDs from IL-1β-induced apoptosis and degeneration by promoting autophagy through mTOR signaling., (© 2020 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

Intervertebral discs are important for maintaining mobility and offer support to the body trunk. If these discs lose their biomechanical features, lower back pain can occur. We previously reported that hepatocyte growth factor (HGF) promotes cell proliferation and suppresses apoptosis, inflammation, and matrix degradation in nucleus pulposus (NP) cells. In the present study, we investigated the molecular mechanisms of how HGF promotes the proliferation of NP cells in hypoxic conditions. Hypoxic stimulation promoted modest cell proliferation, which was further upregulated by HGF. Expression of hypoxia-inducible factor (HIF-1α) protein, which contributes to the maintenance of homeostasis in NP cells, was also upregulated in hypoxia-treated cell groups; HGF further increased HIF-1α expression in NP cells. Additionally, knockdown of HIF-1α expression significantly reduced the proliferation of NP cells. An MAPK inhibitor inhibited the expression of HIF-1α and pERK, as well as cell proliferation in a dose-dependent manner. Similarly, inhibiting the PI3K/Akt and STAT3 pathways also decreased the expression of HIF-1α and cell proliferation. These results show that under hypoxic conditions, HGF promotes NP cell proliferation via HIF-1α-, MAPK-, PI3K/Akt-, and STAT3-mediated signaling which is involved in this pathway. The control of these signaling pathways may be a target for potential therapeutic strategies for the treatment of disc degeneration in hypoxic conditions., (© 2020 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

The extracellular matrix (ECM) degradation of nucleus pulposus cells (NPCs) is mainly induced by metalloproteinases (MMPs). Zn 2+ is an essential component of MMPs, but the effect of Zn 2+ importers in controlling ECM metabolism remains unclear. The purpose of this research was to identify the involvement of Zn 2+ importers in ECM degradation induced by inflammatory stimuli and excessive mechanical stressing. In this study, NPCs from Sprague-Dawley (SD) rats were separated and cultured. FluoZin-3 AM staining was applied to detect [Zn 2+ ]i in NPCs treated with Interleukin-1β (IL-1β) or cyclic tensile strain (CTS) with a Flexcell Strain Unit. We found that intracellular Zn 2+ concentration ([Zn 2+ ]i) elevated dramatically, and ZIP8 is the predominant Zn 2+ importer among all importers in senescent NPCs. The [Zn 2+ ]i and MMP expression level both increased in IL-1β and CTS treated NPCs. Furthermore, the expression of ZIP8 was also markedly increased. However, knockdown of ZIP8 with siRNA alleviated ECM degradation induced by inflammatory stimuli and CTS. Both stimuli activated NF-κB signaling pathway, and knockdown of ZIP8 effectively inhibited NF-κB signaling pathway activation. In conclusion, knockdown of ZIP8 can alleviate NPCs' ECM degradation caused by inflammatory stimuli and excessive mechanical stressing., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The purpose of this study was to identify a specific circular RNA and to investigate its regulatory mechanism in intervertebral disc degeneration (IDD). CircGLCE was selected after microarray analyses and was further analysed by RT-qPCR and FISH. CircGLCE was found to stably exist in the cytoplasm of nucleus pulposus (NP) cells. It was downregulated in IDD. After silencing CircGLCE, its function was assessed with RT-qPCR, immunofluorescence analysis and flow cytometry. Knockdown of CircGLCE promoted apoptosis and induced the expression of matrix-degrading enzymes in NP cells. CircGLCE served as a miR-587 sponge in NP cells. Inhibiting miR-587 counteracted the IDD-enhancing effect caused by silencing CircGLCE. STAP1 served as the miRNA target that mediated the functions of miR-587. In an IDD mouse model, the in vivo effects of overexpressing CircGLCE on IDD were confirmed by imaging techniques, TUNEL staining, FISH, western blotting, H&E staining and immunohistochemistry. Thus, CircGLCE attenuates IDD by inhibiting the apoptosis of NP cells and ECM degradation through the targeting of miR-587/STAP1. CircGLCE may be a potential therapeutic target for IDD treatments.

