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Low back pain (LBP) is a significant global health issue, contributing to disability and socioeconomic burdens worldwide. The degeneration of the human intervertebral disc (IVD) is a critical factor in the pathogenesis of LBP. Recent studies have emphasized the significance of a specific set of genes and extracellular matrix (ECM) in IVD health. In particular, Noggin has emerged as a critical gene due to its high expression levels in healthy nucleus pulposus cells (NPCs) observed in our previous research. In this study, it was hypothesized that decreased Noggin expression in NPCs is associated with IVD degeneration and contributes to LBP development. A lentivirus-mediated RNAi was applied to knock down Noggin expression in primary NPCs from six human donors. The NPCs after transduction were evaluated through cell viability analysis, XTT assay, and cell apoptosis analyses. After two weeks, a colony formation assay was used to examine the anchor-independent growth ability of transduced cells. At the transcript level, anabolic and catabolic markers were quantified using RT-qPCR. The results demonstrated that lentivirus-mediated downregulation of Noggin significantly inhibited cell proliferation, reduced cell viability, and suppressed colony formation, while inducing apoptosis in human NPCs in vitro. Notably, it disrupted cellular anabolic processes and promoted catabolic activity in human NPCs post-transduction. Our findings indicated that the degeneration of human IVD is possibly related to decreased Noggin expression in NPCs. This research provides valuable insights into the role of Noggin in IVD homeostasis and its implications in LBP treatment. [ABSTRACT FROM AUTHOR]

Low back pain is a common clinical symptom of intervertebral disc degeneration (IVDD), which seriously affects the quality of life of the patients. The abnormal apoptosis and senescence of nucleus pulposus cells (NPCs) play important roles in the pathogenesis of IVDD. PHLDA2 is an imprinted gene related to cell apoptosis and tumour progression. However, its role in NPC degeneration is not yet clear. Therefore, this study was set to explore the effects of PHLDA2 on NPC senescence and apoptosis and the underlying mechanisms. The expression of PHLDA2 was examined in human nucleus pulposus (NP) tissues and NPCs. Immunohistochemical staining, magnetic resonance imaging imaging and western blot were performed to evaluate the phenotypes of intervertebral discs. Senescence and apoptosis of NPCs were assessed by SA‐β‐galactosidase, flow cytometry and western blot. Mitochondrial function was investigated by JC‐1 staining and transmission electron microscopy. It was found that the expression level of PHLDA2 was abnormally elevated in degenerated human NP tissues and NPCs. Furthermore, knockdown of PHLDA2 can significantly inhibit senescence and apoptosis of NPCs, whereas overexpression of PHLDA2 can reverse senescence and apoptosis of NPCs in vitro. In vivo experiment further confirmed that PHLDA2 knockdown could alleviate IVDD in rats. Knockdown of PHLDA2 could also reverse senescence and apoptosis in IL‐1β‐treated NPCs. JC‐1 staining indicated PHLDA2's knockdown impaired disruption of the mitochondrial membrane potential and also ameliorated superstructural destruction of NPCs as showed by transmission electron microscopy. Finally, we found the PHLDA2 knockdown promoted Collagen‐II expression and suppressed MMP3 expression in NPCs by repressing wnt/β‐catenin pathway. In conclusion, the results of the present study showed that PHLDA2 promotes IL‐1β‐induced apoptosis and senescence of NP cells via mitochondrial route by activating the Wnt/β‐catenin pathway, and suggested that therapy targeting PHLDA2 may provide valuable insights into possible IVDD therapies. [ABSTRACT FROM AUTHOR]

Background: Intervertebral disc degeneration (IDD) progression involves multiple factors, including loss of nucleus pulposus cells and extracellular matrix as the basic pathological mechanism of degeneration, and is closely related to cellular senescence and immune cell infiltration. The aim of study was to identify critical cellular senescence-related genes and immune cell infiltration characteristics in IDD. Methods: Four datasets, including GSE70362, GSE112216, GSE114169, and GSE150408, were downloaded from the Gene Expression Omnibus database. The senescence-related genes were acquired from the CellAge Database and intersected with differentially expressed genes (DEGs) between IDD and control samples for senescence-related DEGs (SRDEGs). Protein-protein interaction (PPI) network analysis was performed to obtain ten hub SRDEGs. A consensus cluster analysis based on these hub genes was performed to divide the patients into clusters. The functional enrichment, and immune infiltration statuses of the clusters were compared. Weighted gene co-expression network analysis was used to identified key gene modules. The overlapping genes from key modules, DEGs of clusters and hub SRDEGs were intersected to obtain potential biomarkers. To verify the expression of potential biomarkers, quantitative polymerase chain reaction (qPCR) and immunohistochemistry were performed by using human intervertebral disc tissues. Results: In the GSE70362 dataset, a total of 364 DEGs were identified, of which 150 were upregulated and 214 were downregulated, and 35 genes were selected as SRDEGs. PPI analysis revealed ten hub SRDEGs and consensus cluster analysis divided the patients into two clusters. Compared to Cluster 2, Cluster 1 was highly enriched in extracellular matrix organization and various metabolic process. The level of Follicular T helper cells in the Cluster 1 was significantly higher than that in the Cluster 2. IGFBP3 and NQO1 were identified as potential biomarkers. The remaining 3 datasets, and the result of qPCR and immunohistochemistry showed that the expression levels of NQO1 and IGFBP3 in the degenerated group were higher than those in the control or treatment groups. Conclusion: Senescence-related genes play a key role in the development and occurrence of IDD. IGFBP3 and NQO1 are strongly correlated with immune infiltration in the IDD and could become novel therapeutic targets that prevent the progression of IDD. [ABSTRACT FROM AUTHOR]

Objective To explore the role and mechanism of exosomes derived from bone marrow mesenchymal stem cells (BMSCs) overexpressing transglutaminase 2 (TGM2) in lactate-mediated oxidative stress-induced apoptosis in nucleus pulposus cells. Methods Twenty 6-week-old male SD rats (350±50 g) were used to extract nucleus pulposus cells. Lactic acid was utilized to establish a model of oxidative stress-induced apoptosis in primary nucleus pulposus cells. Lentiviral vectors carrying TGM2 were employed to transfect BMSCs to overexpress the protein, and then exosomes were extracted from the BMSCs and identified. Subsequently, to investigate the potential mechanisms of TGM2-overexpressing exosomes against oxidative stress-induced apoptosis in nucleus pulposus cells, flow cytometry was used to detect the production of reactive oxygen species (ROS), and Western blotting and immunofluorescence assay were applied to measure the expression levels of Bax, Bcl-2, Cleaved-Caspase3, p-PI3K, p-Akt, n-Nrf2, c-Nrf2 and HO-1, and Nrf2 nuclear translocation. The rate of oxidative stress-induced apoptosis in nucleus pulposus cells and changes in the PI3K/Akt/Nrf2 pathway were assessed in 4 groups of cells (n=3): control group, lactate treatment group, lactate+control exosome group, and lactate+TGM2-overexpressing exosomes group. Results Lactic acid of 10 mmol/L was found to be more effective in inducing oxidative stress-induced apoptosis in nucleus pulposus cells (P < 0.05). Both control BMSCs and TGM2-overexpressing BMSCs could produce exosomes and stably overexpress TGM2 (P < 0.05, P < 0.01). Compared with the lactate treatment group, the nucleus pulposus cells in the lactate+TGM2-overexpressing exosome group and the lactate+control exosome group showed reduced apoptotic rates (P < 0.05), as well as decreased ROS level (P < 0.05). The lactate+TGM2-overexpressing exosome group exhibited better anti-apoptosis and ROS accumulation effects (P < 0.05). Additionally, the expression of Bax and Cleaved-Caspase3 was decreased (P < 0.05), that of Bcl-2 protein was increased (P < 0.05), the phosphorylation of PI3K/Akt was enhanced (P < 0.05), and both Nrf2 nuclear translocation and the expression of the antioxidant stress factor HO-1 were increased in nucleus pulposus cells. The lactic acid + TGM2-overexpressing exosome group showed stronger anti-apoptotic effects and activation of the PI3K/Akt/Nrf2 pathway (P < 0.05). Conclusion Exosomes derived from BMSCs overexpressing TGM2 can inhibit lactate-mediated oxidative stress-induced apoptosis in nucleus pulposus cells, and the mechanism may be related to the activation of the PI3K/Akt/Nrf2 pathway.

