Nucleus Pulposus Cells In Intervertebral Disc Degeneration Research Articles

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.

Exploration of the mode of death and potential death mechanisms of nucleus pulposus cells.

Intervertebral disc degeneration (IVDD) is a common chronic orthopaedic disease in orthopaedics that imposes a heavy economic burden on people and society. Although it is well established that IVDD is associated with genetic susceptibility, ageing and obesity, its pathogenesis remains incompletely understood. Previously, IVDD was thought to occur because of excessive mechanical loading leading to destruction of nucleus pulposus cells (NPCs), but studies have shown that IVDD is a much more complex process associated with inflammation, metabolic factors and NPCs death and can involve all parts of the disc, characterized by causing NPCs death and extracellular matrix (ECM) degradation. The damage pattern of NPCs in IVDD is like that of some programmed cell death, suggesting that IVDD is associated with programmed cell death. Although apoptosis and pyroptosis of NPCs have been studied in IVDD, the pathogenesis of intervertebral disc degeneration can still not be fully elucidated by using only traditional cell death modalities. With increasing research, some new modes of cell death, PANoptosis, ferroptosis and senescence have been found to be closely related to intervertebral disc degeneration. Among these, PANoptosis combines essential elements of pyroptosis, apoptosis and necroptosis to form a highly coordinated and dynamically balanced programmed inflammatory cell death process. Furthermore, we believe that PANoptosis may also crosstalk with pyroptosis and senescence. Therefore, we review the progress of research on multiple deaths of NPCs in IVDD to provide guidance for clinical treatment.

Identification and validation of ferroptosis-related lncRNA signature in intervertebral disc degeneration

Low back pain influences people of every age and is one of the major contributors to the global cost of illness. Intervertebral disc degeneration (IVDD) is a major contributor to low back pain, but its pathogenesis is unknown. Recently, ferroptosis has been shown to have a substantial role in modulating IVDD progression. However, the function of ferroptosis-related long non-coding RNAs (lncRNAs) has rarely been reported in IVDD. Consequently, the research was conducted to explore the ferroptosis-related lncRNA signature in the IVDD occurrence and development. We analyzed two datasets (GSE167199 and GSE167931) archived in the NCBI Gene Expression Omnibus (GEO) public database. We screened differentially expressed genes (DEGs) and differentially expressed lncRNAs (DELncs) in these datasets using the limma package. Ferroptosis-related genes (FRGs) were derived from the FerrDb V2 website and the intersection of DEGs and FRGs was considered as differentially expressed ferroptosis-related genes (DFGs). These genes were then subjected to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis. Correlations between DFGs and DELncs were shown by Pearson test to determine differential expression of ferroptosis-related lncRNAs. The Pearson test showed that CPEB1-HTR2A-AS1 and ACSL3-DNAJC27-AS1 pairs had correlation coefficients over 0.9. Twenty ferroptosis-related lncRNAs were identified and validated in IVDD. Eight of these lncRNAs were upregulated in IVDD nucleus pulposus cells, including HTR2A-AS1, MIF-AS1, SLC8A1-AS1, LINC00942, DUXAP8, LINC00161, LUCAT1 and LINC01615. Twelve were downregulated in IVDD nucleus pulposus cells, including DNAJC27-AS1, H19, LINC01588, LINC02015, FLNC1, CARMN, PRKG1-AS1, APCDD1L-DT, LINC00839, LINC00536, LINC00710 and LINC01535. Eighteen of the 20 lncRNAs (excluding H19 and LUCAT1) were identified as ferroptosis-related lncRNAs for the first time and verified in IVDD. We have identified a ferroptosis-related lncRNA signature involved in IVDD and revealed a close relationship between CPEB1 and HTR2A-AS1, and between ACSL3 and DNAJC27-AS1. Our findings indicate that ferroptosis-related lncRNAs are a new target set for the early detection and therapy of IVDD.

Necroptosis of nucleus pulposus cells involved in intervertebral disc degeneration through MyD88 signaling.