Our present study investigated whether exosome secretion of nucleus pulposus cells (NPCs) is regulated by autophagy. Different autophagic states of NPCs were induced by rapamycin (Rap), bafilomycin A1 (Baf) and other agents, and it was found that exosomes were secreted in an autophagy-dependent manner. Activation or inhibition of autophagy increased or decreased, respectively, the amount of exosomes that were released into the extracellular space. In addition, in order to confirm that Rap-promoted release of exosomes was mediated by autophagy rather than other pathways, we used autophagy associated gene 5 (ATG5) small-interfering RNA (siRNA) to silence the expression of ATG5 gene, which is indispensable for autophagy. The results showed that siRNA against ATG5 (siATG5) induced an accumulation of intraluminal vesicles (ILVs) in NPCs and a concomitant decrease in the amount of exosomes isolated from supernatant. Ras homolog gene (Rho) and Rho-associated coiled-coil forming protein kinase (ROCK) family molecules are capable of cytoskeletal remodeling and affecting vesicle transport. Therefore, we carried out targeted interventions and evaluated the effects of the RhoC/ROCK2 pathway on the secretion of exosomes within autophagic environment. Knockdown of RhoC and ROCK2 with corresponding siRNA significantly inhibited the secretion of exosomes originating from ILVs in NPCs, even when NPCs were subsequently treated with Rap. Taken together, our findings suggest that autophagy positively regulates expression levels of RhoC and ROCK2, and that the RhoC/ROCK2 pathway exerts a key function on NPCs-derived exosome secretion., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Intervertebral disc degeneration (IDD) is a major contributor to lower back pain, however, the molecular and pathogenetic mechanisms underlying IDD are poorly understood. As a high-risk factor for IDD, compression stress was reported to induce apoptosis of nucleus pulposus (NP) cells and extracellular matrix (ECM) degradation during IDD progression. Circular RNA (circRNA) is a class of endogenous non-coding RNA (ncRNA) and has been reported to function in several diseases. However, whether and how circRNA regulates compression-induced damage of NP cells remains vague. Here, we aimed to investigate the key role of circRNA in compression loading-induced IDD., Methods: We analysed the circRNA expression of three samples from compression-treated NP cells and three control samples using circRNA microarray assays and further investigated the circRNA involved in compression-induced damage of NP cells (circRNA-CIDN). We investigated the effects of circRNA-CIDN on compression-induced cell apoptosis and NP ECM degradation in vitro and ex vivo. We observed that circRNA-CIDN bound to miRNAs as a miRNA sponge based on luciferase and RNA immunoprecipitation (RIP) assays., Findings: CircRNA-CIDN was significantly downregulated in compression-treated human NP cells, as validated by circRNA microarray and qRT-PCR analysis, and overexpressing circRNA-CIDN inhibited compression-induced apoptosis and NP ECM degradation. Further studies demonstrated that circRNA-CIDN served as a sponge for miR-34a-5p, an important miRNA that enhanced compression-induced damage of NP cells via repressing the silent mating type information regulation 2 homolog 1 (SIRT1). CircRNA-CIDN was also verified to contain IDD development in an ex vivo IDD model., Interpretation: Our results revealed that circRNA-CIDN binding to miR-34a-5p played an important role in mitigating compression loading-induced nucleus pulposus cell damage via targeting SIRT1, providing a potential therapeutic strategy for IDD treatment., Funding: National Natural Science Foundation of China (81772391, 81974348), Fundamental Research Funds for the Central Universities (2017KFYXJJ248)., Competing Interests: Declaration of Competing Interest These authors have no conflict of interest to declare., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Intervertebral disc degeneration (IVDD) is a well-known cause of lower back pain, which is induced by multiple factors including increased apoptosis and decreased survival of nucleus pulposus cells. In this study, we evaluate the effect and potential mechanism of miR-660 on the nucleus pulposus cells apoptosis induced by TNF-α., Methods: First, we collected tissue of nucleus pulposus from IVDD and healthy controls. General characteristic of the IVDD and healthy control was also collected. And, we also collected nucleus pulposus cells that stimulated by TNF-α or control. miRNA microarray was performed to identify the differentially expressed miRNAs. Apoptosis rate and miR-660 relative expression was measured after stimulated with different concentration of TNF-α to identify the optimal concentration of TNF-α. Second, we successfully constructed antigomiR-660 to block the miR-660 expression in nucleus pulposus cells and then stimulated with TNF-α (100 ng/ml, 12 h). The apoptosis rates and relative protein expression were then measured again. The target association between miR-660 and SAA1 was confirmed by dual-luciferase reporter., Results: There was no significant difference between the age (IVDD: 39 ± 10 years, healthy controls: 36 ± 7 years), BMI and sex between IVDD and healthy controls. Microarray analysis found that miR-660 was significantly up-regulated in IVDD and TNF-α treated groups, which was further identified by PCR. We found that the rate of apoptosis and miR-660 expression increased with TNF-α concentration increased. Finally, TNF-a with 100 ng/ml was used for further experiment. Compared with TNF-α group, TNF-α + antigomiR-660 could significantly down-regulated the apoptosis rate and relative protein (c-Caspase3 and c-Caspase7). Dual-luciferase reporter revealed that miR-660 could directly binding to the SAA1 at 80-87 sites. Compared with TNF-α alone group, TNF-α + antigomiR-660 significantly up-regulated the SAA1 expression (P < 0.05)., Conclusion: These results indicated that knockdown of miR-660 protected the nucleus pulposus from apoptosis that induced TNF-α via up-regulation of SAA1. Further studies should focus on the role of miR-660 in protecting IVDD in vivo.

Intervertebral disc (IVD) degeneration and consequent lower back pain is a common disease. Micro fragmented adipose tissue (MFAT) is promising for a wide range of applications in regenerative medicine. In this study, MFAT was isolated by a nonenzymatic method and co-cultured with nucleus pulposus cells (NPCs) using an indirect co-culture system in vitro . A pig disc degeneration model was used to investigate the regenerative effect of MFAT on degenerated IVDs in vivo . The mRNA expression of Sox9 , Acan , and Col2 in NPCs was significantly increased, while no significant increase was observed in the mRNA expression of proinflammatory cytokine genes after the NPCs were co-cultured with MFAT. Nucleus pulposus (NP)-specific markers were increased in MFAT cells after co-culture with NPCs. After injection of MFAT, the disc height, water content, extracellular matrix, and structure of the degenerated NP were significantly improved. MFAT promoted the matrix synthesis function of NPCs, and NPCs stimulated the NP-like differentiation of MFAT cells. In addition, MFAT also partly regenerated degenerated IVDs in the pig model.

Intervertebral disk degeneration (IDD) has been widely considered as one of the main causes for low back pain, which can cause a severe impact to human health and huge economic burden to worldwide society. IDD pathogenesis can be affected by extensive degradation of extracellular matrix (ECM) and the hyperproliferation of nucleus pulposus (NP) cells. During the IDD process, expression of the ECM degradation enzymes matrix metalloproteinase and ADAMTS increases, whereas expression of ECM synthesis-related aggrecan and COL2A1 decreases. In addition, the Wnt signaling pathway is reportedly involved in the process of IDD. Bu-Shen-Huo-Xue-Fang (BSHXF), a Chinese traditional medicine formula that contains six Chinese traditional medicinal herbs, is widely used in the treatment of IDD. Herein, we obtained the serum containing BSHXF from BSHXF-fed rat and demonstrated that the BSHXF promoted NP cell proliferation and ECM synthesis through the Wnt signaling pathway. By using DIANA online tools and luciferase reporter gene assays, we confirmed that miR-483-3p and miR-23c regulated CTNNB1 and GSK3B, respectively, through direct targeting, thereby affecting the effect of BSHXF on NP cell proliferation and ECM synthesis through the Wnt signaling pathway. Taken together, we demonstrated the function and mechanism of BSHXF in regulating NP cell proliferation and ECM remodeling through the Wnt signaling pathway during IDD., (© 2018 Wiley Periodicals, Inc.)

miR-129-5p is implicated in many diseases, such as laryngeal cancer and breast cancer. In this study, we studied the mechanism underlying the role of BMP2 in intervertebral disc degeneration (IDD). We used a luciferase assay system to determine the relationship between BMP2 and miR-129-5 expression. In addition, Western blot and real-time PCR were used to confirm the regulatory relationship between miR-129-5p and its targets, while flow cytometry was used to evaluate the effect of miR-129-5p on the apoptosis of neural progenitor cells (NPCs). BMP2 was confirmed as a direct target of miR-129-5p. Furthermore, the expression of miR-129 was downregulated along with upregulated BMP2 expression in IDD patients. Meanwhile, BMP2 was validated as the target of miR-129-5p in cells transfected with miR-129-5p and BMP2 siRNA. Also, compared with NPCs transfected with blank/scramble controls or miR-129-5p inhibitors, the NPCs treated with miR-129-5p mimics or BMP2 siRNA exhibited evidently elevated viability and inhibited apoptosis. The data demonstrated that miR-129-5p was poorly expressed in IDD patients, and the dysregulation of miR-129-5p might contribute to the development of IDD by targeting BMP2 expression., (© 2019 Wiley Periodicals, Inc.)