Abstract Background Cellular senescence features irreversible growth arrest and secretion of multiple proinflammatory cytokines. Cyclic GMP-AMP synthase (cGAS) detects DNA damage and activates the DNA-sensing pathway, resulting in the upregulation of inflammatory genes and induction of cellular senescence. This study aimed to investigate the effect of cGAS in regulating senescence of nucleus pulposus (NP) cells under inflammatory microenvironment. Methods The expression of cGAS was evaluated by immunohistochemical staining in rat intervertebral disc (IVD) degeneration model induced by annulus stabbing. NP cells were harvested from rat lumbar IVD and cultured with 10ng/ml IL-1β for 48 h to induce premature senescence. cGAS was silenced by cGAS specific siRNA in NP cells and cultured with IL-1β. Cellular senescence was evaluated by senescence-associated beta-galactosidase (SA-β-gal) staining and flow cytometry. The expression of senescence-associated secretory phenotype including IL-6, IL-8, and TNF-a was evaluated by ELISA and western blotting. Results cGAS was detected in rat NP cells in cytoplasm and the expression was significantly increased in degenerated IVD. Culturing in 10ng/ml IL-1β for 48 h induced cellular senescence in NP cells with attenuation of G1-S phase transition. In senescent NP cells the expression of cGAS, p53, p16, NF-kB, IL-6, IL-8, TNF-α was significantly increased while aggrecan and collagen type II was reduced than in normal NP cells. In NP cells with silenced cGAS, the expression of p53, p16, NF-kB, IL-6, IL-8, and TNF-α was reduced in inflammatory culturing with IL-1β. Conclusion cGAS was increased by NP cells in degenerated IVD promoting cellular senescence and senescent inflammatory phenotypes. Targeting cGAS may alleviate IVD degeneration by reducing NP cell senescence.

Cunxin Zhang,1,&ast; Qian Wang,1,&ast; Kang Li,1 Maoqing Fu,1 Kai Gao,2 Chaoliang Lv1 1Department of Spine Surgery, Jining No. 1 People’s Hospital, Jining, 272011, People’s Republic of China; 2Department of Orthopaedics, Jining No. 1 People’s Hospital, Jining, 272011, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Chaoliang Lv, Department of Spine Surgery, Jining No. 1 People’s Hospital, No. 6 Healthy Road, Rencheng District, Jining, 272011, People’s Republic of China, Tel +86 15265775628, Fax +86-0537- 2253252, Email lvchaolianggk@163.com Kai Gao, Department of Orthopaedics, Jining No. 1 People’s Hospital, Jining, No. 6 Healthy Road, Rencheng District, Jining, 272011, People’s Republic of China, Tel +86 15853760330, Fax +86-0537- 2253252, Email gaohaikai88@126.comBackground: Intervertebral disc degeneration (IDD) underlies the pathogenesis of degenerative diseases of the spine; however, its exact molecular mechanism is unclear.Purpose: To explore the molecular mechanism of mechanical pressure (MP)-induced IDD and to assess the role and mechanism of Rosuvastatin (RSV) inhibits MP-induced IDD.Methods: SD rat nucleus pulposus cells (NPCs) were cultured in vitro and an apoptosis model of NPCs was constructed using MP. Proliferative activity, reactive oxygen species content, apoptosis, and wound healing were detected in each group of NPCs, respectively. The expression of relevant proteins was detected by qPCR and Western Blot techniques. 18 SD rats were randomly divided into control, pressure and RSV groups. Elisa, qPCR, Western Blot and immunohistochemical staining techniques were used to detect changes in the content of related proteins in the intervertebral discs of each group. HE staining and Modified Saffron-O and Fast Green Stain Kit were used to assess IDD in each group.Results: MP treatment at 1.0 MPa could significantly induce apoptosis of NPCs after 24 h. MP could significantly inhibit the proliferative activity and wound healing ability of NPCs, and increase the intracellular reactive oxygen species content and apoptosis rate; pretreatment with RSV could significantly activate the Nrf2/HO-1 signaling pathway and reverse the cellular damage caused by MP; when inhibit the Nrf2/HO-1 signaling pathway activation, the protective effect of RSV was reversed. In vivo MP could significantly increase the content of inflammatory factors within the IVD and promote the degradation of extracellular matrix, leading to IDD. When the intervention of RSV was employed, it could significantly activate the Nrf2/HO-1 signaling pathway and improve the above results.Conclusion: RSV may inhibit MP-induced NPCs damage and IDD by activating the Nrf2/HO-1 signaling pathway.Keywords: rosuvastatin, intervertebral disc, nucleus pulposus cells, oxidative stress, nuclear factor E2-related factor 2, heme oxygenase-1, quinone oxidoreductase 1

Nucleus pulposus cells survive in a hypoxic, acidic, nutrient-poor, and hypotonic microenvironment. Consequently, they maintain low proliferation and undergo autophagy to protect themselves from cellular stress. Therefore, we aimed to identify autophagy-related biomarkers involved in intervertebral disc degeneration pathogenesis. Autophagy-related differentially expressed genes were derived from the intersection between the public GSE147383 microarray data set to identify differentially expressed genes and online databases to identify autophagy-related genes. Furthermore, we assessed their biological functions with gene annotation and enrichment analysis in the Metscape portal. Then, the STRING database and Cytoscape software allowed inferring a protein-protein interaction (PPI) network and identifying hub genes. In addition, to predict transcription factors that may regulate the hub genes, we used the GeneMANIA website. Finally, the competing endogenous RNA prediction tools and Cytoscape were also used to construct an mRNA-miRNA-lncRNA network. A total of 123 autophagy-related differentially expressed genes were identified, they were mainly involved in phosphoinositide 3-kinase-Akt signaling, autophagy animal, and apoptosis pathways. Nine were identified as hub genes (PTEN, MYC, CTNNB1, JUN, BECN1, ERBB2, FOXO3, ATM, and FN1) and 36 transcription factors were associated with them. Finally, an autophagy-associated competing endogenous RNA network was constructed based on the 9 hub genes. Nine hub genes were identified and a network of competing endogenous RNA associated with autophagy was established. They can be used as autophagy-related biomarkers of intervertebral disc degeneration and for further exploration. [ABSTRACT FROM AUTHOR]

Background: Cellular senescence features irreversible growth arrest and secretion of multiple proinflammatory cytokines. Cyclic GMP-AMP synthase (cGAS) detects DNA damage and activates the DNA-sensing pathway, resulting in the upregulation of inflammatory genes and induction of cellular senescence. This study aimed to investigate the effect of cGAS in regulating senescence of nucleus pulposus (NP) cells under inflammatory microenvironment. Methods: The expression of cGAS was evaluated by immunohistochemical staining in rat intervertebral disc (IVD) degeneration model induced by annulus stabbing. NP cells were harvested from rat lumbar IVD and cultured with 10ng/ml IL-1β for 48 h to induce premature senescence. cGAS was silenced by cGAS specific siRNA in NP cells and cultured with IL-1β. Cellular senescence was evaluated by senescence-associated beta-galactosidase (SA-β-gal) staining and flow cytometry. The expression of senescence-associated secretory phenotype including IL-6, IL-8, and TNF-a was evaluated by ELISA and western blotting. Results: cGAS was detected in rat NP cells in cytoplasm and the expression was significantly increased in degenerated IVD. Culturing in 10ng/ml IL-1β for 48 h induced cellular senescence in NP cells with attenuation of G1-S phase transition. In senescent NP cells the expression of cGAS, p53, p16, NF-kB, IL-6, IL-8, TNF-α was significantly increased while aggrecan and collagen type II was reduced than in normal NP cells. In NP cells with silenced cGAS, the expression of p53, p16, NF-kB, IL-6, IL-8, and TNF-α was reduced in inflammatory culturing with IL-1β. Conclusion: cGAS was increased by NP cells in degenerated IVD promoting cellular senescence and senescent inflammatory phenotypes. Targeting cGAS may alleviate IVD degeneration by reducing NP cell senescence. [ABSTRACT FROM AUTHOR]

Intervertebral disc degeneration (IDD) is a common cause of low back pain, causing a huge emotional and economic burden on patients and society. Reduction of nucleus pulposus cells (NPC) and extracellular matrix (ECM) is the main feature of IDD, and NPC is the main source of ECM. Thermal apoptosis is a newly discovered form of cell death in recent years that differs significantly from apoptosis in terms of molecular mechanisms and cellular morphological changes. Diacetoxy-6-gingerdiol(D-6-G), a type of gingerol, has anti-inflammatory and antioxidant effects, but whether it has an inhibitory effect on cellular pyroptosis is not clear. Therefore, in the present study, we investigated the effect of D-6-G on the ECM of the nucleus pulposus oblongata under IL-1β treatment, as well as the mechanism of its effect on NLRP3 inflammasome and cellular focal death. In vitro cellular experiments demonstrated that D-6-G could bind to and inhibit the activity of NLRP3 inflammasome, and interestingly, D-6-G could also inhibit cellular pyroptosis and protect the nucleus pulposusry cellular microenvironment by activating the Nrf2/HO-1 axis. In conclusion, we found that D-6-G could inhibit NLRP3 inflammatory vesicle activity as well as cellular pyroptosis in NPCs and protect the ECM, suggesting the potential of D-6-G to delay IDD.

