Palmitic acid induces lipid droplet accumulation and senescence in nucleus pulposus cells via ER-stress pathway.

Intervertebral disc degeneration (IDD) is a highly prevalent musculoskeletal disorder affecting millions of adults worldwide, but a poor understanding of its pathogenesis has limited the effectiveness of therapy. In the current study, we integrated untargeted LC/MS metabolomics and magnetic resonance spectroscopy data to investigate metabolic profile alterations during IDD. Combined with validation via a large-cohort analysis, we found excessive lipid droplet accumulation in the nucleus pulposus cells of advanced-stage IDD samples. We also found abnormal palmitic acid (PA) accumulation in IDD nucleus pulposus cells, and PA exposure resulted in lipid droplet accumulation and cell senescence in an endoplasmic reticulum stress-dependent manner. Complementary transcriptome and proteome profiles enabled us to identify solute carrier transporter (SLC) 43A3 involvement in the regulation of the intracellular PA level. SLC43A3 was expressed at low levels and negatively correlated with intracellular lipid content in IDD nucleus pulposus cells. Overexpression of SLC43A3 significantly alleviated PA-induced endoplasmic reticulum stress, lipid droplet accumulation and cell senescence by inhibiting PA uptake. This work provides novel integration analysis-based insight into the metabolic profile alterations in IDD and further reveals new therapeutic targets for IDD treatment.

Viscoelastic hydrogels regulate adipose-derived mesenchymal stem cells for nucleus pulposus regeneration.

Low back pain is a leading cause of disability worldwide, often attributed to intervertebral disc (IVD) degeneration with loss of the functional nucleus pulposus (NP). Regenerative strategies utilizing biomaterials and stem cells are promising for NP repair. Human NP tissue is highly viscoelastic, relaxing stress rapidly under deformation. However, the impact of tissue-specific viscoelasticity on the activities of adipose-derived stem cells (ASC) remains largely unexplored. Here, we investigated the role of matrix viscoelasticity in regulating ASC differentiation for IVD regeneration. Viscoelastic alginate hydrogels with stress relaxation time scales ranging from 100 s to 1000s were developed and used to culture human ASCs for 21 days. Our results demonstrated that the fast-relaxing hydrogel significantly enhanced ASCs long-term cell survival and NP-like extracellular matrix secretion of aggrecan and type-II collagen. Moreover, gene expression analysis revealed a substantial upregulation of the mechanosensitive ion channel marker TRPV4 and NP-specific markers such as SOX9, HIF-1α, KRT18, CDH2 and CD24 in ASCs cultured within the fast-relaxing hydrogel, compared to slower-relaxing hydrogels. These findings highlight the critical role of matrix viscoelasticity in regulating ASC behavior and suggest that viscoelasticity is a key parameter for novel biomaterials design to improve the efficacy of stem cell therapy for IVD regeneration. STATEMENT OF SIGNIFICANCE: Systematically characterized the influence of tissue-mimetic viscoelasticity on ASC. NP-mimetic hydrogels with tunable viscoelasticity and tissue-matched stiffness. Long-term survival and metabolic activity of ASCs are substantially improved in the fast-relaxing hydrogel. The fast-relaxing hydrogel allows higher rate of cell protrusions formation and matrix remodeling. ASC differentiation towards an NP-like cell phenotype is promoted in the fast-relaxing hydrogel, with more CD24 positive expression indicating NP committed cell fate. The expression of TRPV4, a molecular sensor of matrix viscoelasticity, is significantly enhanced in the fast-relaxing hydrogel, indicating ASC sensing matrix viscoelasticity during cell development. The NP-specific ECM secretion of ASC is considerably influenced by matrix viscoelasticity, where the deposition of aggrecan and type-II collagen are significantly enhanced in the fast-relaxing hydrogel.

Characterization of the Nucleus Pulposus Progenitor Cells via Spatial Transcriptomics.

Loss of refreshment in nucleus pulposus (NP) cellularity leads to intervertebral disc (IVD) degeneration. Nevertheless, the cellular sequence of NP cell differentiation remains unclear, although an increasing body of literature has identified markers of NP progenitor cells (NPPCs). Notably, due to their fragility, the physical enrichment of NP-derived cells has limited conventional transcriptomic approaches in multiple studies. To overcome this limitation, a spatially resolved transcriptional atlas of the mouse IVD is generated via the 10x Genomics Visium platform dividing NP spots into two clusters. Based on this, most reported NPPC-markers, including Cathepsin K (Ctsk), are rare and predominantly located within the NP-outer subset. Cell lineage tracing further evidence that a small number of Ctsk-expressing cells generate the entire adult NP tissue. In contrast, Tie2, which has long suggested labeling NPPCs, is actually neither expressed in NP subsets nor labels NPPCs and their descendants in mouse models; consistent with this, an in situ sequencing (ISS) analysis validated the absence of Tie2 in NP tissue. Similarly, no Tie2-cre-mediated labeling of NPPCs is observed in an IVD degenerative mouse model. Altogether, in this study, the first spatial transcriptomic map of the IVD is established, thereby providing a public resource for bone biology.