Objective: Intradiscal biologic therapy is a promising strategy for managing intervertebral disc degeneration. However, these therapies require a rich nutrient supply, which may be limited by the transport properties of the cartilage endplate (CEP). This study investigated how fluctuations in CEP transport properties impact nutrient diffusion and disc cell survival and function., Design: Human CEP tissues harvested from six fresh cadaveric lumbar spines (38-66 years old) were placed at the open sides of diffusion chambers. Bovine nucleus pulposus (NP) cells cultured inside the chambers were nourished exclusively by nutrients diffusing through the CEP tissues. After 72 h in culture, depth-dependent NP cell viability and gene expression were measured, and related to CEP transport properties and biochemical composition determined using fluorescence recovery after photobleaching and Fourier transform infrared (FTIR) spectroscopy., Results: Solute diffusivity varied nearly 4-fold amongst the CEPs studied, and chambers with the least permeable CEPs appeared to have lower aggrecan, collagen-2, and matrix metalloproteinase-2 gene expression, as well as a significantly shorter viable distance from the CEP/nutrient interface. Increasing chamber cell density shortened the viable distance; however, this effect was lost for low-diffusivity CEPs, which suggests that these CEPs may not provide enough nutrient diffusion to satisfy cell demands. Solute diffusivity in the CEP was associated with biochemical composition: low-diffusivity CEPs had greater amounts of collagen and aggrecan, more mineral, and lower cross-link maturity., Conclusions: CEP transport properties dramatically affect NP cell survival/function. Degeneration-related CEP matrix changes could hinder the success of biologic therapies that require increased nutrient supply., (Copyright © 2019 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Emerging evidence suggests that microRNAs (miRNAs, miRs) play important roles in the development of intervertebral disc degeneration (IVDD). Nonetheless, the expression level and biological function of miR-499a-5p in IVDD are still unclear. In this study, we found that miR-499a-5p was significantly downregulated in degenerative tissues of the human nucleus pulposus (NP) compared with healthy tissues. Knockdown of miR-499a-5p promoted NP cell (NPC) apoptosis, stimulated caspase activation, enhanced MMP3 and MMP13 expression, and downregulated aggrecan and type II collagen. Furthermore, TNF-α-treated NPCs showed increased apoptosis and induced an imbalance between anabolism and catabolism of the extracellular matrix (ECM); these changes were attenuated by miR-499a-5p overexpression. Research into possible mechanisms revealed that miR-499a-5p suppressed the expression of SOX4 both at mRNA and protein levels and directly bound to the 3' untranslated region of SOX4 mRNA. Ectopic expression of SOX4 attenuated the negative effect of miR-499a-5p on NPC apoptosis and the positive effect on ECM synthesis. Taken together, these results indicate that miR-499a-5p may attenuate TNF-α-induced NPC apoptosis and an imbalance between anabolism and catabolism of the ECM by downregulating SOX4., (Copyright © 2019 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: The aims of this study were to investigate the gene expression of CCN family members in rat intervertebral disc (IVD) cells and to examine whether Wnt-β-catenin signaling regulates the expression of CCN family 2 (CCN2)/connective tissue growth factor (CTGF) in rat nucleus pulposus (NP) cells., Methods: The gene expression of CCN family members were assessed in rat IVD cells using real-time reverse transcription polymerase chain reaction (RT-PCR). The expression pattern of CCN2 was also assessed in rat IVD cells using western blot and immunohistochemical analyses. Gain-of-function and loss-of-function experiments were performed to identify the mechanisms by which Wnt-β-catenin signaling influences the activity of the CCN2 promoter. To further determine if the mitogen-activated protein kinase (MAPK) pathway is required for the Wnt-β-catenin signaling-induced regulation of CCN2 expression in the NP cells, CCN2 expression was analyzed by reporter assay, RT-PCR and western blot analysis., Results: CCN2 messenger RNA (mRNA) and protein were expressed in rat IVDs. Expression of CCN2 was significantly higher than for mRNA of other CCN family members in both rat NP and annulus fibrosus (AF) cells. The relative activity of the CCN2 promoter decreased 24 h after treatment with 6-bromoindirubin-3'-oxime (1.0 μM) (0.773 (95% 0.735, 0.812) P = 0.0077) in NP cells. In addition, treatment with the WT-β-catenin vector (500 ng) significantly decreased CCN2 promoter activity (0.688 (95% 0.535, 0.842) P = 0.0063), whereas β-catenin small interfering RNA (500 ng) significantly increased CCN2 promoter activity (1.775 (95% 1.435, 2.115) P < 0.001). Activation of Wnt-β-catenin signaling decreased the expression of CCN2 mRNA and protein by NP cells. Regulation of CCN2 by Wnt-β-catenin signaling involved the MAPK pathway in rat NP cells., Conclusions: This study shows that Wnt-β-catenin signaling regulates the expression of CCN2 through the MAPK pathway in NP cells. Understanding the balance between Wnt-β-catenin signaling and CCN2 is necessary for developing therapeutic alternatives for the treatment of IVD 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/cm 2 ) 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/cm 2 ), 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.

Aim: To improve nucleus pulposus cell-targeted therapy for intervertebral disc degeneration (IDD) by fabricating a novel kind of ultrasound (US)-mediated poly(lactic-co-glycolic acid) nanobubbles (NBs) as a means of targeted drug delivery., Materials & Methods: The resveratrol (RES)-embedded NBs were synthesized using a double-emulsion method. The active NP cell-targeting biomarker CDH2 antibody (AbCDH2) was further conjugated to the NBs using a carbodiimide method. Then, this RES/AbCDH2 NBs were examined by physical properties, specifc cell-targeting ability, anticatabolism effect in vitro and in vivo., Results: RES/AbCDH2 NBs exhibited high RES-loading efficiency, and US triggered accelerated RES release. Furthermore, RES/AbCDH2 NB treatment exhibited excellent anticatabolic ability in vitro; and in an IDD rabbit model, US-mediated RES/AbCDH2 NB injection effectively retarded the degenerative process of the intervertebral disc in vivo., Conclusion: The combination of US irradiation and drug delivery through RES/AbCDH2 NBs can be considered as a novel treatment option for IDD.