Objective To investigate the effect of Yes-associated protein (YAP) on intervertebral disc nucleus pulposus cells and its possible mechanism. Methods The relatively normal and degenerative intervertebral disc tissues of patients who underwent lumbar surgery in our hospital from March 2021 to July 2022 were harvested, and then the expression of YAP in the tissues were detected by immunohistochemistry assay and Western blotting. Human primary nucleus pulposus cells were isolated and primarily cultured, and treated with IL-1β to induce degeneration. Then the cells was divided into control group, IL-1β group, IL-1β+LV-YAP group, IL-1β+YAP-siRNA group, and IL-1β+LV-YAP+3-MA group. Western blot analysis was used to detect the expression of the proteins related to extracellular matrix catabolism and autophagy in each group. Finally, a rat model of disc degeneration was established, and the expression of YAP and LC3 and disc degeneration were observed with MRI, Alcian blue staining and immunohistochemistry. Results The expression level of YAP was significantly lower in the degraded disc tissues than the relatively normal disc tissues (P < 0.05). The IL-1β+LV-YAP group had significantly increased protein levels of Collagen Ⅱ, Aggrecan, and LC3-Ⅱ (P < 0.05), and decreased levels of MMP-3 and MMP-13 (P < 0.05) when compared with the cells after IL-1β treatment, whereas the IL-1β+YAP-siRNA group showed the exact opposite effects. What's more, pretreatment with autophagy inhibitor 3-MA resulted in decreased number of GFP-LC3 positive particles and protein levels of Collagen Ⅱ, Aggrecan and LC3-Ⅱ (P < 0.05), and increased protein expression of MMP-3 and MMP-13 (P < 0.05) in comparison with the conditions in the IL-1β+LV-YAP group. Furthermore, YAP overexpression promoted LC3 expression and inhibited disc degeneration in rat model of disc degeneration. Conclusion YAP overexpression can inhibit extracellular matrix degradation by promoting autophagy in human nucleus pulposus cells and thus delaying disc degeneration.

Yongxiong Huang,1,2,&ast; Xianshuai Qiu,3,&ast; Jinlian Liu,4 Jiangtao Wan,5 Cheng Yu,1 Chun Liu,1 Yang Duan,1 Chong Chen,2 Jingxing Dai,6 Jun Ouyang,6 Ming Liu,3 Shaoxiong Min,1,5 Sujun Qiu1 1Department of Spine Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou, 510280, People’s Republic of China; 2Department of Spine Surgery, Guangdong Provincial People’s Hospital, Southern Medical University, Guangzhou, 510000, People’s Republic of China; 3Department of Orthopedics and Sports Medicine Center, Heyou Hospital, Foshan, 528333, People’s Republic of China; 4Department of Pathophysiology, Guangdong Provincial Key Laboratory of Shock and Microcirculation, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, People’s Republic of China; 5Shenzhen Key Laboratory of Spine Surgery, Department of Spine Surgery, Peking University Shenzhen Hospital, Shenzhen, 518036, People’s Republic of China; 6Guangdong Provincial Key Laboratory of Medical Biomechanics & National Key Discipline of Human Anatomy, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Sujun Qiu, Department of Spine Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou, 510280, People’s Republic of China, Email qiusjspine@126.com Shaoxiong Min, Shenzhen Key Laboratory of Spine Surgery, Department of Spine Surgery, Peking University Shenzhen Hospital, Shenzhen, 518036, People’s Republic of China, Email msxbear24@163.comBackground: Intervertebral disc degeneration (IDD) is the leading cause of low back pain (LBP). The mechanism of IDD development and progression is not fully understood. Peripheral biomarkers are increasingly vital non-radioactive methods in early detection and diagnosis for IDD. Nevertheless, less attention has been paid to the role of mitophagy genes in the progress of IDD. This study aimed to identify the mitophagy disease-causing genes in the process of IDD and mitophagy diagnostic biomarkers for IDD.Methods: Mitophagy-related differentially expressed genes (MRDEGs) related to IDD were investigated by analyzing the microarray datasets of IDD cases from GEO, PathCards and Molecular Signatures Databases. We used R software, WGCNA, PPI, mRNA-miRNA, mRNA-TF, GO, KEGG, GSEA, GSVA and Cytoscape to analyze and visualize the data. We further used ssGSEA for immunoinfiltration analysis to obtain different immune cell infiltration. LASSO model was developed to screen for genes that met the diagnostic gene model requirements. Finally, qRT-PCR, Western blotting and HE were used to verify hub genes and their expression from clinical IDD samples.Results: We identified 14 MRDEGs and 12 hub genes. GO, KEGG, GSEA and GSVA analyses demonstrated that hub genes were critical for the development of IDD. LASSO diagnostic model consisted of six hub genes, among which SQSTM1, ATG7 and OPTN were significantly different between the two IDD disease subtypes. At the same time, SQSTM1 also had a high correlation with immune characteristic subtypes. The results of qRT-PCR and Western blotting also indicated that these genes were significantly differentially expressed in nucleus pulposus cells (NPCs) of the IDD group.Conclusion: We explored an association between MRDEGs-associated signature in IDD and validated that hub genes like SQSTM1 might serve as biomarkers for diagnostic and therapeutic targets for IDD. Meanwhile, this study can provide new insights into the functional characteristics and mechanism of mitophagy in the development of IDD. Keywords: intervertebral disc degeneration, mitophagy-related differentially expressed genes, immune infiltration, diagnostic model, SQSTM1, nucleus pulposus cells

Mechanical environment worsening is an important predisposing factor that accelerates intervertebral disc degeneration (IDD), but its specific regulatory mechanisms remain unclear. In this study, we reveal the molecular mechanisms of WTAP/YTHDF2‐mediated m6A modification in abnormal stress‐induced intervertebral disc (IVD) matrix degradation. WTAP expression in human nucleus pulposus cells was elevated under tension. Similarly, high WTAP expression was detected in severe degenerated human and rat nucleus pulposus tissues. Functionally, WTAP was found to increase the TIMP3 transcript methylation level under tension, resulting in YTHDF2 recognition, binding, and induction of its degradation. Reduction in TIMP3 caused increases in active matrix metalloproteinases, ultimately inducing extracellular matrix degradation in nucleus pulposus cells. Macroscopically, this promotes IDD. Additionally, in vitro and in vivo inhibition of WTAP expression or TIMP3 overexpression significantly increased stress resistance in the nucleus pulposus, thereby alleviating IDD. Our results show that abnormal stress disrupts IVD matrix stability through WTAP/YTHDF2‐dependent TIMP3 m6A modification. [ABSTRACT FROM AUTHOR]

Intervertebral disc degeneration (IDD) poses a significant health burden, necessitating a deeper understanding of its molecular underpinnings. Transcriptomic analysis reveals 485 differentially expressed genes (DEGs) associated with IDD, underscoring the importance of immune regulation. Weighted gene co‐expression network analysis (WGCNA) identifies a yellow module strongly correlated with IDD, intersecting with 197 DEGs. Protein–protein interaction (PPI) analysis identifies ITGAX, MMP9 and FCGR2A as hub genes, predominantly expressed in macrophages. Functional validation through in vitro and in vivo experiments demonstrates the pivotal role of FCGR2A in macrophage polarization and IDD progression. Mechanistically, FCGR2A knockdown suppresses M1 macrophage polarization and NF‐κB phosphorylation while enhancing M2 polarization and STAT3 activation, leading to ameliorated IDD in animal models. This study sheds light on the regulatory function of FCGR2A in macrophage polarization, offering novel insights for IDD intervention strategies. Key points: This study unveils the role of FCGR2A in intervertebral disc (IVD) degeneration (IDD).FCGR2A knockdown mitigates IDD in cellular and animal models.Single‐cell RNA‐sequencing uncovers diverse macrophage subpopulations in degenerated IVDs.This study reveals the molecular mechanism of FCGR2A in regulating macrophage polarization.This study confirms the role of the NF‐κB/STAT3 pathway in regulating macrophage polarization in IDD. [ABSTRACT FROM AUTHOR]

Objective Interaction of tumor cells with the surrounding environment is essential for tumor growth and progression that eventually leads to metastasis. Growing evidence shows that extracellular vesicles also known as exosomes play a crucial role in signaling between the tumor and its microenvironment. Tumor-derived exosomes have generally protumorigenic effects such as metastasis, hypoxia, angiogenesis, and epithelial-mesenchymal transition. Methods In this study, exosomes were isolated from a chordoma cell line, MUG-Chor1, and characterized subsequently. The number of exosomes was determined and introduced into the healthy nucleus pulposus (NP) cells for 140 days. The protumorigenic effects of a chordoma cell line-derived exosomes that initiate the tumorigenesis on NP cells were investigated. The impact of tumor-derived exosomes on various cellular events including cell cycle, migration, proliferation, apoptosis, and viability has been studied by treating NP cells with chordoma cell-line-derived exosomes cells. Results Upon treatment with exosomes, the NP cells not only gained a chordoma-like morphology but also molecular characteristics such as alterations in the levels of certain gene expressions. The migratory and angiogenic capabilities of NP cells increased after treatment with chordoma-derived exosomes. Conclusion Based on our findings, we can conclude that exosomes carry information from tumor cells and may exert tumorigenic effects on nontumorous cells. [ABSTRACT FROM AUTHOR]