Acidic Environment Induces Pyroptosis of NLRP3 Inflammatory Vesicle-Mediated Nucleus Pulposus Cells Through Up-Regulation of POSTN Expression

SUMMARY: Intervertebral disc degeneration (IVDD) is induced by nucleus pulposus (NP) dysfunction as a result of massive loss of NP cells. It has been reported that the acidic microenvironment of the intervertebral disc (IVD) can induce NP cell pyroptosis, and that up-regulation of periostin (POSTN) expression has a negative effect on NP cell survival. However, the relationship between the acidic environment, POSTN expression level and NP cell pyroptosis is unclear. Therefore, the aim of this study was to explore the relationship between acidic environment and POSTN expression level in NP cells, as well as the effect of POSTN in acidic environment on NP cell pyroptosis. NP cells were obtained from the lumbar vertebrae of Sprague Dawley (SD) male rats. These cells were divided into normal and acidic groups according to whether they were exposed to 6 mM lactic acid solution. And NP cells in the acidic group were additionally divided into three groups: (1) Blank group: no transfection; (2) NC group: cells transfected with empty vector plasmid; (3) sh-POSTN group: cells transfected with sh-POSTN plasmid to knock down the expression level of POSTN. Quantitative real-time PCR (qRT-PCR) and western blot was performed to assess the expression of POSTN at the mRNAand protein levels. CCK8 was used to evaluate cell survival. Western blot, in addition, was performed to examine acid-sensing ion channels (ASIC)-related proteins. And pyroptosis was detected by ELISA and western blot. The expression level of POSTN was significantly increased in NP cells in acidic environment. Knockdown of POSTN expression promoted the survival of NP cells in acidic environment and reduced the protein levels of ASIC3 and ASIC1a in NP cells. Moreover, knockdown of POSTN expression decreased the pyroptosis proportion of NP cells and the levels of pro-inflammatory cytokines interleukin (IL)-1β and IL-18. The levels of pyroptosis-related proteins NLRP3, ASC, cleaved-Caspase-1, and cleaved-GSDMD were also affected by the decreased POSTN expression. The extracellular acidic environment created by lactic acid solution activated NLRP3 inflammatory vesicle-induced caspase-1 to get involved in NP cell pyroptosis by up-regulating POSTN expression.

La degeneración del disco intervertebral (DDIV) es inducida por una disfunción del núcleo pulposo (NP) como resultado de una pérdida masiva de células NP. Se ha informado que el microambiente ácido del disco intervertebral (DIV) puede inducir la piroptosis de las células NP y que la regulación positiva de la expresión de periostina (POSTN) tiene un efecto negativo en la supervivencia de las células NP. Sin embargo, la relación entre el ambiente ácido, el nivel de expresión de POSTN y la piroptosis de las células NP es poco clara. Por lo tanto, el objetivo de este estudio fue explorar la relación entre el ambiente ácido y el nivel de expresión de POSTN en células NP, así como el efecto de POSTN en ambiente ácido sobre la piroptosis de las células NP. Las células NP se obtuvieron de las vertebras lumbares de ratas macho Sprague Dawley (SD). Estas células se dividieron en grupos normales y ácidos según se expusieron a una solución de ácido láctico 6 mM. Las células NP en el grupo ácido se dividieron adicionalmente en tres grupos: (1) Grupo en blanco: sin transfección; (2) grupo NC: células transfectadas con plásmido vector vacío; (3) grupo sh-POSTN: células transfectadas con plásmido sh-POSTN para reducir el nivel de expresión de POSTN. Se realizó una PCR cuantitativa en tiempo real (qRT-PCR) y una transferencia Western para evaluar la expresión de POSTN en los niveles de ARNm y proteína. Se utilizó CCK8 para evaluar la supervivencia celular. Además, se realizó una transferencia Western para examinar las proteínas relacionadas con los canales iónicos sensibles al ácido (ASIC). La piroptosis se detectó mediante ELISA y Western blot. El nivel de expresión de POSTN aumentó significativamente en células NP en ambiente ácido. La eliminación de la expresión de POSTN promovió la supervivencia de las células NP en un ambiente ácido y redujo los niveles de proteína de ASIC3 y ASIC1a en las células NP. Además, la eliminación de la expresión de POSTN disminuyó la proporción de piroptosis de las células NP y los niveles de citocinas proinflamatorias interleucina (IL) - 1β e IL-18. Los niveles de proteínas relacionadas con la piroptosis NLRP3, ASC, Caspasa-1 escindida y GSDMD escindida también se vieron afectados por la disminución de la expresión de POSTN. El ambiente ácido extracelular creado por la solución de ácido láctico activó la caspasa-1 inducida por vesículas inflamatorias NLRP3 para involucrarse en la piroptosis de las células NP mediante la regulación positiva de la expresión de POSTN.