Background: Mechanical overloading can lead to disc degeneration. Nucleus pulposus (NP) cell senescence is aggravated within the degenerated disc. This study was designed to investigate the effects of high compression on NP cell senescence and the underlying molecular mechanism of this process., Methods: Rat NP cells seeded in decalcified bone matrix were subjected to non-compression (control) or compression (2% or 20% deformation, 1.0 Hz, 6 hours/day). The reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) and the p38 MAPK inhibitor SB203580 were used to investigate the roles of the ROS and p38 MAPK pathway under high-magnitude compression. Additionally, we studied the effects of compression (0.1 or 1.3 MPa, 1.0 Hz, 6 hours/day) in a rat disc organ culture., Results: Both in scaffold and organ cultures, high-magnitude compression (20% deformation or 1.3 MPa) increased senescence-associated β-galactosidase (SA-β-Gal) activity, senescence marker (p16 and p53) expression, G1 cell cycle arrest, and ROS generation, and decreased cell proliferation, telomerase activity and matrix (aggrecan and collagen II) synthesis. Further analysis of the 20% deformation group showed that NAC inhibited NP cell senescence but had no obvious effect on phospho-p38 MAPK expression and that SB203580 significantly attenuated ROS generation and NP cell senescence., Conclusions: High-magnitude compression can accelerate NP cell senescence through the p38 MAPK-ROS pathway.

Objective: To investigate the gene expression profiles of long non-coding RNAs (lncRNA)in human degenerated intervertebral disc degeneration (IDD). Methods: An lncRNA-mRNA microarray analysis of human nucleus pulposus (NP) was employed. Bioinformatics prediction was also applied to delineate the functional roles of the differentially expressed lncRNAs. Several lncRNAs and mRNAs were chosen for quantitative real-time PCR (qRT-PCR) validation. Results: A total of 1 570 lncRNAs expressed in degenerate group compared with the nondegenerate group. Of these, the expression level of 428 lncRNAs was upregulated >2-fold compared with nondegenerate group while that of 584 was downregulated. Bioinformatics analysis (GO and pathway analyses) revealed that some classical pathways participating in extracellular matrix (ECM) and cell apoptosis were aberrantly expressed in the intervertebral disc ( P <0.05). Enhancer-like lncRNAs and their nearby coding genes were analyzed. Three lncRNAs were identified as potential enhancers. Several lncRNAs were validated in the intervertebral disc using RT-qPCR. Conclusion: The lncRNAs express differentially in the intervertebral disc. LncRNAs may therefore be novel candidate biomarkers and potential targets for intervertebral disc degeneration therapy in the future.

We developed a nucleus pulposus cell (NPC)-modulated decellularized small intestinal submucosa (SIS) scaffold, and assessed the ability of this material to prevent Intervertebral disc degeneration (IVD) degeneration. Decellularized porcine SIS was squashed into particles and the biological safety and efficiency of these particles were evaluated. Next, SIS particles were seeded with rabbit NPCs, cultured for two months in vitro, decellularized again and suspended for intervertebral injection. We demonstrated that use of the decellularization protocol resulted in the removal of cellular components with maximal retention of extracellular matrix. The xenogeneic decellularized SIS did not display cytotoxicity in vitro and its application prevented NPC degradation. Furthermore, the xenogeneic SIS microparticles were effective in preventing IVD progression in vivo in a rabbit disc degeneration model. In conclusion, our study describes an optimized method for decellularized SIS preparation and demonstrated that the material is safe and effective for treating IVD degeneration.

Background: The stem cell-based therapies for intervertebral disc degeneration have been widely studied. However, the mechanisms of mesenchymal stem cells interacting with intervertebral disc cells, such as nucleus pulposus cells (NPCs), remain unknown. Exosomes as a vital paracrine mechanism in cell-cell communication have been highly focused on. The purpose of this study was to detect the role of exosomes derived from bone marrow mesenchymal stem cells (BM-MSCs) and NPCs in their interaction with corresponding cells., Methods: The exosomes secreted by BM-MSCs and NPCs were purified by differential centrifugation and identified by transmission electron microscope and immunoblot analysis of exosomal marker proteins. Fluorescence confocal microscopy was used to examine the uptake of exosomes by recipient cells. The effects of NPC exosomes on the migration and differentiation of BM-MSCs were determined by transwell migration assays and quantitative RT-PCR analysis of NPC phenotypic genes. Western blot analysis was performed to examine proteins such as aggrecan, sox-9, collagen II and hif-1α in the induced BM-MSCs. Proliferation and the gene expression profile of NPCs induced by BM-MSC exosomes were measured by Cell Counting Kit-8 and qRT-PCR analysis, respectively., Results: Both the NPCs and BM-MSCs secreted exosomes, and these exosomes underwent uptake by the corresponding cells. NPC-derived exosomes promoted BM-MSC migration and induced BM-MSC differentiation to a nucleus pulposus-like phenotype. BM-MSC-derived exosomes promoted NPC proliferation and healthier extracellular matrix production in the degenerate NPCs., Conclusion: Our study indicates that the exosomes act as an important vehicle in information exchange between BM-MSCs and NPCs. Given a variety of functions and multiple advantages, exosomes alone or loaded with specific genes and drugs would be an appropriate option in a cell-free therapy strategy for intervertebral disc degeneration.

Accumulating evidence indicates that miRNAs, a class of small non-coding RNAs, are implicated in the pathogenesis of various diseases such as cancer and intervertebral disc degeneration. The aim of this study was to investigate the expression and the biological function of microRNA-34a in intervertebral disc degeneration. In this study, microRNA-34a expression was assessed in nucleus pulposus specimens and in IL-1β-stimulated nucleus pulposus cells by real-time polymerase chain reaction. microRNA-34a functions were investigated by using gain and loss of function experiments in nucleus pulposus cells and a dual luciferase reporter assay in 293T cells. microRNA-34a was dramatically up-regulated in degenerative nucleus pulposus tissues and in IL-1β-stimulated nucleus pulposus cells when compared with controls. Furthermore, growth differentiation factor 5 was identified as a target of microRNA-34a. Aberrant expression of microRNA-34a inhibited growth differentiation factor 5 expression by direct binding to its 3'-untranslated region. This inhibition was abolished by mutation of the microRNA-34a binding sites. In addition, microRNA-34a silencing reversed IL-1β-induced decrease in type II collagen and aggrecan expression in nucleus pulposus cells. This effect was substantially suppressed by growth differentiation factor 5 silencing. Our results suggested that microRNA-34a inhibition prevents IL-1β-induced extracellular matrix degradation in human nucleus pulposus by increasing growth differentiation factor 5 expression. microRNA-34a inhibition may be a novel molecular target for intervertebral disc degeneration treatment through the prevention of nucleus pulposus extracellular matrix degradation., (© 2016 by the Society for Experimental Biology and Medicine.)