Background contextLow back pain, affecting nearly 40% of adults, mainly results from intervertebral disc degeneration (IVDD), while the pathogenesis of IVDD is still not fully elucidated. Recently, some researches have revealed that necroptosis, a programmed necrosis, participated in the progression of IVDD, nevertheless, the underlying mechanism remains unclear.PurposeTo study the mechanism of necroptosis of Nucleus Pulposus (NP) cells in IVDD, focusing on the role of MyD88 signaling.Study designThe expression and co-localization of necroptotic indicators and MyD88 were examined in vivo, and MyD88 inhibitor was applied to determine the role of MyD88 signaling in necroptosis of NP cells in vitro.MethodsHuman disc specimens were collected from patients receiving diskectomy for lumbar disc herniation (LDH) or traumatic lumbar fractures after MRI scanning. According to the Pfirrmann grades, they were divided into normal (Grades 1, 2) and degenerated groups (4, 5). Tissue slides were prepared for immunofluorescence to assess the co-localization of necroptotic indicators (RIP3, MLKL, p-MLKL) and MyD88 histologically. The combination of TNFα, LPS and Z-VAD-FMK was applied to induce necroptosis of NP cells. Level of ATP, reactive oxygen species (ROS), live-cell staining and electron microscope study were employed to study the role of MyD88 signaling in necroptosis of NP cells.Results In vivo, the increased expression and co-localization of necroptotic indicators (RIP3, MLKL, p-MLKL) and MyD88 were found in NP cells of degenerated disc, while very l low fluorescence intensity in tissue of traumatic lumbar fractures. In vitro, the MyD88 inhibitor effectively rescued the necroptosis of NP cells, accompanied by increased viability, ATP level, and decreased ROS level. The effect of MyD88 inhibition on necroptosis of NP cells was further confirmed by ultrastructure of mitochondria shown by Transmission Electron Microscope (TEM).ConclusionOur results indicated that the involvement of MyD88 signaling in the necroptosis of NP cells in IVDD, which will replenish the pathogenesis of IVDD and provide a novel potential therapeutic target for IVDD.

Panax notoginseng saponin reduces IL-1β-stimulated apoptosis and endoplasmic reticulum stress of nucleus pulposus cells by suppressing miR-222-3p.

BackgroundIt is well documented that the malignant biological behaviors of nucleus pulposus cells (NPCs) could trigger intervertebral disc degeneration (IDD). Panax notoginseng saponin (PNS) is a traditional Chinese medicine that inhibits osteoclastogenesis. However, its effects on the phenotypes of NPCs in IDD remains largely unknown. This study sought to examine the role of PNS in IDD and its regulatory mechanism.MethodsFirst, human NPCs (hNPCs) were treated with interleukin-1 beta (IL-1β) to induce an IDD cell model. Cell proliferation and apoptosis were estimated by Cell Counting Kit-8 (CCK-8) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays. Western blot was employed to examine the levels of proteins related to apoptosis and endoplasmic reticulum (ER) stress. Enzyme-linked immunosorbent assays (ELISAs) were used to test inflammatory factors levels. Immunofluorescence (IF) assays were used to determine the nuclear translocation of nuclear factor-kappa beta (NF-κB) p65. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect miR-222-3p expression.ResultsWe discovered that PNS enhanced the viability but reduced the apoptosis, inflammation, and ER stress response of IL-1β-induced hNPCs in a concentration-dependent manner. Additionally, PNS significantly reduced miR-222-3p expression in the IL-1β-induced hNPCs. Notably, these PNS effects were reversed by the upregulation of miR-222-3p.ConclusionsIn summary, PNS appears to facilitate the proliferation and attenuate the apoptosis, inflammatory response, and ER stress response of IL-1β-induced hNPCs by inhibiting miR-222-3p expression. Our findings provide a theoretical basis for a novel drug application in IDD research.

Cyanidin attenuates the apoptosis of rat nucleus pulposus cells and the degeneration of intervertebral disc via the JAK2/STAT3 signal pathway in vitro and in vivo

Context Cyanidin has been shown to have therapeutic potential in osteoarthritis. However, it is unclear whether cyanidin prevents the progression of intervertebral disc degeneration (IVDD). Objective This study evaluates the effects of cyanidin on IVDD in vitro and in vivo. Materials and methods Nucleus pulposus cells (NPCs) isolated from lumbar IVD of 4-week-old male Sprague-Dawley (SD) rats were exposed to 20 ng/mL IL-1β, and then treated with different doses (0-120 µM) of cyanidin for 24 h. SD rats were classified into three groups (n = 8) and treated as follows: control (normal saline), IVDD (vehicle), IVDD + cyanidin (50 mg/kg). Cyanidin was administered intraperitoneally for 8 weeks. Results The IC50 of cyanidin for NPCs was 94.78 µM, and cyanidin had no toxicity at concentrations up to 500 mg/kg in SD rats. Cyanidin inhibited the apoptosis of NPCs induced by IL-1β (12.73 ± 0.61% vs. 18.54 ± 0.60%), promoted collagen II (0.82-fold) and aggrecan (0.81-fold) expression, while reducing MMP-13 (1.02-fold) and ADAMTS-5 (1.40-fold) expression. Cyanidin increased the formation of autophagosomes in IL-1β-induced NPCs, and promoted LC3II/LC3I (0.83-fold) and beclin-1 (0.85-fold) expression, which could be reversed by chloroquine. Cyanidin inhibited the phosphorylation of JAK2 (0.47-fold) and STAT3 (0.53-fold) in IL-1β-induced NPCs. The effects of cyanidin could be enhanced by AG490. Furthermore, cyanidin mitigated disc degeneration in IVDD rats in vivo. Discussion and conclusions Cyanidin improved the function of NPCs in IVDD by regulating the JAK2/STAT3 pathway, which may provide a novel alternative strategy for IVDD. The mechanism of cyanidin improving IVDD still needs further work for in-depth investigation.