DJ-1-mediated p62 degradation delays intervertebral disc degeneration by inhibiting apoptosis of nucleus pulposus cells.

Intervertebral disc degeneration (IDD) is the most important pathological basis of degenerative spinal diseases, for which effective interventions are still lacking. Oxidative stress is considered to be one of the leading pathological mechanisms contributing to IDD. However, the exact role of DJ-1 as an essential member of the antioxidant defense system in IDD is still unclear. Therefore, the aim of this study was to investigate the role played by DJ-1 in IDD and to reveal its potential molecular mechanisms. Western blot and immunohistochemical staining assays were performed to detect the expression of DJ-1 in degenerative nucleus pulposus cells (NPCs). After overexpression of DJ-1 in NPCs by lentiviral transfection, DCFH-DA and MitoSOX fluorescent probes were used to evaluate the levels of reactive oxygen species (ROS); while western blot, TUNEL staining, and Caspase-3 activity were used to assess apoptosis. Immunofluorescence staining was used to demonstrate the relationship between DJ-1 and p62. After inhibition of lysosomal degradation function with chloroquine, p62 degradation and apoptosis in DJ-1 overexpressing NPCs were further examined. In vivo, we assessed the therapeutic effect of upregulated DJ-1 on IDD by X-ray, MRI and Safranin O-Fast green staining. The protein expression of DJ-1 was significantly decreased in degenerated NPCs, accompanied by increased apoptosis. However, overexpression of DJ-1 significantly inhibited the elevated ROS levels and apoptosis in NPCs under oxidative stress. Mechanistically, our results showed that upregulation of DJ-1 promoted p62 degradation via the autophagic lysosomal pathway and that the protective effect of DJ-1 on NPCs under oxidative stress was partially mediated by promoting lysosomal pathway degradation of p62. Moreover, intradiscal injection of adeno-associated virus for overexpression of DJ-1 mitigated the progression of IDD in rats. This study reveals that DJ-1 maintains the homeostasis of NPCs by promoting the degradation of p62 through the autophagic lysosomal pathway, suggesting that DJ-1 is a promising new target for IDD intervention.

Three-dimensional culture model to study the biology of vacuolated notochordal cells from mouse nucleus pulposus explants.

Intervertebral disc degeneration (IDD) involves cellular changes in the nucleus pulposus (NP) characterised by a decline of the large vacuolated notochordal cells (vNCs) and a rise of smaller vacuole-free mature chondrocyte-like NP cells. An increasing number of studies demonstrate that notochordal cells (NCs) exert disease-modifying effects, establishing that NC-secreted factors are essential for the maintenance of a healthy intervertebral disc (IVD). However, understanding the role of the NCs is hampered by a restricted reserve of native cells and the lack of robust ex vivo cell model. A precise dissection enabled the isolation of NP cells from 4 d post-natal stage mouse spines and their culture into self-organised micromasses. The maintenance of cells' phenotypic characteristics was demonstrated by the presence of intracytoplasmic vacuoles and the immuno-colocalisation of the NC-markers (brachyury; SOX9) after 9 d of culture both in hypoxic and normoxic conditions. A significant increase of the size of the micromass was observed under hypoxia, consistent with a higher level of Ki-67+ immunostained proliferative cells. Furthermore, several proteins of interest for the study of vNCs phenotype (CD44; caveolin-1; aquaporin 2; patched-1) were successfully detected at the plasma membrane of NP-cells cultured in micromasses under hypoxic condition. IHC was performed on mouse IVD sections as control staining. An innovative 3D culture model of vNCs derived from mouse postnatal NP is proposed, allowing future ex vivo exploration of their basic biology and of the signalling pathways involved in IVD homeostasis that may be relevant for disc repair.

TAK-715 alleviated IL-1ß-induced apoptosis and ECM degradation in nucleus pulposus cells and attenuated intervertebral disc degeneration ex vivo and in vivo.

BACKGROUND: Intervertebral disc degeneration (IDD) is one of the most common disorders related to the spine. Inflammation, apoptosis and extracellular matrix (ECM) degradation contribute to disc degeneration in nucleus pulposus cells (NPCs). This study focused on the role and mechanism of the p38 inhibitor TAK-715 in intervertebral disc degeneration. METHODS: NPCs were treated with IL-1ß to mimic apoptosis, followed by the addition of TAK-715. It was determined that apoptosis, inflammatory mediators (COX-2), inflammatory cytokines (HMGB1), and ECM components (collagen II, MMP9, ADAMTS5, and MMP3) existed in NPCs. In addition, the p38MAPK signaling pathways were examined. The role of TAK-715 in vivo was determined by acupuncture-induced intervertebral disc degeneration. Following an intradiscal injection of TAK-715, MRI and a histopathological analysis were conducted to assess the degree of degeneration. RESULTS: IL-1ß-induced apoptosis was alleviated by TAK-715 in vitro, and antiapoptotic proteins were upregulated. Furthermore, TAK-715 blocked IL-1ß-induced inflammatory mediator production (COX-2) and inflammatory cytokine production (HMGB1) and degraded the ECM (collagen II, MMP9, ADAMTS5, and MMP3). By inhibiting the phosphorylation of p38, TAK-715 exerted its effects. In a rat tail model, TAK-715 ameliorates puncture-induced disc degeneration based on MRI and histopathology evaluations. CONCLUSION: TAK-715 attenuated intervertebral disc degeneration in vitro and in vivo, suggesting that it might be an effective treatment for IDD.