Transplantation of mesenchymal stem cells (MSCs) into the degenerated intervertebral disc (IVD) has shown promise for decelerating or arresting IVD degeneration. Cellular mechanical properties play crucial roles in regulating cell-matrix interactions, potentially reflecting specific changes that occur based on cellular phenotype and behavior. However, the effect of co-culturing of MSCs with nucleus pulposus cells (NPCs) on the mechanical properties of NPCs remains unknown. In our study, we demonstrated that co-culture of degenerated NPCs with MSCs resulted in significantly decreased mechanical moduli (elastic modulus, relaxed modulus, and instantaneous modulus) and increased biological activity (proliferation and expression of matrix genes) in degenerated NPCs, but not normal NPCs. SDF-1, CXCR4 ligand, was highly expressed in MSCs when co-cultured with degenerated NPCs. Inhibition of SDF-1 using CXCR4 antagonist AMD3100 or knocking-down CXCR4 in degenerated NPCs abolished the MSCs-induced decrease in the mechanical moduli and increased biological activity of degenerated NPCs, suggesting a crucial role for SDF-1/CXCR4 signaling. AKT and FAK inhibition attenuated the MSCs- or SDF-1-induced decrease in the mechanical moduli of degenerated NPCs. In conclusion, it was demonstrated in vitro that MSCs regulate the mechanical properties of degenerated NPCs through SDF-1/CXCR4/AKT signaling. These findings highlight a possible mechanical mechanism for MSCs-induced modulation with degenerated NPCs, which may be applicable to MSCs-based therapy for disc degeneration., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Intervertebral disc degeneration proceeds with age and is one of the major causes of lumbar pain and degenerative lumbar spine diseases. However, studies in the field of intervertebral disc biology have been hampered by the lack of reliable cell lines that can be used for in vitro assays. In this study, we show that a chordoma-derived cell line U-CH1-N cells highly express the nucleus pulposus (NP) marker genes, including T (encodes T brachyury transcription factor), KRT19, and CD24. These observations were further confirmed by immunocytochemistry and flow cytometry. Reporter analyses showed that transcriptional activity of T was enhanced in U-CH1-N cells. Chondrogenic capacity of U-CH1-N cells was verified by evaluating the expression of extracellular matrix (ECM) genes and Alcian blue staining. Of note, we found that proliferation and synthesis of chondrogenic ECM proteins were largely dependent on T in U-CH1-N cells. In accordance, knockdown of the T transcripts suppressed the expression of PCNA, a gene essential for DNA replication, and SOX5 and SOX6, the master regulators of chondrogenesis. On the other hand, the CD24-silenced cells showed no reduction in the mRNA expression level of the chondrogenic ECM genes. These results suggest that U-CH1-N shares important biological properties with notochordal NP cells and that T plays crucial roles in maintaining the notochordal NP cell-like phenotype in this cell line. Taken together, our data indicate that U-CH1-N may serve as a useful tool in studying the biology of intervertebral disc. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 34:1341-1350, 2016., (© 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society.)

Resveratrol (RSV) is known to play a role of anti-TNF-α in a number of cell types. However, whether RSV modulates the effects of TNF-α on human nucleus pulposus (NP) cells is unknown. The purpose of this study is to investigate whether RSV regulates TNF-α-induced matrix metalloproteinase-3 (MMP-3) expression. Via quantitative real-time polymerase chain reaction (qRT-PCR) analysis, we found that MMP-3 expression induced by TNF-α was inhibited by RSV treatment. Depending on Western blot and qRT-PCR assay, we found that RSV induced autophagy in human NP cells, whereas inhibition of autophagy remarkably abolished the restraining role of RSV in the TNF-α-mediated up-regulation of MMP-3. Furthermore, RSV increased SIRT1 expression and SIRT1 knockdown significantly suppressed RSV-induced autophagy in NP cells. RSV also activated AMP-activated protein kinase (AMPK), while inhibition of AMPK notably abolished RSV-induced SIRT1 expression. Our data showed that RSV attenuated TNF-α-induced MMP-3 expression in human NP cells by activating autophagy via AMPK/SIRT1 signaling pathway. This new finding suggested that RSV might act as a novel preventive and therapeutic role in intervertebral disc degeneration., (© 2016 by the Society for Experimental Biology and Medicine.)

To ascertain the optimal dosage of CO within LWDHD for enhancing its therapeutic efficacy in treating IVDD, through a comparison of its effects across varied dosages using a mouse IVDD model. Methods: Eight-week-old male C57BL/6J mice were subjected to a lumbar spine instability surgery to induce an IVDD model and received a modified LWDHD formulation containing varied dosages of CO (original dose of CO, or 5- or 10-time dose of CO (referred to as 1 × CO, 5 × CO, and 10 × CO)) for 8 weeks. The therapeutic efficacy on IVDD was evaluated through changes in lumbar spine function, histopathological morphology, extracellular matrix metabolism, nucleus pulposus cell viability, sensory nerve ingrowth, and nucleus pulposus (NP) cell pyroptosis. Results: Augmenting CO levels in LWDHD led to a dose-dependent increase in the levels of CO-sourced active compounds in the plasma of mice. The modified LWDHD formulations, particularly the 5 × CO, exhibited a favorable pharmacological effect on lumbar function, structural integrity, ECM composition, NP cell viability, and sensory nerve ingrowth. Importantly, all 3 formulations notably mitigated NP cell pyroptosis by activating NRF2/KEAP1 pathway, with the 5 × CO formulation exhibiting superior efficacy. Additionally, a comprehensive score analysis indicated that 5 × CO formulation achieved the highest score. Conclusion: These data underscore that elevating the dosage of CO to a specific threshold can enhance the effectiveness of LWDHD in treating IVDD. [ABSTRACT FROM AUTHOR]