The study aimed to investigate the effect of ginsenoside Rg1 on intervertebral disc degeneration (IVDD) in rats and IL-1β-induced nucleus pulposus (NP) cells, and explore its underlying mechanism. Forty IVDD rat models were divided into the IVDD group, low-dose (L-Rg1) group (intraperitoneal injection of 20 mg/kg/d ginsenoside Rg1), medium-dose (M-Rg1) group (intraperitoneal injection of 40 mg/kg/d ginsenoside Rg1), and high-dose (H-Rg1) group (intraperitoneal injection of 80 mg/kg/d ginsenoside Rg1). The pathological change was observed by HE and safranin O-fast green staining. The expression of IL-1β, IL-6, TNF-α, MMP3, aggrecan, and collagen II was detected. The expression of NF-κB p65 in IVD tissues was detected. Rat NP cells were induced by IL-1β to simulate IVDD environment and divided into the control group, IL-1β group, and 20, 50, and 100 µmol/L Rg1 groups. The cell proliferation activity, the apoptosis, and the expression of IL-6, TNF-α, MMP3, aggrecan, collagen II, and NF-κB pathway–related protein were detected. In IVDD rats, ginsenoside Rg1 improved the pathology of IVD tissues; suppressed the expression of IL-1β, IL-6, TNF-α, aggrecan, and collagen II; and inhibited the expression of p-p65/p65 and nuclear translocation of p65, to alleviate the IVDD progression. In the IL-1β-induced NP cells, ginsenoside Rg1 also improved the cell proliferation and inhibited the apoptosis and the expression of IL-6, TNF-α, aggrecan, collagen II, p-p65/p65, and IκK in a dose-dependent manner. Ginsenoside Rg1 alleviated IVDD in rats and inhibited apoptosis, inflammatory response, and ECM degradation in IL-1β-induced NP cells. And Rg1 may exert its effect via inhibiting the activation of NF-κB signaling pathway. [ABSTRACT FROM AUTHOR]

Objective To observe the effects of dexmedetomidine (Dex) on apoptosis and inflammatory response of human nucleus pulposus cells induced by interleukin-1β (IL-1β). Methods Human nucleus pulpocytes were cultured in vitro and divided into the Control group (without any treatment), IL-1β group (cultured with 10 ng/mL IL-1β for 24 h), IL1β+Dex-L group (cultured with 25 nmol/L dexmedetomidine and 10 ng/mL IL-1β for 24 h), IL-1β+Dex-M group (cultured with 50 nmol/L dexmedetomidine and 10 ng/mL IL-1β for 24 h), and IL-1β+Dex-H group (cultured with 100 nmol/L dex⁃ medetomidine and 10 ng/mL IL-1β for 24 h), respectively. The cell survival rate was measured by MTT, the apoptosis rate was detected by flow cytometry, and the tumor necrosis factor-α (TNF-α), interleukin-8 (IL-8), nitric oxide (NO) and in⁃ terleukin-6 (IL-6) were detected by enzyme-linked immunosorbent assay (ELISA). The expression levels of B-cell lympho⁃ ma/leukemia 2 (Bcl-2), Bcl-2 associated X protein (Bax), inducible-nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were detected by Western blotting. Results Compared with the Control group, the cell survival rates at 24 h and 48 h and Bax protein expression in the IL-1β group decreased, while the apoptosis rate, Bax protein expression, TNFα, IL-6, IL-8, COX-2, iNOS protein expression and NO expression increased, with statistically significant difference (all P <0. 05). Compared with the IL-1β group, the survival rates of cells at 24 h and 48 h were higher, and the expression of Bcl2 protein was higher, the apoptotic rate, the expression levels of Bax protein, TNF-α, IL-6, IL-8, COX-2, iNOS protein, and NO were lower in the IL-1β+Dex-L group, IL-1β+Dex-M group, and IL-1β+Dex-H group, and the differences were all statistically significant (all P<0. 05). The survival rates of cells at 24 h and 48 h and the expression of Bcl-2 protein in⁃ creased successively, the apoptotic rate, the expression levels of Bax protein, TNF-α, IL-6, IL-8, COX-2, iNOS protein, and NO decreased successively in the IL-1β+Dex-L group, IL-1β+Dex-M group, and IL-1β+Dex-H group, and the differ⁃ ences were all statistically significant (all P<0. 05). Conclusion Dexmedetomidine can inhibit the apoptosis of human nucleus pulposus cells induced by IL-1β and reduce the inflammatory response in a concentration-dependent manner. [ABSTRACT FROM AUTHOR]

Abstract Objective To explore the potential mechanism of Yougui Wan on deformed lumbar intervertebral disk structure in rats. Methods Thirty male Sprague–Dawley rats were randomly divided into 3 groups, with 10 rats in each group. The animals in the blank control group were healthy rats without specific treatment, and those in the model group and traditional Chinese medicine (TCM) group were used to establish the intervertebral disk degeneration (IDD) model by puncturing the annulus. Four weeks after modeling, rats in the TCM group were administered Yougui Wan by gavage for 2 consecutive weeks. Serum interleukin-6 (IL-10), macrophage migration inhibitory factor (MIF) and tumor necrosis factor alpha (TNF-α) levels were measured by ELISA, and the protein expression levels of collagen II and Notch1 in intervertebral disk tissues were examined by Western blotting. Apoptosis was detected by the TUNEL method. Results Compared with those in the blank group, IL-10, MIF and TNF-α levels in the model group and TCM group were increased (P

Chunran Pan,&ast; Wenjie Hou,&ast; Xiaofeng Deng, Jiawei Liu, Ruimin Chi, Xingru Shang, Tao Xu, Xiaoxia Hao Department of Rehabilitation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Xiaoxia Hao, Department of Rehabilitation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095&num;, Jie-Fang Avenue, Qiaokou District, Wuhan, 430030, People’s Republic of China, Tel +86 1-597-296-0593, Fax +86- 27-83662640, Email haoxiaoxia@hust.edu.cnAbstract: Intervertebral disc degeneration (IDD) is considered as a dominant contributor to low back pain (LBP), causing severe pain, limited range of lumbar motion, physical dysfunction, and restriction of social activity. However, the specific pathological mechanisms underlying IDD remain elusive, and effective strategies to delay the pathogenesis of IDD are still unclear and limited. In recent years, some studies have found that nuclear factor erythroid 2-related factor 2 (Nrf2), an important antioxidant transcription factor, may play crucial roles in the pathogenesis and progression of age-related diseases including IDD. Nrf2 can maintain redox homeostasis and protecting nucleus pulposus (NP) cells against oxidative stress, inflammatory response, extracellular matrix (ECM) catabolism, cell senescence and cell death involving in the progression of IDD. In this review, we aim to systematically describe the vital roles and pathological mechanism of Nrf2 signaling axis in the pathogenesis of IDD, which may put forward potential therapeutic strategies for the prevention and treatment of IDD by targeting Nrf2.Keywords: Nrf2, intervertebral disc degeneration, nucleus pulposus cells, oxidative stress

Lower back pain (LBP) is a worldwide health problem associated with significant economic and social burden. Intervertebral disc degeneration (IVDD) is a leading cause of LBP. Several studies show that the death of nucleus pulposus cells (NPCs), abnormal metabolism of the extracellular matrix (ECM), and inflammatory response are the key mechanisms behind the pathogenesis of IVDD. Circular RNAs (circRNAs) are key regulators of gene expression and play a significant role in regulating NPCs death, ECM homeostasis, and inflammatory response by acting as microRNAs (miRNAs) sponges in IVDD. However, the regulatory role of circRNAs in mediating IVDD remains unknown. This review comprehensively describes the normal anatomic structure and function of IVD, the pathogenesis of IVDD, the characteristics, synthesis, mechanisms, and function of circRNAs. Moreover, we highlighted the 23 circRNAs that mediate ECM metabolism, 16 circRNAs that mediate NPCs apoptosis, circ_0004354 and circ_0040039 that mediate NPCs pyroptosis, and 5 circRNAs that mediate inflammatory response in IVDD. In addition, this review presents suggestions for future studies, such as the need for further investigation on ferroptosis-related circRNAs in IVDD. This review could provide novel insights into the pathogenesis and treatment of IVDD.