In Vitro Characterization of a Tissue Renin-Angiotensin System in Human Nucleus Pulposus Cells.

Low back pain is a clinically highly relevant musculoskeletal burden and is associated with inflammatory as well as degenerative processes of the intervertebral disc. However, the pathophysiology and cellular pathways contributing to this devastating condition are still poorly understood. Based on previous evidence, we hypothesize that tissue renin-angiotensin system (tRAS) components, including the SARS-CoV-2 entry receptor angiotensin-converting enzyme 2 (ACE2), are present in human nucleus pulposus (NP) cells and associated with inflammatory and degenerative processes. Experiments were performed with NP cells from four human donors. The existence of angiotensin II, angiotensin II type 1 receptor (AGTR1), AGTR2, MAS-receptor (MasR), and ACE2 in human NP cells was validated with immunofluorescent staining and gene expression analysis. Hereafter, the cell viability was assessed after adding agonists and antagonists of the target receptors as well as angiotensin II in different concentrations for up to 48 h of exposure. A TNF-α-induced inflammatory in vitro model was employed to assess the impact of angiotensin II addition and the stimulation or inhibition of the tRAS receptors on inflammation, tissue remodeling, expression of tRAS markers, and the release of nitric oxide (NO) into the medium. Furthermore, protein levels of IL-6, IL-8, IL-10, and intracellular as well as secreted angiotensin II were assessed after exposing the cells to the substances, and inducible nitric oxide synthase (iNOS) levels were evaluated by utilizing Western blot. The existence of tRAS receptors and angiotensin II were validated in human NP cells. The addition of angiotensin II only showed a mild impact on gene expression markers. However, there was a significant increase in NO secreted by the cells. The gene expression ratios of pro-inflammatory/anti-inflammatory cytokines IL-6/IL-10, IL-8/IL-10, and TNF-α/IL-10 were positively correlated with the AGTR1/AGTR2 and AGTR1/MAS1 ratios, respectively. The stimulation of the AGTR2 MAS-receptor and the inhibition of the AGTR1 receptor revealed beneficial effects on the gene expression of inflammatory and tissue remodeling markers. This finding was also present at the protein level. The current data showed that tRAS components are expressed in human NP cells and are associated with inflammatory and degenerative processes. Further characterization of the associated pathways is warranted. The findings indicate that tRAS modulation might be a novel therapeutic approach to intervertebral disc disease.

Microfluidic Electroceuticals Platform for Therapeutic Strategies of Intervertebral Disc Degeneration: Effects of Electrical Stimulation on Human Nucleus Pulposus Cells under Inflammatory Conditions.

The degeneration of an intervertebral disc (IVD) is a major cause of lower back pain. IVD degeneration is characterized by the abnormal expression of inflammatory cytokines and matrix degradation enzymes secreted by IVD cells. In addition, macrophage-mediated inflammation is strongly associated with IVD degeneration. However, the precise pathomechanisms of macrophage-mediated inflammation in IVD are still unknown. In this study, we developed a microfluidic platform integrated with an electrical stimulation (ES) array to investigate macrophage-mediated inflammation in human nucleus pulposus (NP). This platform provides multiple cocultures of different cell types with ES. We observed macrophage-mediated inflammation and considerable migration properties via upregulated expression of interleukin (IL)-6 (p < 0.001), IL-8 (p < 0.05), matrix metalloproteinase (MMP)-1 (p < 0.05), and MMP-3 (p < 0.05) in human NP cells cocultured with macrophages. We also confirmed the inhibitory effects of ES at 10 µA due to the production of IL-6 (p < 0.05) and IL-8 (p < 0.01) under these conditions. Our findings indicate that ES positively affects degenerative inflammation in diverse diseases. Accordingly, the microfluidic electroceutical platform can serve as a degenerative IVD inflammation in vitro model and provide a therapeutic strategy for electroceuticals.

Upregulation of SIRT1 by Evodiamine activates PI3K/AKT pathway and blocks intervertebral disc degeneration.