BACKGROUND: Intervertebral disk degeneration is a pathological change caused by a series of complex molecular mechanisms that result in the aging and damage of intervertebral discs, ultimately leading to severe clinical symptoms. Traditional Chinese medicine has unique advantages in the treatment of intervertebral disk degeneration due to its low cost, non-addictive nature, multi-target effects, minimally toxic and side effects, and high patient acceptance. OBJECTIVE: To review the latest research results of traditional Chinese medicine monomer intervention-related signaling pathways in the treatment of intervertebral disk degeneration, describe and analyze the action mechanism of traditional Chinese medicine monomer on intervertebral disk degeneration, and provide a new approach and theoretical basis for future basic research and clinical treatment. METHODS: The first author searched for relevant literature from January 2018 to February 2023 in CNKI, PubMed, VIP, and WanFang using the search terms “intervertebral disc, signal pathway”. The articles that did not meet the criteria were excluded after preliminary screening of the titles and abstracts. Finally, 72 articles were selected for review and analysis. RESULTS AND CONCLUSION: Traditional Chinese medicine monomers can regulate multiple classical signaling pathways such as Wnt/β-catenin, PI3K/Akt, mTOR, NF-κB, and MAPK. They achieve this by regulating oxidative stress, adjusting the expression of pro/anti-apoptotic proteins in cells, stimulating cellular autophagy function, reducing stimulation of cell inflammatory factors, increasing the expression of extracellular matrix markers, reducing the production of matrix-degrading enzymes, maintaining the synthesis and stability of extracellular matrix, inducing differentiation of mesenchymal stem cells in the nucleus pulposus into nucleus pulposus cells, promoting endogenous repair and reconstruction, controlling apoptosis and aging of nucleus pulposus cells, and increasing the activity of nucleus pulposus cells. These actions improve the microenvironment within the intervertebral disc, maintain the normal physiological function of the intervertebral disc, and delay intervertebral disc degeneration. [ABSTRACT FROM AUTHOR]

Nucleus pulposus cell (NPC) senescence is a major cause of intervertebral disc degeneration (IVDD). Oxidative stress and reactive oxygen species (ROS) play critical roles in regulating cell senescence. Selenophosphate synthetase 1 (SEPHS1) was reported to play an important role in mitigating oxidative stress in an osteoarthritis (OA) model by reducing the production of ROS, thereby, delaying the occurrence and development of osteoarthritis. In this study, we explored the, hitherto unknown, role of SEPHS1 in IVDD in vitro and in vivo using an interleukin-1b (IL-1β)-induced NPC senescence model and a rat needle puncture IVDD model, respectively. SEPHS1 delayed NPC senescence in vitro by reducing ROS production. Age-related dysfunction was also ameliorated by the overexpression of SEPHS1 and inhibition of the Hippo-Yap/Taz signaling pathway. In vivo experiments revealed that the overexpression of SEPHS1 and inhibition of Hippo-Yap/Taz alleviated IVDD in rats. Moreover, a selenium (Se)-deficient diet and lack of SEPHS1 synergistically aggravated IVDD progression. Taken together, our results demonstrate that SEPHS1 plays a significant role in NPC senescence. Overexpression of SEPHS1 and inhibition of Hippo-Yap/Taz can delay NPC senescence, restore the balance of extracellular matrix metabolism, and attenuate IVDD. SEPHS1 could be a promising therapeutic target for IVDD. NEW & NOTEWORTHY Selenophosphate synthetase 1 (SEPHS1) deficiency leads to an increase in reactive oxygen species levels and in the subsequent activation of the Hippo-Yap/Taz signaling pathway. In the rat model of intervertebral disc degeneration (IVDD), overexpression of SEPHS1 and inhibition of Hippo-YAP/Taz mitigated the progression of disc degeneration indicating the involvement of SEPHS1 in IVDD. SEPHS1 is a promising therapeutic target for IVDD. [ABSTRACT FROM AUTHOR]

BACKGROUND: Salvianolic acid B can inhibit cell damage induced by H2O2, effectively remove excess reactive oxygen species, and exert antioxidant properties. It has been used in the treatment of many diseases. However, there are relatively few studies on the role and mechanism of salvianolic acid B in intervertebral disc degeneration. OBJECTIVE: To observe the effect and mechanism of salvianolic acid B on oxidative stress-induced intervertebral disc degeneration by using gelatin methacryloyl hydrogel as a carrier through the in vitro cell experiment and the in vivo animal experiment. METHODS: The gelatin methacryloyl hydrogel (drug-loaded hydrogel) loaded with salvianolic acid B was prepared. (1) In vitro cell experiment: The lumbar nucleus pulposus cells of adult SD rats were isolated and extracted, and passage 3 nucleus pulposus cells were selected and divided into groups: Group A was added complete medium. In group B, a complete medium containing H2O2 was added. Group C was inoculated on methylacrylylated gelatin hydrogel and added with a complete medium containing H2O2. Group D was inoculated on methyl acrylyl gelatin hydrogel loaded with salvianolic acid B and added into a complete medium containing H2O2. The E group was inoculated on the methylacrylyl gelatin hydrogel loaded with salvianolic acid B, and the complete medium containing H2O2 and the complete medium containing TLR4 signaling pathway inhibitor were added. Cell proliferation, oxidative stress, inflammatory response, gene expression of cell matrix-associated proteins and the protein expression of TLR4/nuclear factor-kB signaling pathway were detected. (2) Animal in vivo experiment: Sixty adult SD rats were randomly divided into normal group, acupuncture group, acupuncture + salvianolic acid group, acupuncture + hydrogel group and acupuncture + loading potion gel group, with 12 rats in each group. The last four groups were treated with acupuncture to establish models of intervertebral disc degeneration and then injected with normal saline, salvianolic acid B solution, non-drug loaded gel and drug-loaded gel in turn. Imaging examination and pathological observation were performed 4 weeks after surgery. RESULTS AND CONCLUSION: (1) In vitro cell experiment: Compared with group A, the cell proliferation was decreased; the oxidative stress reaction and inflammation reaction were enhanced; the expression of extracellular matrix degrading enzymes (matrix metalloproteinase 3, matrix metalloproteinase 13, ADAMTS4, ADAMTS5) was increased in group B (P < 0.05), and the synthesis of extracellular matrix (type II collagen, proteoglycan) was decreased (P < 0.05). The protein expression of the TLR4/nuclear factor-kB signaling pathway was increased (P < 0.05). Compared with group B, the cell proliferation of groups D and E was increased, the oxidative stress response and inflammatory response were weakened, and the expression of extracellular matrix degrading enzymes (matrix metalloproteinase 3, matrix metalloproteinase 13, ADAMTS4, ADAMTS5) was decreased (P < 0.05), and the synthesis of extracellular matrix was increased (P < 0.05). The protein expression of TLR4/nuclear factor-kB signaling pathway was decreased (P < 0.05), and the effect was more significant in group E. (2) Animal in vivo experiment: 4 weeks after surgery, intervertebral disc height index, index of MRI and pathological and histological grading of the intervertebral disc had improved significantly in the acupuncture+drug-loaded hydrogel group, and simply injecting hydrogel or salvianolic acid B solution can to a certain extent improve the intervertebral disc degeneration, but they are not as good as the injection of the drug-loaded hydrogel. (3) It is concluded that gelatin methacryloyl hydrogel loaded with salvianolic acid B can inhibit oxidative stress and inflammation in the degenerated intervertebral disc tissue, inhibit the degradation of extracellular matrix, and alleviate the process of intervertebral disc degeneration, which may be accomplished by inhibiting the TLR4/nuclear factor-kB signaling pathway. [ABSTRACT FROM AUTHOR]