MiR-98 Protects Nucleus Pulposus Cells against Apoptosis by Targeting TRAIL in Cervical Intervertebral Disc Degeneration.

The excessive apoptosis of nucleus pulposus (NP) cells is a major risk factor in the progress of cervical intervertebral disc degeneration (IVDD). In this study, we investigated the impact of miR-98 on apoptosis of NP cells and the potential molecular mechanisms. Lipopolysaccharide (LPS) was used to establish an NP cell IVDD model. The sponging effect of miR-98 on TRAIL 3′UTR was predicted by ENCORI and assessed by the dual-luciferase reporter gene system. The expression levels of miR-98, TRAIL, and TRAIL pathway-related genes were tested by qRT-PCR, Western blot, and immunofluorescence analysis. Cell apoptosis was analyzed by Hoechst 33258 staining and flow cytometry. Cell viability was analyzed by MTT assay. It was found that the expression level of miR-98 was downregulated, while the level of TRAIL was upregulated in IVDD tissues, and their levels were negatively and positively associated with the clinical MRI grade, respectively. The LPS treatment resulted in a significant decrease of the miR-98 expression level and an increase of the TRAIL expression level in NP cells. miR-98 reduced NP cell apoptosis under LPS treatment in vitro. miR-98 directly targeted TRAIL. Moreover, the mRNA and protein levels of DR5, FADD, cleaved caspase8, cleaved caspase3, and cleaved PARP were downregulated by miR-98 overexpression. Overexpression of TRAIL partially reversed the suppressive roles of miR-98 on cell apoptosis and activation of the TRAIL pathway. We concluded that miR-98 inhibited apoptosis of NP cells by inactivating the TRAIL pathway via targeting TRAIL in IVDD NP cells. These results indicated that miR-98 might be a therapeutic target for IVDD.

MicroRNA-155-3p attenuates intervertebral disc degeneration via inhibition of KDM3A and HIF1α.

Intervertebral disc degeneration (IDD) is a key element resulting in low back pain, but the mechanisms underlying IDD remain largely unknown. The purpose of the study was to investigate the influence of microRNA-155-3p (miR-155-3p) on proliferation and autophagy of nucleus pulposus (NP) cells in IDD with the involvement of hypoxia-inducible factor 1 α (HIF1α)/histone lysine demethylase 3A (KDM3A) axis. IDD NP tissues of patients with lumbar disc herniation and traumatic intervertebral disc NP tissues from patients with traumatic lumbar fracture were collected. Apoptosis in NP tissues was observed, and autophagy marker proteins in NP tissues were detected. NP cells in IDD were transfected with miR-155-3p mimic or KDM3A-siRNA to explore their roles in cell proliferation, autophagy and apoptosis. MiR-155-3p, KDM3A and HIF1α expression in NP tissues and cells were detected. Decreased miR-155-3p, and elevated HIF1α and KDM3A were presented in NP tissues and cells of IDD. Elevated miR-155-3p or silenced KDM3A promoted the proliferation and autophagy, and inhibited the apoptosis of NP cells of IDD. Moreover, elevated miR-155-3p decreased KDM3A and HIF1α expression, while silenced KDM3A decreased HIF1α expression in NP cells with IDD. The study concludes that up-regulated miR-155-3p or silenced KDM3A promotes the proliferation, autophagy, and restrains the apoptosis of NP cells of IDD via inhibition of HIF1α, which may be a promising approach for the treatment of IDD.

Anisodamine Maintains the Stability of Intervertebral Disc Tissue by Inhibiting the Senescence of Nucleus Pulposus Cells and Degradation of Extracellular Matrix via Interleukin-6/Janus Kinases/Signal Transducer and Activator of Transcription 3 Pathway.