Intervertebral disc degeneration (IDD) is a major cause of a number of spinal diseases, resulting in serious public health problems. Evodiamine (Evo) is an indole quinazoline alkaloid extracted from Evodia rutaecarpa, which has antioxidant, anti­apoptosis and anti­inflammatory effects. The purpose of the present study was to investigate lipopolysaccharide (LPS)­induced IDD progression in human nucleus pulposus cells (NPCs) and its potential mechanism. The viability and apoptosis of NPCs were detected by Cell Counting Kit­8 (CCK­8) and TUNEL staining, respectively. Western blotting was used to detect the expression levels of proteins, cell transfection was performed to knockdown Sirtuin 1 (SIRT1) and the expression of tumor necrosis factor­alpha (TNF­α) and interleukin 6 (IL­6) was detected by enzyme­linked immunosorbent assay kits. The results showed that Evo effectively alleviated LPS­induced NPCs apoptosis and caspase­3 activation and Evo treatment reversed the upregulation of matrix metalloproteinase­13, as well as the downregulation of collagen type II (collagen II), Sry­type high­mobility­group box 9 and aggrecan and reduced the production of pro­inflammatory factors TNF­α and IL­6 in LPS­stimulated NPCs. In addition, treatment with Evo upregulated SIRT1 and activated the PI3K/Akt pathway, knockdown of SIRT1 inhibited the phosphorylation of Akt and PI3K in LPS­stimulated NPCs. In general, Evo upregulated SIRT1 and inhibited LPS­induced NPCs apoptosis, extracellular matrix degradation and inflammation by activating the PI3K/Akt pathway.

SIAH1 promotes senescence and apoptosis of nucleus pulposus cells to exacerbate disc degeneration through ubiquitinating XIAP.

BACKGROUND: Using clinical samples and database analysis, this study aimed to investigate the signaling pathways that mediated degeneration of nucleus pulposus cells (NPCs) in patients with intervertebral disc degeneration (IDD). METHODS: NPCs were extracted from enucleated intervertebral discs of IDD patients, and the senescence, apoptosis, and extracellular matrix (ECM) synthesis levels of cells were confirmed by ß-galactosidase (SA-ß-gal), Western blot, and measurement of superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione (GSH). The microarray expression profile of GSE56081 was downloaded to screen differentially expressed mRNAs. CO-IP and ubiquitination assays were used to determine the targeted regulation of XIAP by SIAH1. Methylation of mRNA was verified by m6A RIP and actinomycin D assays. RESULTS: NPCs extracted from the enucleated intervertebral discs of IDD patients exhibited marked senescence, apoptosis, elevated levels of inflammation, and decreased ECM synthesis. The expression of SIAH1 was significantly elevated in NPCs of IDD patients, and SIAH1 knockdown reversed senescence, apoptosis, elevated levels of inflammation, and decreased ECM synthesis in NPCs of IDD patients. CO-IP and ubiquitination assays indicated that SIAH1 can target and ubiquitinate XIAP. Besides, MeRIP-qPCR and actinomycin experiments showed that METTL3-mediated m6A can methylate SIAH1 mRNA. CONCLUSION: In IDD patients, SIAH1 can target and ubiquitinate XIAP, thereby mediating senescence, apoptosis, increased inflammation, and decreased ECM synthesis of NPCs, while METTL3-mediated m6A can methylate SIAH1 mRNA, producing harmful effects.

Naringin Inhibits Apoptosis Induced by Cyclic Stretch in Rat Annular Cells and Partially Attenuates Disc Degeneration by Inhibiting the ROS/NF-κB Pathway.

Oxidative stress and apoptosis play important roles in the pathogenesis of various degenerative diseases. Previous studies have shown that naringin can exert therapeutic effects in multiple degenerative diseases by resisting oxidative stress and inhibiting apoptosis. Although naringin is effective in treating degenerative disc disease, the underlying mechanism remains unclear. This study is aimed at investigating the effects of naringin on oxidative stress, apoptosis, and intervertebral disc degeneration (IVDD) induced by cyclic stretch and the underlying mechanisms in vitro and in vivo. Abnormal cyclic stretch was applied to rat annulus fibrosus cells, which were then treated with naringin, to observe the effects of naringin on apoptosis, oxidative stress, mitochondrial function, and the nuclear factor- (NF-) κB signaling pathway. Subsequently, a rat model of IVDD induced by dynamic and static imbalance was established to evaluate the effects of naringin on the degree of degeneration (using imaging and histology), apoptosis, and oxidative stress in the serum and the intervertebral disc. Naringin inhibited the cyclic stretch-induced apoptosis of annulus fibrosus cells, reduced oxidative stress, improved mitochondrial function, enhanced the antioxidant capacity, and suppressed the activation of the NF-κB signaling pathway. Additionally, it reduced the degree of IVDD (evaluated using magnetic resonance imaging) and the level of oxidative stress and inhibited apoptosis and p-P65 expression in the intervertebral discs of rats. Thus, naringin can inhibit cyclic stretch-induced apoptosis and delay IVDD, and the underlying mechanism may be related to the inhibition of oxidative stress and activation of the NF-κB signaling pathway. Naringin may be an effective drug for treating degenerative disc disease.