BACKGROUND: Intervertebral disc degeneration is the basis of spinal degenerative diseases; however, there is no effective treatment. OBJECTIVE: To investigate whether sinomenine can inhibit interleukin-1β-induced apoptosis in nucleus pulposus cells and its molecular mechanism. METHODS: Rat nucleus pulposus cells were cultured in vitro by trypsin combined with type II collagenase digestion, and the cell growth curve was plotted. An appropriate sinomenine concentration was determined using the cell counting kit-8 kit. Nucleus pulposus cells were divided into control group, sinomenine group, interleukin-1β group, sinomenine+interleukin-1β group, zinc protoporphyrin group, zinc protoporphyrin+sinomenine group, zinc protoporphyrin+interleukin-1β group, and sinomenine+zinc protoporphyrin+interleukin-1β group. Proliferative activity, reactive oxygen species content, apoptosis rate, and heme oxygenase-1 expression in nucleus pulposus cells were detected. RESULTS AND CONCLUSION: The rat nucleus pulposus cells cultured in vitro were polygonal, triangular, and short wedge-shaped, and the cell growth showed an “S” curve. The cells grew slowly in the first 3 days of culture, rapidly in 4-6 days, and slowly again in 7-8 days. The cells then entered the “platform stage” where the number of cells no longer increased. The proliferative activity of myeloid cells showed no significant changes when the concentration of sinomenine was ≤ 80 μmol/L (P > 0.05). Interleukin-1β significantly reduced the proliferative activity of nucleus pulposus cells, increased the content of reactive oxygen species and led to apoptosis (P < 0.01). Sinomenine intervention not only promoted heme oxygenase-1 expression (P < 0.05) but also inhibited interleukin-1βinduced decrease in proliferative activity and increase in reactive oxygen species content and apoptosis rate in nucleus pulposus cells (P < 0.05). These effects could be reversed by zinc protoporphyrin (P < 0.01). [ABSTRACT FROM AUTHOR]

Background: The method of action of Bushen Formula (BSHXF) in the treatment of intervertebral disc degeneration (IVDD) was uncovered in this work using in vivo and in vitro tests. To clarify the mechanism of action of BSHXF, we validated the rat intervertebral disc degeneration model and the nucleus pulposus cell degeneration model.Methods: In an in vivo model of IVDD the study explores the impact of BSHXF on mitochondrial function, pro-inflammatory cytokines, pro-apoptotic factors, and matrix metalloproteinases. Additionally, it evaluates the induced degeneration of nucleus pulposus (NP) cells in an in vitro model stimulated by interleukin-1 β (IL-1β). The study measures the effects of BSHXF on both the inflammatory response and mitochondrial function.Results: The MRI results showed that BSHXF reduced intervertebral disc volume reduction and degradation of NP tissue. HE, SO-FG and immunofluorescence further confirmed the protective effect of BSHXF on degenerative intervertebral discs. BSHXF reduced the inflammatory levels of IL-6 IL-1β and TNF-α in degenerative intervertebral disc tissue. Meanwhile, JC-1, mPTP and ROS detection revealed that BSHXF can restore mitochondrial function by regulating the expression of antioxidant proteins, playing a protective role in NP cells. Finally, the WB results showed that BSHXF can alleviate IL-1β mediate the degeneration of NP cells. BSHXF can alleviate NP cell apoptosis by inhibiting the expression of bax, cleaved caspase-3, caspase-3, and cyt-c, and increasing the expression of Bcl-2.Conclusion: This study reveals that BSHXF inhibits the development of inflammatory factors, which may play a significant role in intervertebral disc degeneration. This implies that BSHXF is a suitable herbal medication for future research into inflammatory cytokine treatment. [ABSTRACT FROM AUTHOR]

BACKGROUND: Ferroptosis is a new type of programmed cell death, distinguished from apoptosis, autophagy and pyroptosis, which is characterized by the iron-dependent accumulation of polyunsaturated fatty acid peroxidation. Studies in recent years have shown that ferroptosis is closely associated with the development of intervertebral disc degeneration and osteoarthritis. OBJECTIVE: To review the action mechanism of ferroptosis and its role in intervertebral disc degeneration and osteoarthritis progression, and provide new therapeutic strategies for intervertebral disc degeneration and osteoarthritis. METHODS: PubMed, WanFang and CNKI databases were used as the literature sources. The search terms were “intervertebral disc degeneration; osteoarthritis; nucleus pulposus; chondrocytes; articular cartilage; ferroptosis” in English and Chinese. Finally, 67 articles were screened for this review. RESULTS AND CONCLUSION: (1) Ferroptosis is a newly discovered programmed cell death, whose mechanism is closely related to the factors such as abnormal iron ion accumulation, polyunsaturated fatty acid peroxidation, abnormal amino acid metabolism, mitochondrial dysfunction, and ferritin autophagy. (2) Various stimulation factors such as homocysteine, tert-buty1 hydroperoxide and ferric ammonium citrate mainly through inhibiting glutathione peroxidase 4 activity cause ferroptosis of nucleus pulposus cells, annulus fibrosus cells, and chondrocytes of the endplate chondrocytes, thus promoting intervertebral disc degeneration. In addition, the role of ferroptosis in intervertebral disc degeneration is regulated by miRNA. (3) Ferroptosis promotes the progression of osteoarthritis by participating in chondrocyte loss, extracellular matrix lysis, and synovitis. Chondrocyte ferroptosis is inhibited by stigmasterol, D-mannose, astaxanthin, metformin and icariin, while it is exacerbated by interleukin-1β, ferric ammonium citrate and RNA binding protein SND1. (4) The therapeutic effects of some drugs targeting ferroptosis, such as deferoxamine, ferroptosis inhibitor, on intervertebral disc degeneration and osteoarthritis, have been verified by experiment, but have not been applied clinically. With the deepening of research, inhibiting the occurrence of ferroptosis can provide a new and effective strategy for the treatment and prevention of intervertebral disc degeneration and osteoarthritis. [ABSTRACT FROM AUTHOR]