Aim: Low back pain (LBP) is one of the most common musculoskeletal diseases in the world. Apoptosis of nucleus pulposus (NP) cells and structural changes in the intervertebral disc (IVD) matrix cause its degeneration and are directly related to LBP. Because of exosomes derived from mesenchymal stem cells (MSC-Exo) are high therapeutic potential and may be valuable options for decreasing intervertebral disc degeneration (IDD). The present study evaluates changes in the expression level of genes responsible for repairing NP cells (collagen and aggrecan) and tumor necrosis factor-alpha (TNF-α) gene and the effectiveness of exosomes in inhibiting excessive apoptosis of NP cells under inflammation. Material and Methods: Exosomes were separated by ultracentrifuge. They were identified with the help of transmission electron microscopy (TEM), atomic force microscope (AFM), and dynamic light scattering (DLS). Cell viability was evaluated by MTT assay and DAPI staining. Real-time PCR measured the expression of collagen, aggrecan, and TNF-α genes. Results: Exosomes are vesicles with a diameter of about 35.5 to 100 nm. Based on the findings of the MTT assay, the survival rate of the inflammatory cells treated with exosomes was significantly different from the inflammation group without treatment (*P<0.05 at the dose of 100 μg/ml, **P<0.01 at the doses of 25 and 50 μg/ml). DAPI results showed a decrease in cell death in the exosome-treated group. Real-time PCR results showed increased expression of collagen (*P<0.05) and aggrecan (***P<0.001) genes and decreased the TNF-α (**P<0.01) gene in inflammatory cells treated with exosomes. Conclusion: Exosomes derived from mesenchymal stem cells can increase collagen and aggrecan gene expression and decrease TNF-α gene expression in treated cells. [ABSTRACT FROM AUTHOR]

Objective: To explore the potential mechanism of Yougui Wan on deformed lumbar intervertebral disk structure in rats. Methods: Thirty male Sprague–Dawley rats were randomly divided into 3 groups, with 10 rats in each group. The animals in the blank control group were healthy rats without specific treatment, and those in the model group and traditional Chinese medicine (TCM) group were used to establish the intervertebral disk degeneration (IDD) model by puncturing the annulus. Four weeks after modeling, rats in the TCM group were administered Yougui Wan by gavage for 2 consecutive weeks. Serum interleukin-6 (IL-10), macrophage migration inhibitory factor (MIF) and tumor necrosis factor alpha (TNF-α) levels were measured by ELISA, and the protein expression levels of collagen II and Notch1 in intervertebral disk tissues were examined by Western blotting. Apoptosis was detected by the TUNEL method. Results: Compared with those in the blank group, IL-10, MIF and TNF-α levels in the model group and TCM group were increased (P < 0.05), the protein expression levels of collagen II were decreased, and the protein expression levels of Notch1 were increased. Compared with those in the model group, the levels of IL-10 in the TCM group were increased (P < 0.05), the levels of MIF and TNF-α were decreased (P < 0.05), the protein expression levels of collagen II were increased, and the protein expression levels of Notch1 were decreased. Conclusion: Yougui Wan can inhibit the inflammatory response in IDD rats, reduce the degradation of extracellular matrix, reduce apoptosis in nucleus pulposus cells, and alleviate intervertebral disk degeneration. The mechanism may be related to the regulation of the Notch signaling pathway. [ABSTRACT FROM AUTHOR]

OBJECTIVE: The aim of the study was to explore the expression of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT)/hypoxia-induced factor-1 alpha (HIF-1α) signaling pathway and the apoptosis of nucleus pulposus cells (NPCs) under different oxygen concentrations to clarify the biological characteristics of NPCs and the molecular mechanism of intervertebral disc degeneration (IVDD). MATERIALS AND METHODS: Normal and degenerated human NPCs were collected. Leibovitz's medium with 100 μmol/L CoCl2 was used to establish a hypoxic culture environment, and 100 μmol/L H2O2 was used to establish an oxidative stress culture environment. Third-generation NPCs were divided into 6 groups: normal NPCs + hypoxia, normal NPCs + normoxia, normal NPCs + oxidative stress, degenerated NPCs + hypoxia, degenerated NPCs + normoxia, and degenerated NPCs + oxidative stress. Normal NPCs + hypoxia was used as the experimental control group. Cell viability and proliferation were detected by using the Cell Counting Kit- 8 (CCK-8) method. Cell apoptosis rate was assessed by flow cytometry, and expression levels of PI3K, AKT, and HIF-1α were determined by Real-Time-Polymerase Chain Reaction (RT-PCR) and Western blotting. RESULTS: The cell proliferation rate of both normal and degenerated NPCs decreased with increasing oxygen concentration. Conversely, the apoptosis rate increased as the oxygen concentration increased (p<0.05). Compared with the control group, whether the cells degenerated had a very significant impact on the apoptosis rate (p<0.001), and oxygen concentration also had a highly significant impact on both the cell proliferation rate and apoptosis rate (both p<0.001). The interaction between cell degeneration and oxygen concentration significantly affected both cell proliferation and apoptosis rates (p<0.05). Considering the expression levels of PI3K, AKT, and HIF-1α, normal NPCs had the highest levels under low oxygen concentrations, followed by oxidative stress and normoxia. In degenerated NPCs, the expression levels also decrease as the oxygen concentration increases. CONCLUSIONS: The PI3K/AKT/HIF-1α signaling pathway plays a significant role in inhibiting oxidative stress, antagonizing NPC apoptosis, and consequently delaying IVDD. [ABSTRACT FROM AUTHOR]

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

Accumulation of advanced glycation end products (AGEs) causes apoptosis in human nucleus pulposus cells (NPCs), contributing to intervertebral disc degeneration (IVDD). The purpose of this study was to determine the roles of thioredoxin‐interacting protein (TXNIP) in the mechanisms underlying AGE‐induced apoptosis of NPCs. TXNIP was silenced or overexpressed in HNPCs exposed to AGEs. Glycolysis was assessed using extracellular acidification rate (ECAR), ATP level, GLUT1, and GLUT4 measurements. AGEs, TXNIP, GLUT1, and GLUT4 levels in IVDD patients were measured as well. In NPCs, AGEs reduced cell viability, induced apoptosis, inhibited glycolysis, and increased TXNIP expression. Silencing TXNIP compromised the effects of AGEs on cell viability, apoptosis, and glycolysis in NPCs. Furthermore, TXNIP overexpression resulted in decreased cell viability, increased apoptotic cells, and glycolysis suppression. Furthermore, co‐treatment with a glycolysis inhibitor improved TXNIP silencing's suppressive effects on AGE‐induced cell injury in NPCs. In IVDD patients with Pfirrmann Grades II–V, increasing trends in AGEs and TXNIP were observed, while decreasing trends in GLUT1 and GLUT4. AGE levels had positive correlations with TXNIP levels. Both AGE and TXNIP levels correlated negatively with GLUT1 and GLUT4. Our study indicates that TXNIP plays a role in mediating AGE‐induced cell injury through suppressing glycolysis. The accumulation of AGEs, the upregulation of TXNIP, and the downregulation of GLUT1 and GLUT4 are all linked to the progression of IVDD. [ABSTRACT FROM AUTHOR]

Objective: Intervertebral disc degeneration (IVDD) is a chronic degenerative orthopedic illness that causes lower back pain as a typical clinical symptom, severely reducing patients' quality of life and work efficiency, and imposing a significant economic burden on society. IVDD is defined by rapid extracellular matrix breakdown, nucleus pulposus cell loss, and an inflammatory response. It is intimately related to the malfunction or loss of myeloid cells among them. Many mechanisms have been implicated in the development of IVDD, including inflammatory factors, oxidative stress, apoptosis, cellular autophagy, and mitochondrial dysfunction. In recent years, mitochondrial dysfunction has become a hot research topic in age-related diseases. As the main source of adenosine triphosphate (ATP) in myeloid cells, mitochondria are essential for maintaining myeloid cell survival and physiological functions. Methods: We searched the PUBMED database with the search term "intervertebral disc degeneration and mitochondrial dysfunction" and obtained 82 articles, and after reading the abstracts and eliminating 30 irrelevant articles, we finally obtained 52 usable articles. Results: Through a review of the literature, it was discovered that IVDD and cellular mitochondrial dysfunction are also linked. Mitochondrial dysfunction contributes to the advancement of IVDD by influencing a number of pathophysiologic processes such as mitochondrial fission/fusion, mitochondrial autophagy, cellular senescence, and cell death. Conclusion: We examine the molecular mechanisms of IVDD-associated mitochondrial dysfunction and present novel directions for quality management of mitochondrial dysfunction as a treatment approach to IVDD. [ABSTRACT FROM AUTHOR]