GATA4 promotes the senescence of nucleus pulposus cells via NF-κB pathway.

PURPOSE: Cell senescence plays a vital role in intervertebral disc degeneration. The regulatory mechanism of the cellular senescence of nucleus pulposus cells has not been fully elucidated. A recent study identified GATA4 as an emerging regulator of IMR90 cellular senescence. However, whether GATA4 controls senescence in nucleus pulposus cells still needs to be explored. METHODS: Nucleus pulposus cells were exposed to acidified medium mimic the acid environment of intervertebral disc degeneration. RESULTS: We found that GATA4 protein expression was significantly upregulated in older rats and nucleus pulposus cells undergoing stress-induced aging. Moreover, the data indicated that inhibition of GATA4 significantly inhibited the senescence of nucleus pulposus cells cultured under acidic conditions and that over expression of GATA4 promoted a senescence phenotype. The NF-κB pathway has been confirmed in this study to play a role in the regulation of nucleus pulposus cell senescence by GATA4. By using the NF-κB pathway inhibitor, PDTC (100 µmol/L), significantly decreased the IL-6, matrix metallopeptidase (MMP)-2, MMP-3, MMP-9, MMP-13, ADAM metallopeptidase with thrombospondin type 1 motif (ADAMTS)-4, ADAMTS-5 expression level, and increased Aggrecan and typeⅡcollagen expression level in GATA4 transfected nucleus pulposus cells compared with the group in the absence of PDTC. CONCLUSION: This outcome suggested that GATA4 might play a significant role in nucleus pulposus cell senescence through the NF-κB signaling pathway, and GATA4 is a promising target for intervertebral disc degeneration treatment in the future.

lncRNA LINC00284 promotes nucleus pulposus cell proliferation and ECM synthesis via regulation of the miR­205­3p/Wnt/ß­catenin axis.

Intervertebral disc degeneration (IDD) is a leading cause of degenerative spinal disease. Long non­coding RNA (lncRNA) LINC00284 is overexpressed in multiple types of cancer and promotes cancer cell proliferation and inhibits apoptosis; however, its role in human IDD and nucleus pulposus (NP) remain unclear. In the present study, intervertebral disc (IVD) tissues were collected from IDD patients for detection of LINC00284 expression using reverse transcription­quantitative PCR, the binding effect between miR­205­3p and LINC00284 was validated by dual­luciferase reporter assay. miR­205­3p and small interfering RNA (siRNA) was used for LINC00240 knockdown to investigate the proliferation, apoptosis of cells in the NP cells measured by Cell Counting Kit (CCK)­8 assay and Annexin V­FITC/Propidium Iodide (PI) staining with flow cytometry receptivity. IDD animal models were constructed for in vivo study of the role LINC00284 in IDD improvement. The results showed that LINC00284 expression was upregulated in IDD tissue and IL­1ß­induced NP cells. LINC00284 knockdown resulted in an increase in IL­1ß­induced NP cell proliferation, a decrease in apoptosis and matrix metalloproteinase­3 expression and an increase in expression of extracellular matrix (ECM) markers aggrecan and collagen II. In vivo experiments and histomorphometric analysis confirmed the protective effect of LINC00284 knockdown in IDD. LINC00284 was also shown to be a target of microRNA (miR)­205­3p, and there was a negative correlation between LINC00284 and miR­205­3p levels in IDD tissue. Additionally, LINC00284 knockdown or miR­205­3p upregulation resulted in inhibition of Wnt/ß­catenin signaling and subsequent degradation of the ECM. The present study demonstrated that LINC00284 activated the Wnt/ß­catenin signaling via sponging miR­205­3p, resulting in inhibition of NP cell proliferation and ECM synthesis. These results suggested that targeting LINC00284 to rescue miR­205­3p expression may be a potential method for IDD management.

Tetrandrine reduces oxidative stress, apoptosis, and extracellular matrix degradation and improves intervertebral disc degeneration by inducing autophagy.