Study Design. Experimental analysis of circular RNA in intervertebral disk degeneration (IDD). Objective. This study aimed to explore the roles of hsa_ circ_0001946 (circ-CDR1as) in mechanical stress-induced nucleus pulposus cell injury in IDD. Summary of Background Data. Mechanical stress is an important pathogenic factor for IDD. Excessive compression stress leads to nucleus pulposus (NP) cell apoptosis and extracellular matrix (ECM) degradation and accelerated IDD. Circ-CDR1as is associated with various degenerative conditions, but its role in IDD is not clear. Herein, we explored the roles and mechanisms of circ-CDR1as in IDD in vitro. Materials and Methods. An in vitro model of IDD was constructed by treating NP cells with 1.0 MPa compression stress. Quantitative real-time polymerase chain reaction assay was used for detecting the expression of circ-CDR1as and miR-432-5p. Immunofluorescent analysis was performed for MMP13 detection. Western blot assay was performed for detecting apoptosis and ECM-related protein expression. Flow cytometry analysis was used for cell apoptosis analysis. The dual-luciferase reporter was used to analyze the interaction between miR-432-5p and circ-CDR1as or SOX9. Differences in means between groups were evaluated using the Student t test or one-way analysis of variance. Results. In compression-treated human NP cells, we found that circ-CDR1as was significantly downregulated. Functional experiments showed that circ-CDR1as overexpression reduced the compression-induced apoptosis and ECM degradation in NP cells. Further research indicated that circ-CDR1as could act as a molecular sponge for miR-432-5p, a miRNA that enhanced compression-induced damage of NP cells by inhibiting the expression of SOX9. The luciferase reporter experiments also showed that the mutual dialogue between circ-CDR1as and miR-432-5p regulated the expression of SOX9. Conclusions. Circ-CDR1as binds to miR-432-5p and plays a protective role in mitigating compression-induced NP cell apoptosis and ECM degradation by targeting SOX9. Circ-CDR1as may provide a novel therapeutic target for the clinical management of IDD in the future. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Ferroptosis, a novel type of cell death mediated by the iron‐dependent lipid peroxidation, contributes to the pathogenesis of the intervertebral disc degeneration (IDD). Increasing evidence demonstrated that melatonin (MLT) displayed the therapeutic potential to prevent the development of IDD. Current mechanistic study aims to explore whether the downregulation of ferroptosis contributes to the therapeutic capability of MLT in IDD. Current studies demonstrated that conditioned medium (CM) from the lipopolysaccharide (LPS)‐stimulated macrophages caused a series of changes about IDD, including increased intracellular oxidative stress (increased reactive oxygen species and malondialdehyde levels, but decreased glutathione levels), upregulated expression of inflammation‐associated factors (IL‐1β, COX‐2 and iNOS), increased expression of key matrix catabolic molecules (MMP‐13, ADAMTS4 and ADAMTS5), reduced the expression of major matrix anabolic molecules (COL2A1 and ACAN), and increased ferroptosis (downregulated GPX4 and SLC7A11 levels, but upregulated ACSL4 and LPCAT3 levels) in nucleus pulposus (NP) cells. MLT could alleviate CM‐induced NP cell injury in a dose‐dependent manner. Moreover, the data substantiated that intercellular iron overload was involved in CM‐induced ferroptosis in NP cells, and MLT treatment alleviated intercellular iron overload and protected NP cells against ferroptosis, and those protective effects of MLT in NP cells further attenuated with erastin and enhanced with ferrostatin‐1(Fer‐1). This study demonstrated that CM from the LPS‐stimulated RAW264.7 macrophages promoted the NP cell injury. MLT alleviated the CM‐induced NP cell injury partly through inhibiting ferroptosis. The findings support the role of ferroptosis in the pathogenesis of IDD, and suggest that MLT may serve as a potential therapeutic approach for clinical treatment of IDD. [ABSTRACT FROM AUTHOR]

The study aims to explore the medical prospect of melatonin (MLT) and the underlying therapeutic mechanism of MLT-mediated macrophage (Mφ) polarization on the function of nucleus pulposus (NP) in intervertebral disc degeneration (IDD). RAW 264.7 Mφs were induced by lipopolysaccharide (LPS) to simulate Mφ polarization and the inflammatory reaction of Mφs with or without MLT were detected. Conditioned medium (CM) collected from these activated Mφs with or without MLT treatment were further used to incubate NP cells. The oxidative stress, inflammation and extracellular matrix (ECM) metabolism in NP cells were determined. Then, the changes in SIRT1/Notch signaling were detected. The agonist (SRT1720) and inhibitor (EX527) of SIRT1 were used to further explore the association among MLT. The interaction between SIRT1 and NICD was detected by immunoprecipitation (IP). Finally, puncture-induced rat IDD models were established and IDD degrees were clarified by X-ray, MRI, H&E staining and immunofluorescence (IF). The results of flow cytometry and inflammation detection indicated that LPS could induce M1-type Mφ polarization with pro-inflammatory properties. MLT significantly inhibited the aforementioned process and inhibited M1-type Mφ polarization, accompanied by the alleviation of inflammation. Compared with those without MLT, the levels of oxidative stress, pro-inflammatory cytokines and ECM catabolism in NP cells exposed to CM with MLT were markedly downregulated in a dose-dependent manner. The inhibition of SIRT1 and the enhancement of Notch were observed in activated Mφs and they can be reversed after MLT treatment. This prediction was further confirmed by using the SRT1720 and EX527 to activate or inhibit the signaling. The interaction between SIRT1 and NICD was verified by IP. In vivo study, the results of MRI, Pfirrmann grade scores and H&E staining demonstrated the degree of disc degeneration was significantly lower in the MLT-treated groups when compared with the IDD control group. The IF data showed M1-type Mφ polarization decreased after MLT treatment. MLT could inhibit M1-type Mφ polarization and ameliorate the NP cell injury caused by inflammation in vitro and vivo, which is of great significance for the remission of IDD. The SIRT1/Notch signaling pathway is a promising target for MLT to mediate Mφ polarization. [ABSTRACT FROM AUTHOR]