Gongbiao Lu,1,2,&ast; Cunxin Zhang,2,&ast; Kang Li,2 Kai Gao,3 Maoqing Fu,2 Chaoliang Lyu,2 Zhengxue Quan1 1Department of Spine Surgery, the First Affiliated Hospital of Chongqing Medical University, Chongqing, 400042, People’s Republic of China; 2Department of Spine Surgery, Jining No.1 People’s Hospital, Jining, 272011, People’s Republic of China; 3Department of Orthopaedics, Jining No.1 People’s Hospital, Jining, 272011, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Zhengxue Quan, Department of Spine Surgery, the First Affiliated Hospital of Chongqing Medical University, No. 1 Youyi Road, Yuzhong District, Chongqing, 400042, People’s Republic of China, Tel +86 13608321800, Fax +86-023-68811360, Email Quanzx18@126.com Chaoliang Lyu, Department of Spine Surgery, Jining No.1 People’s Hospital, No. 6 Healthy Road, Rencheng District, Jining, 272011, People’s Republic of China, Tel +86 15265775628, Fax +86-0537- 2253252, Email lvchaolianggk@163.comPurpose: To investigate the molecular mechanism underlying the inhibitory effect of sinomenine (SN) on interleukin-1β (IL-1β)-induced apoptosis in nucleus pulposus cells (NPCs), and to evaluate the potential role of SN in preventing intervertebral disk degeneration (IDD).Methods: The Rat NPCs were cultured in vitro and identified using Hematoxylin-Eosin (HE) staining, toluidine blue staining, and immunofluorescence analysis. NPCs were pretreated with or without SN, then induced with IL-1β to assess cell viability, ROS levels, apoptotic rates, and wound healing ability. Relevant protein expression was detected using Elisa, qPCR and Western Blot techniques. NPCs were pretreated with SN, either alone or in combination with Nrf2-IN-1 or SC, before being induced to undergo apoptosis by IL-1β. Apoptosis was detected using Hoechst staining, while qPCR and Western Blot techniques assessed protein expression. Rat caudal intervertebral discs were induced with IDD, with or without SN injection, and then co-injected with IL-1β. The levels of IDD were evaluated using HE staining and modified saffron-O-fix green cartilage staining. Relevant protein expression was detected using Elisa, qPCR, and Western Blot techniques.Results: IL-1β significantly reduced NPC activity, induced ROS accumulation and apoptosis, decreased cell healing rate, promoted the expression and secretion of inflammatory factors, and inhibited extracellular matrix synthesis. However, pretreatment with SN effectively reversed these effects. Inhibition of the Keap1/Nrf2 signaling pathway or activation of the NF-κB signaling pathway significantly attenuated the cytoprotective effects of SN and increased apoptosis. Acupuncture combined with IL-1β injection markedly induced intervertebral disc degeneration in rat caudal spine, upregulated inflammatory factors expression and secretion, and downregulated extracellular matrix synthesis. SN intervention notably enhanced antioxidant enzyme expression and reversed these outcomes.Conclusion: SN can prevent IL-1β-induced apoptosis of NPCs and ameliorate IDD by activating the Keap1/Nrf2 pathway and inhibiting the NF-κB signaling pathway.Keywords: sinomenine, intervertebral disc degeneration, nucleus pulposus cells, apoptosis, Nrf2, NF-κB

Objective To explore the effect and mechanism of zinc finger protein A20 in the nucleus pulposus cells of intervertebral disc with hypoxic conditions. Methods Rat intervertebral discs were cultured in normoxic (21% O2) and hypoxic (1% O2) conditions treated with dimethyloxalylglycine (DMOG) and divided into normoxia group, normoxia+DMOG group, hypoxia group and hypoxia+DMOG group. Histological staining and Western blotting were used to detect the histomorphological changes of the nucleus pulposus and the differences in expression of HIF-1α and A20. A20 interfering adenovirus were used to treat human nucleus pulposus cells with hypoxic and normoxic condition respectively, and divided into hypoxia+empty adenovirus group, hypoxia+A20 interference adenovirus group, normoxia+empty adenovirus group and normoxia+A20 interference adenovirus group. Flow cytometry, JC-1 detection, EdU detection and Western blotting were used to detect the differences in apoptosis, mitochondrial membrane potential, proliferation, extracellular matrix and inflammatory cytokine expression of nucleus pulposus cells. Results Compared with the normoxia group, the morphological degeneration of the other 3 groups was milder, and the protein levels of HIF-1α and A20 were significantly increased (P < 0.05); Compared with the hypoxia+empty adenovirus group, the hypoxia+A20 interference adenovirus group showed the increased protein levels of TNF-α and P-p65 in nucleus pulposus cells (P < 0.05), increased apoptosis (P < 0.05), decreased cell proliferation (P < 0.05) and decreased protein levels of extracellular matrix proteins (P < 0.05). In the normoxia+empty adenovirus group and the normoxia+A20 interference adenovirus group, there was no significant difference of the indicators mentioned above. Conclusion Hypoxia can inhibit the apoptosis and attenuate inflammation and promote the proliferation and the anabolism of extracellular matrix in nucleus pulposus cells by inducing the expression of A20 protein.

Abstract Background Nucleus pulposus cell (NPC) death and progressive reduction play important roles in intervertebral disc degeneration (IVDD). As part of a damage-associated molecular pattern, mitochondrial DNA (mtDNA) can be recognized by TLR9 and triggers the expression of NF-κB and NLRP3 inflammasomes, inducing pyroptosis and inflammatory response. However, whether mtDNA induces NPC pyroptosis via the TLR9-NF-κB-NLRP3 axis and promotes IVDD remains uncertain. Methods We constructed an in vitro NPC oxidative stress injury model to clarify the mechanism of mtDNA release, TLR9-NF-κB signaling pathway activation, and NPC injury. We further verified the mechanism of action underlying the inhibition of mtDNA release or TLR9 activation in NPC injury in vitro. We then constructed a rat punctured IVDD model to understand the mechanism inhibiting mtDNA release and TLR9 activation in IVDD. Results We used human NP specimen assays to show that the expression levels of TLR9, NF-κB, and NLRP3 inflammasomes correlated with the degree of IVDD. We demonstrated that mtDNA mediated TLR9-NF-κB-NLRP3 axis activation in oxidative stress-induced human NPC pyroptosis in vitro. Oxidative stress can damage the mitochondria of NPCs, causing the opening of the mitochondrial permeability transition pores (mPTP) and leading to the release of mtDNA into the cytosol. Furthermore, inhibition of mPTP opening or TLR9 activation blocked TLR9-NF-κB-NLRP3 axis activation and thereby mediated NPC pyroptosis and IVDD. Conclusion mtDNA plays a key role in mediating NPC pyroptosis and IVDD via the TLR9-NF-κB-NLRP3 axis. Our findings provide new potential targets for IVDD.

Abstract Background Intervertebral disk degeneration (IDD) is a degenerative disease that underlies various musculoskeletal and spinal disorders and is positively correlated with age. tRNA-derived small RNAs (tsRNA), as a new small noncoding RNAs, its function in IDD is unclear. Herein, our goal was to find the key tsRNA that affects IDD independently of age and explore the underlying mechanisms. Methods Small RNA sequencing was performed in nucleus pulposus (NP) tissues of traumatic lumbar fracture individuals, young IDD (IDDY) patients, and old IDD (IDDO) patients. The biological functions of tsRNA-04002 in NP cells (NPCs) were investigated by qRT-PCR, western blot, and flow cytometry analysis. The molecular mechanism of tsRNA-04002 was demonstrated by luciferase assays and rescue experiments. Furthermore, the therapeutic effects of tsRNA-04002 on IDD rat model were used and evaluated in vivo. Results Compared with fresh traumatic lumbar fracture patients, a total of 695 disordered tsRNAs is obtained (398 down-regulated tsRNAs and 297 up-regulated tsRNAs). These disordered tsRNAs were mainly involved in Wnt signaling pathway and MAPK signaling pathway. tsRNA-04002 was an age-independent key target in IDD, which was both lower expressed in IDDY and IDDO groups than control group. Overexpression of tsRNA-04002 restrained inflammatory cytokines IL-1β and TNF-α expression, increased the COL2A1, and inhibited the NPCs apoptosis. Furthermore, we determined that PRKCA was the target gene of tsRNA-04002 and was negatively regulated by tsRNA-04002. The rescue experiment results suggested that the high expression of PRKCA reversed the inhibitory effect of tsRNA-04002 mimics on NPCs inflammation and apoptosis, and promotive effect of COL2A1. Moreover, tsRNA-04002 treatment dramatically ameliorated the IDD process in the puncture-induced rat model, together with the blockade of PRKCA in vivo. Conclusion Collectively, our results substantiated that tsRNA-04002 could alleviate IDD by targeting PRKCA to inhibit apoptosis of NPCs. tsRNA-04002 may be a novel therapeutic target of IDD progression.