Tetrandrine (TET) was reported to be an autophagy agonist, and the activating autophagy could delay intervertebral disc degeneration (IDD). Our study focused on exploring whether TET attenuated tert butyl hydrogen peroxide (TBHP)-induced nucleus pulposus (NP) cell injury and delayed rat IDD by inducing autophagy. In vitro, cytotoxicity was detected by MTT assay, ROS was measured with DCFH-DA probe, MDA, and SOD content was evaluated through ELISA, NP cell apoptosis was tested by flow cytometry, protein expression was detected by Western blot, in particular, LC3 expression was assessed by immunofluorescence. In vivo, pathological changes were estimated by HE and safranin-O staining, related protein expression was measured by immunohistochemistry, and the apoptosis was detected by TUNEL. Compared with the control group, oxidative stress, apoptosis, and extracellular matrix (ECM) degradation were increased, the expression of cleaved caspase-3,9, aggrecan and collagen II were reduced, and the expression of MMP13 and ADAMTS5 were up-regulated in TBHP-treated NP cells. Moreover, TET could reverse the effect of TBHP on NP cells. Further, TET enhanced autophagy in NP cells by amplifying the LC3 II/LC3 I/ratio and reducing p62 expression, which attenuated oxidative stress, apoptosis, and ECM degradation in TBHP-treated NP cells. In addition, in vivo, TET delayed rat IDD, increased the expression of LC3 and collagen II, and weakened apoptosis. TET inhibited oxidative stress, apoptosis, and ECM degradation in TBHP-treated NP cells by inducing autophagy, and alleviated IDD. These indicated that TET might be a potential candidate drug for the treatment of IDD.

High hydrostatic pressure (30 atm) enhances the apoptosis and inhibits the proteoglycan synthesis and extracellular matrix level of human nucleus pulposus cells via promoting the Wnt/ß-catenin pathway.

Hydrostatic pressure is known to regulate bovine nucleus pulposus cell metabolism, but its mechanism in human nucleus pulposus cells (HNPCs) remains obscure, which attracts our attention and becomes the focus in this study. Specifically, HNPCs were treated with SKL2001 (an agonist in the Wnt/ß-catenin pathway) or XAV-939 (an inhibitor of the Wnt/ß-catenin pathway), and pressurized under the hydrostatic pressure of 1, 3 and 30 atm. The viability, apoptosis and proteoglycan synthesis of treated HNPC were assessed by CCK-8, flow cytometry and radioisotope incorporation assays. The levels of extracellular matrix, Collagen-II, matrix metalloproteinase 3 (MMP3), Wnt-3a and ß-catenin were measured by toluidine blue staining, immunocytochemistry and Western blot. Appropriate hydrostatic stimulation (3 atm) enhanced the viability and proteoglycan synthesis yet inhibited the apoptosis of HNPCs, which also up-regulated extracellular matrix and Collagen-II levels, and down-regulated MMP3, Wnt-3a and ß-catenin levels in treated HNPCs. Furthermore, high hydrostatic pressure (30 atm) inhibited the viability and proteoglycan synthesis, and promoted the morphological change and apoptosis of HNPCs, which also down-regulated extracellular matrix and Collagen-II levels and up-regulated MMP3, Wnt-3a and ß-catenin levels. Besides, SKL2001 reversed the effects of hydrostatic pressure (3 atm) on inhibiting Wnt-3a, ß-catenin, and MMP3 levels and promoting Collagen-II level in HNPC; whereas, XAV-939 reversed the effects of high hydrostatic pressure (30 atm) on promoting MMP3, Wnt-3a, and ß-catenin levels and inhibiting Collagen-II level and proteoglycan synthesis of HNPCs. Collectively, high hydrostatic pressure promoted the apoptosis and inhibited the viability of HNPCs via activating the Wnt/ß-catenin pathway.

The protective effects of dezocine on interleukin-1ß-induced inflammation, oxidative stress and apoptosis of human nucleus pulposus cells and the possible mechanisms.

Intervertebral disc degeneration (IDD) is a natural problem linked to the inflammation. We aimed to investigate the role of dezocine (DEZ) in the development of IDD. Human nucleus pulposus cells (HNPCs) induced by interleukin (IL)-1ß was used as a cellular model of IDD. After treatment with DEZ, HNPCs viability was evaluated with a CCK-8 assay. Then, the levels of inflammatory factors, including IL-6 and tumor necrosis factor-α (TNF-α), and oxidative stress-related markers, including reactive oxygen species (ROS), malondialdehyde (MDA) and reduced glutathione (GSH), were tested by RT-qPCR or kits. TUNEL staining was employed to detect cell apoptosis and Western blot was used to determine the expression of proteins related to inflammation, oxidative stress, apoptosis, endoplasmic reticulum stress (ERS) and MAPK signaling. Afterward, PMA, a MAPK signaling pathway agonist, was adopted for exploring the regulatory effects of DEZ on MAPK pathway. Results indicated that DEZ enhanced cell viability of HNPCs after IL-1ß exposure. DEZ alleviated the inflammation and oxidative stress, evidenced by decreased levels of IL-6, TNF-α, ROS, MDA, p-NF-κB p65, NF-κB p65 in nucleus, cox-2 and increased levels of NF-κB p65 in cytoplasm, GSH, SOD1 and SOD2. Moreover, DEZ notably inhibited IL-1ß-induced apoptosis of HNPCs. Furthermore, DEZ suppressed the levels of ERS-related proteins. The levels of related proteins in MAPK signaling including p-P38 and p-ERK1/2 were remarkably reduced after DEZ administration. By contrast, PMA crippled the impacts of DEZ on inflammation, oxidative stress and apoptosis of HNPCs induced by IL-1ß. Collectively, DEZ ameliorates IL-1ß-induced HNPCs injury via inhibiting MAPK signaling.