Objectives: Anisodamine (ANI) has been used to treat a variety of diseases. However, the study of ANI in intervertebral disc degeneration (IVDD) is unclear. This study investigated the effects of ANI on degenerative nucleus pulposus cells (NPCs) and IVDD rats, and its possible mechanisms. Methods: Human nucleus pulposus cells (HNPCs) were treated with IL-1β (20 ng/ml) to simulate IVDD, and an IVDD rat model was constructed. IL-1β-induced HNPCs were treated with different concentrations (10, 20, or 40 μM) of ANI, and IVDD rats were also treated with ANI (1 mg/kg). Results: ANI treatment significantly reduced the apoptosis, caspase-3 and SA-β-gal activities, and p53 and p21 proteins expression, while promoted telomerase activity and aggrecan and collagen II synthesis in IL-1β-induced HNPCs. Moreover, the introduction of ANI inhibited the expression of IL-6, phosphorylation of JAK and STAT3, and nuclear translocation of p-STAT3 in Degenerated HNPCs. Additionally, the application of ANI abolished the effects of IL-6 on apoptosis, SA-β-gal and telomerase activity, and the expression of p53, p21, aggrecan and collagen II proteins in degenerated HNPCs. Simultaneously, ANI treatment enhanced the effects of AG490 (inhibitor of JAK/STAT3 pathway) on IL-1β-induced apoptosis, senescence and ECM degradation in HNPCs. Furthermore, ANI treatment markedly inhibited the apoptosis and senescence in the nucleus pulposus of IVDD rats, while promoted the synthesis of aggrecan and collagen II. ANI treatment obviously inhibited JAK and STAT3 phosphorylation and inhibited nuclear translocation of p-STAT3 in IVDD rats. Conclusion: ANI inhibited the senescence and ECM degradation of NPCs by regulating the IL-6/JAK/STAT3 pathway to improve the function of NPCs in IVDD, which may provide new ideas for the treatment of IVDD.

LncRNA SNHG6 can regulate the proliferation and apoptosis of rat degenerate nucleus pulposus cells via regulating the expression of miR-101-3p.

Intervertebral disc (IVD) degeneration (IDD) is a well-known consequence of low back pain, as characterized by aberrant cell proliferation and apoptosis of nucleus pulposus (NP) cells. In the present study, we aimed to investigate the effect of lncRNA small nucleolar RNA host gene 6 (SNHG6) on deregulated functions of degenerative NP cells. After the establishment of rat IDD models, the mRNA and protein levels of collagen-I (Col-I) and collagen II (Col-II), and mRNA level of SNHG6 were detected by using reverse transcription quantitative Real Time-PCR (RT-qPCR) and Western blot. We further investigated the role and molecular mechanisms of SNHG6 by overexpressing or silencing it in degenerative NP cells. Cell proliferation was measured by MTT assay and EdU staning, and apoptosis was measured by flow cytometry. The target of SNHG6 was identified by starBase and Dual-Luciferase reporter assay. Upregulation of SNHG6 was found in IDD NP cells than in normal cells, associated with higher level of Col-I and lower level of Col-II. Overexpression of SNHG6 inhibited cell proliferation and enhanced apoptosis, accompanied by increased expression of Bax, caspase-3, and p21, as well as decreased expression of Bcl-2, which was in reverse to the treatment of SNHG6 silencing. Moreover, miR-101-3p was indicated as a target of SNHG6, and inhibition of miR-101-3p reversed the effects on proliferation and apoptosis induced by SNHG6. SNHG6 suppressed cell proliferation and induced apoptosis by increasing expression of Bax, caspase-3, p21 and decreasing Bcl-2 through targeting miR-101-3p, which suggested that SNHG6 could be a potential target in the treatment of IDD.

RETRACTED: circ_001653 Silencing Promotes the Proliferation and ECM Synthesis of NPCs in IDD by Downregulating miR-486-3p-Mediated CEMIP

Functional changes of nucleus pulposus cells (NPCs) are considered to be the initiating factors of intervertebral disc degeneration (IDD). In this study, we investigated whether circular RNA homo sapiens (hsa)_circ_001653 (circ_001653) could bind to microRNA-486-3p (miR-486-3p) to regulate the biological properties of NPCs and the synthesis of extracellular matrix (ECM) in IDD. Initially, circ_001653 was highly expressed in isolated NPCs and degenerative NP tissues in close relation to the severity of IDD. To evaluate the effects of circ_001653 on cellular processes, we performed experiments in vitro and in vivo with altered expression of circ_001653 and miR-486-3p. An increased expression of circ_001653 in the NPCs and the degenerative NP tissues was directly associated with elevated apoptosis and an imbalance between anabolic and catabolic factors of the ECM. miR-486-3p regulated NPC proliferation and inhibited the expression of CEMIP, the cell migration-inducing hyaluronan binding protein. circ_001653 regulated miR-486-3p expression, functioning in NPCs to upregulate CEMIP, whereas circ_001653 silencing alleviated IDD in the mouse model. Altogether, circ_001653 downregulation could potentially alleviate NPC apoptosis and the metabolic imbalance of the ECM through the miR-486-3p/CEMIP axis. These mechanistic insights may present new therapeutic targets for the treatment of IDD.