In regenerative medicine, experimental animal models are commonly used to study potential effects of human cells as therapeutic candidates. Although some studies describe certain cells, such as mesenchymal stromal cells (MSC) or human primary cells, as hypoimmunogenic and therefore unable to trigger strong inflammatory host responses, other studies report antibody formation and immune rejection following xenotransplantation. Accordingly, the goal of our study was to test the cellular retention and survival of human-induced pluripotent stem cell (iPSCs)-derived MSCs (iMSCs) and primary nucleus pulposus cells (NPCs) following their xenotransplantation into immune-privileged knee joints (14 days) and intervertebral discs (IVD; 7 days) of immunocompromised Nude and immunocompetent Sprague Dawley (SD) rats. At the end of both experiments, we could demonstrate that both rat types revealed comparably low levels of systemic IL-6 and IgM inflammation markers, as assessed via ELISA. Furthermore, the number of recovered cells was with no significant difference between both rat types. Conclusively, our results show that xenogeneic injection of human iMSC and NPC into immunoprivileged knee and IVD sites did not lead to an elevated inflammatory response in immunocompetent rats when compared to immunocompromised rats. Hence, immunocompetent rats represent suitable animals for xenotransplantation studies targeting immunoprivileged sites. [ABSTRACT FROM AUTHOR]

Intervertebral disc degeneration is a leading cause of disability in the elderly population. Rigid extracellular matrix is a critical pathological feature of disc degeneration, leading to aberrant nucleus pulposus cells (NPCs) proliferation. However, the underlying mechanism is unclear. Here, we hypothesize that increased matrix stiffness induces proliferation and thus degenerative phenotypes of NPCs through YAP/TEAD1 signaling pathway. We established hydrogel substrates to mimic stiffness of degenerated human nucleus pulposus tissues. RNA-sequencing identified differentially expressed genes between primary rat NPCs cultured on rigid and soft hydrogels. Dual luciferase assay and gain- and loss-function experiments evaluated the correlation between YAP/TEAD1 and Cyclin B1. Furthermore, single-cell RNA-sequencing of human NPCs was performed to identify specific cell clusters with high YAP expression. Matrix stiffness increased in severely degenerated human nucleus pulposus tissues (p < 0.05). Rigid substrate enhanced rat NPCs proliferation mainly through Cyclin B1, which was directly targeted and positively regulated by YAP/TEAD1. Depletion of YAP or Cyclin B1 arrested G2/M phase progression of rat NPCs and reduced fibrotic phenotypes including MMP13 and CTGF (p < 0.05). Fibro NPCs with high YAP expression were identified in human tissues and responsible for fibrogenesis during degeneration. Furthermore, inhibition of YAP/TEAD interaction by verteporfin suppressed cell proliferation and alleviated degeneration in the disc needle puncture model (p < 0.05). Our results demonstrate that elevated matrix stiffness stimulates fibro NPCs proliferation through YAP/TEAD1-Cyclin B1 axis, indicating a therapeutic target for disc degeneration. [ABSTRACT FROM AUTHOR]

Aims: This study was performed to explore the effect of melatonin on pyroptosis in nucleus pulposus cells (NPCs) and the underlying mechanism of that effect. Methods: This experiment included three patients diagnosed with lumbar disc herniation who failed conservative treatment. Nucleus pulposus tissue was isolated from these patients when they underwent surgical intervention, and primary NPCs were isolated and cultured. Western blotting, reverse transcription polymerase chain reaction, fluorescence staining, and other methods were used to detect changes in related signalling pathways and the ability of cells to resist pyroptosis. Results: Western blot analysis confirmed the expression of cleaved CASP-1 and melatonin receptor (MT-1A-R) in NPCs. The cultured NPCs were identified by detecting the expression of CD24, collagen type II, and aggrecan. After treatment with hydrogen peroxide, the pyroptosis-related proteins NLR family pyrin domain containing 3 (NLRP3), cleaved CASP-1, N-terminal fragment of gasdermin D (GSDMD-N), interleukin (IL)-18, and IL-1β in NPCs were upregulated, and the number of propidium iodide (PI)-positive cells was also increased, which was able to be alleviated by pretreatment with melatonin. The protective effect of melatonin on pyroptosis was blunted by both the melatonin receptor antagonist luzindole and the nuclear factor erythroid 2–related factor 2 (Nrf2) inhibitor ML385. In addition, the expression of the transcription factor Nrf2 was up- or downregulated when the melatonin receptor was activated or blocked by melatonin or luzindole, respectively. Conclusion: Melatonin protects NPCs against reactive oxygen species-induced pyroptosis by upregulating the transcription factor Nrf2 via melatonin receptors. Cite this article: Bone Joint Res 2023;12(3):202–211.

Abstract Background Excessive oxidative stress plays a critical role in the progression of various diseases, including intervertebral disk degeneration (IVDD). Recent studies have found that anemonin (ANE) possesses antioxidant and anti-inflammatory effects. However, the role of ANE in IVDD is still unclear. Therefore, this study investigated the effect and mechanism of ANE on H2O2 induced degeneration of nucleus pulposus cells (NPCs). Methods NPCs were pretreated with ANE, and then treated with H2O2. NOX4 was upregulated by transfection of pcDNA-NOX4 into NPCs. Cytotoxicity was detected by MTT, oxidative stress-related indicators and inflammatory factors were measured by ELISA, mRNA expression was assessed by RT-PCR, and protein expression was tested by western blot. Results ANE attenuated H2O2-induced inhibition of NPCs activity. H2O2 enhanced oxidative stress, namely, increased ROS and MDA levels and decreased SOD level. However, these were suppressed and pretreated by ANE. ANE treatment repressed the expression of inflammatory factors (IL-6, IL-1β and TNF-α) in H2O2-induced NPCs. ANE treatment also prevented the degradation of extracellular matrix induced by H2O2, showing the downregulation of MMP-3, 13 and ADAMTS-4, 5 and the upregulation of collagen II. NOX4 is a key factor regulating oxidative stress. Our study confirmed that ANE could restrain NOX4 and p-NF-κB. In addition, overexpression of NOX4 counteracted the antioxidant and anti-inflammatory activities of ANE in H2O2-induced NPCs, and the inhibition of the degradation of extracellular matrix induced by ANE was also reversed by overexpression of NOX4. Conclusion ANE repressed oxidative stress, inflammation and extracellular matrix degradation in H2O2-induced NPCs by inhibiting NOX4/NF-κB pathway. Our study indicated that ANE might be a candidate drug for the treatment of IVDD.

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.

Modulating a favorable inflammatory microenvironment that facilitates the recovery of degenerated discs is a key strategy in the treatment of intervertebral disc (IVD) degeneration (IDD). More interestingly, well-mechanized tissue-engineered scaffolds have been proven in recent years to be capable of sensing mechanical transduction to enhance the proliferation and activation of nucleus pulposus cells (NPC) and have demonstrated an increased potential in the treatment and recovery of degenerative discs. Additionally, existing surgical procedures may not be suitable for IDD treatment, warranting the requirement of new regenerative therapies for the restoration of disc structure and function. In this study, a light-sensitive injectable polysaccharide composite hydrogel with excellent mechanical properties was prepared using dextrose methacrylate (DexMA) and fucoidan with inflammation-modulating properties. Through numerous in vivo experiments, it was shown that the co-culture of this composite hydrogel with interleukin-1β-stimulated NPCs was able to promote cell proliferation whilst preventing inflammation. Additionally, activation of the caveolin1-yes-associated protein (CAV1-YAP) mechanotransduction axis promoted extracellular matrix (ECM) metabolism and thus jointly promoted IVD regeneration. After injection into an IDD rat model, the composite hydrogel inhibited the local inflammatory response by inducing macrophage M2 polarization and gradually reducing the ECM degradation. In this study, we propose a fucoidan-DexMA composite hydrogel, which provides an attractive approach for IVD regeneration. [ABSTRACT FROM AUTHOR]

Oxidative stress can lead to nucleus pulposus cell (NPC) apoptosis, which is considered to be one of the main contributors to intervertebral disc degeneration (IVDD). Procyanidin B2 is a natural antioxidant that protects against oxidative stress. However, whether procyanidin B2 protects NPCs from oxidative stress remains unknown. In this study, we demonstrated that procyanidin B2 could reduce tert‐butyl hydroperoxide‐induced reactive oxygen species in rat NPCs and attenuate rat NPC apoptosis. Further experiments revealed that procyanidin B2 upregulated the expression of both nuclear factor erythroid 2‐related factor 2 (Nrf2) and phosphorylation of protein kinase B (Akt). We then used silencing of Nrf2 and LY294002 to silence Nrf2 expression and block the phosphatidylinositol 3‐kinase (PI3K)/Akt pathway, respectively, and found that the protective roles of procyanidin B2 in NPCs were inhibited. Therefore, we demonstrated that procyanidin B2 alleviated rat NPC apoptosis induced by oxidative stress by upregulating Nrf2 via activation of the PI3K/Akt signaling pathway. This study provides a potential therapeutic approach for procyanidin B2 in IVDD, which might help in the development of new drugs for IVDD treatment. [ABSTRACT FROM AUTHOR]