CircRNA RERE Promotes the Oxidative Stress-Induced Apoptosis and Autophagy of Nucleus Pulposus Cells through the miR-299-5p/Galectin-3 Axis.

Intervertebral disc degeneration (IDD) is widely accepted as a cause of low back pain and related degenerative musculoskeletal disorders. Nucleus pulposus (NP) cell loss is closely related to IDD progression. Thus, investigating the specifically targeted therapeutic agents against NP cell loss depends on understanding the molecular mechanisms. In this study, human NP cells were treated with hydrogen peroxide (H2O2). Cell viability was assessed by using the Cell Counting Kit-8 (CCK-8) kit. The expression of circRNA arginine-glutamic acid dipeptide repeats (hsa_circ_RERE) and miR-299-5p was analyzed by real-time quantitative PCR. Western blot analysis was used to assess the protein expression levels. The autophagy levels in NP cells were detected by using an electronic microscope, LC3B protein immunofluorescence, and western blot. The apoptosis levels of NP cells were detected by flow cytometry and western blot. Dual-luciferase reporter assay analyzed the miR-299-5p bound to circ_RERE and galectin-3. Our results revealed that H2O2 significantly inhibited the viability of NP cells, promoted apoptosis and autophagy, and upregulated galectin-3 expression. miR-299-5p was reduced in IDD and H2O2-induced NP cells. The overexpression of miR-299-5p promoted cell viability and attenuated apoptosis and autophagy under H2O2 treatment. Besides, circ_RERE was upregulated in IDD and H2O2-induced NP cells. However, knockdown of circ_RERE reversed the effects of miR-299-5p overexpression on cell viability, apoptosis, and autophagy in NP cells. We propose that circ_RERE promotes the H2O2-induced apoptosis and autophagy of NP cells through the miR-299-5p/galectin-3 axis and may provide a new target for the clinical treatment of IDD.

Exosomes Derived from Bone Mesenchymal Stem Cells Alleviate Compression-Induced Nucleus Pulposus Cell Apoptosis by Inhibiting Oxidative Stress.

Oxidative stress is relevant in compression-induced nucleus pulposus (NP) cell apoptosis and intervertebral disc (IVD) degeneration. Exosomes derived from bone mesenchymal stem cells (BMSCs-Exos) are key secretory products of MSCs, with important roles in tissue regeneration. This research is aimed at studying the protective impact of BMSCs-Exos on NP cell apoptosis caused by compression and investigating the underlying mechanisms. Our results indicated that we isolated BMSCs successfully. Exosomes were isolated from the BMSCs and found to alleviate the inhibitory effect that compression has on proliferation and viability in NP cells, decreasing the toxic effects of compression-induced NP cells. AnnexinV/PI double staining and TUNEL assays indicated that the BMSCs-Exos reduced compression-induced apoptosis. In addition, our research found that BMSCs-Exos suppressed compression-mediated NP oxidative stress by detecting the ROS and malondialdehyde level. Furthermore, BMSCs-Exos increased the mitochondrial membrane potential and alleviated compression-induced mitochondrial damage. These results indicate that BMSCs-Exos alleviate compression-mediated NP apoptosis by suppressing oxidative stress, which may provide a promising cell-free therapy for treating IVD degeneration.

The REDD1/TXNIP Complex Accelerates Oxidative Stress-Induced Apoptosis of Nucleus Pulposus Cells through the Mitochondrial Pathway.

The death of nucleus pulposus (NP) cells is an important cause of intervertebral disc (IVD) degeneration. Redox disturbance caused by dysfunctional mitochondria has been considered as a vital risk for NP cell survival. It is valuable to identify key proteins maintaining mitochondrial function in NP cells. A previous study found that regulated in development and DNA damage response 1 (REDD1) are upregulated during intervertebral disc degeneration and that REDD1 can cause NP cell apoptosis. Thus, the present study further explores the effect of REDD1 on IVD degeneration. Our results showed that REDD1 promotes NP cell apoptosis via the mitochondrial pathway. Importantly, REDD1 formed a complex with TXNIP to strengthen its own action, and the combination was consolidated under H2O2-induced oxidative stress. The combined inhibition of the REDD1/TXNIP complex was better than that of REDD1 or TXNIP alone in restoring cell proliferation and accelerating apoptosis. Moreover, p53 acts as the transcription factor of REDD1 to regulate the REDD1/TXNIP complex under oxidative stress. Altogether, our results demonstrated that the REDD1/TXNIP complex mediated H2O2-induced human NP cell apoptosis and IVD degeneration through the mitochondrial pathway. Interferences on these sites to achieve mitochondrial redox homeostasis may be a novel therapeutic strategy for oxidative stress-associated IVD degeneration.