Background: Nucleus pulposus cell (NPC) death and progressive reduction play important roles in intervertebral disc degeneration (IVDD). As part of a damage-associated molecular pattern, mitochondrial DNA (mtDNA) can be recognized by TLR9 and triggers the expression of NF-κB and NLRP3 inflammasomes, inducing pyroptosis and inflammatory response. However, whether mtDNA induces NPC pyroptosis via the TLR9-NF-κB-NLRP3 axis and promotes IVDD remains uncertain. Methods: We constructed an in vitro NPC oxidative stress injury model to clarify the mechanism of mtDNA release, TLR9-NF-κB signaling pathway activation, and NPC injury. We further verified the mechanism of action underlying the inhibition of mtDNA release or TLR9 activation in NPC injury in vitro. We then constructed a rat punctured IVDD model to understand the mechanism inhibiting mtDNA release and TLR9 activation in IVDD. Results: We used human NP specimen assays to show that the expression levels of TLR9, NF-κB, and NLRP3 inflammasomes correlated with the degree of IVDD. We demonstrated that mtDNA mediated TLR9-NF-κB-NLRP3 axis activation in oxidative stress-induced human NPC pyroptosis in vitro. Oxidative stress can damage the mitochondria of NPCs, causing the opening of the mitochondrial permeability transition pores (mPTP) and leading to the release of mtDNA into the cytosol. Furthermore, inhibition of mPTP opening or TLR9 activation blocked TLR9-NF-κB-NLRP3 axis activation and thereby mediated NPC pyroptosis and IVDD. Conclusion: mtDNA plays a key role in mediating NPC pyroptosis and IVDD via the TLR9-NF-κB-NLRP3 axis. Our findings provide new potential targets for IVDD. Highlights: NPC pyroptosis mechanism via mtDNA mediated TLR9-NF-κB-NLRP3 axis activation is unclear. We demonstrate that TLR9, NF-κB, and NLRP3 inflammasomes correlate with IVDD. mtDNA via the TLR9-NF-κB-NLRP3 axis mediates NPC pyroptosis and IVDD. [ABSTRACT FROM AUTHOR]

Background: Intervertebral disk degeneration (IDD) is a degenerative disease that underlies various musculoskeletal and spinal disorders and is positively correlated with age. tRNA-derived small RNAs (tsRNA), as a new small noncoding RNAs, its function in IDD is unclear. Herein, our goal was to find the key tsRNA that affects IDD independently of age and explore the underlying mechanisms. Methods: Small RNA sequencing was performed in nucleus pulposus (NP) tissues of traumatic lumbar fracture individuals, young IDD (IDDY) patients, and old IDD (IDDO) patients. The biological functions of tsRNA-04002 in NP cells (NPCs) were investigated by qRT-PCR, western blot, and flow cytometry analysis. The molecular mechanism of tsRNA-04002 was demonstrated by luciferase assays and rescue experiments. Furthermore, the therapeutic effects of tsRNA-04002 on IDD rat model were used and evaluated in vivo. Results: Compared with fresh traumatic lumbar fracture patients, a total of 695 disordered tsRNAs is obtained (398 down-regulated tsRNAs and 297 up-regulated tsRNAs). These disordered tsRNAs were mainly involved in Wnt signaling pathway and MAPK signaling pathway. tsRNA-04002 was an age-independent key target in IDD, which was both lower expressed in IDDY and IDDO groups than control group. Overexpression of tsRNA-04002 restrained inflammatory cytokines IL-1β and TNF-α expression, increased the COL2A1, and inhibited the NPCs apoptosis. Furthermore, we determined that PRKCA was the target gene of tsRNA-04002 and was negatively regulated by tsRNA-04002. The rescue experiment results suggested that the high expression of PRKCA reversed the inhibitory effect of tsRNA-04002 mimics on NPCs inflammation and apoptosis, and promotive effect of COL2A1. Moreover, tsRNA-04002 treatment dramatically ameliorated the IDD process in the puncture-induced rat model, together with the blockade of PRKCA in vivo. Conclusion: Collectively, our results substantiated that tsRNA-04002 could alleviate IDD by targeting PRKCA to inhibit apoptosis of NPCs. tsRNA-04002 may be a novel therapeutic target of IDD progression. [ABSTRACT FROM AUTHOR]

Background: Intervertebral disc degeneration (IVDD) is the initiating factor of adult degenerative scoliosis (ADS), and ADS further accelerates IVDD, creating a vicious cycle. Nevertheless, the role of the Wnt/β-Catenin pathway in ADS combined with IVDD (ADS-IVDD) remains a mystery. Accordingly, this study was proposed to investigate the effect of axial stress on the Wnt/β-Catenin pathway in nucleus pulposus cells (NPCs) isolated from DS-IVDD patients. Methods: Normal NPCs (N-NPC) were purchased and the NPCs of young (25–30 years; Y-NPC) and old (65–70 years; O-NPC) from ADS-IVDD patients were primary cultured. After treatment of NPC with overloaded axial pressure, CCK-8 and Annexin V-FITC kits were applied for detecting proliferation and apoptosis of N-NPC, Y-NPC and O-NPC, and western blotting was performed to assess the expression of Wnt 3a, β-Catenin, NPC markers and apoptosis markers (Bax, Bcl2 and Caspase 3). Results: N-NPC, Y-NPC and O-NPC were mainly oval, polygonal and spindle-shaped with pseudopods, and the cell morphology tended to be flattened with age. N-NPC, Y-NPC and O-NPC were capable of synthesizing proteoglycans and expressing the NPC markers (Collagen II and Aggrecan). Notably, the expression of Wnt 3a, β-Catenin, Collagen II and Aggrecan was reduced in N-NPC, Y-NPC and O-NPC in that order. After overload axial stress treatment, cell viability of N-NPC and Y-NPC was significantly reduced, and the percentage of apoptosis and expression of Wnt 3a and β-Catenin were significantly increased. Conclusions: Overloaded axial pressure activates the Wnt/β-Catenin pathway to suppress proliferation and facilitate apoptosis of NPC in ADS-IVDD patients. [ABSTRACT FROM AUTHOR]

Abstract Background Myricetin (MYR) is a common plant flavonoid with antioxidant and anticancer properties. However, the anti-aging effect of MYR on nucleus pulposus cells (NPCs) is still unknown. The study aimed to explore the effect of MYR on the senescence of NPCs. Methods Methyl-thiazolyl tetrazolium assay was used to detect NPCs viability. Senescence level was evaluated by senescence-associated β-galactosidase (SA-β-Gal) staining and the expression levels of P21, P16, IL-6 and IL-8. RNA-Sequencing (RNA-seq) technology was used to identify differentially expressed genes (DEGs) between hydrogen peroxide + MYR (HO + MYR) group and HO group, and Gene Ontology (GO) functional was performed to analyze DEGs. A Venn diagram was generated to screen overlapping DEGs related to aging and inflammation, and the role of the promising validated DEG was selected for further investigation by gene functional assays. Results HO inhibited NPCs viability and stimulated the senescent phenotype of NPCs, whereas MYR treatment significantly reversed SA-β-gal activity in NPCs. MYR also reduced the expression of p21 and p16 and the secretion of IL-6 and IL-8 induced by HO. RNA-seq screened 421 DEGs. The GO enrichment results showed DEGs were mainly enriched in terms such as "sterol biosynthetic process". We also found SERPINE1 has the highest log2FC abs. Silence of SERPINE1 inhibited HO-induced NPCs senescence, and overexpression of SERPINE1 could limit the anti-aging effect of MYR. Conclusions MYR alleviated HO-induced senescence of NPCs by regulating SERPINE1 in vitro.

Cunxin Zhang,1,2 Ziang Lu,3 Chaoliang Lyu,2 Shanshan Zhang,4 Dechun Wang1 1Department of Spine Surgery, Qingdao Municipal Hospital, Shandong University, Qingdao, 266061, People’s Republic of China; 2Department of Spine Surgery, Jining No.1 People’s Hospital, Jining, 272011, People’s Republic of China; 3Jining Medical University, Jining, 272067, People’s Republic of China; 4Department of Neurology, Jining No.1 People’s Hospital, Jining, 272011, People’s Republic of ChinaCorrespondence: Dechun Wang, Department of Spine surgery, Qingdao Municipal Hospital, Shandong University, 5&num; Donghai Road, Shinan District, Qingdao, 266061, People’s Republic of China, Tel +86+18661809296, Fax +86-0532-82716868, Email dechun-w@163.comPurpose: To explore the molecular mechanism by which andrographolide (ADR) inhibits static mechanical pressure-induced apoptosis in nucleus pulposus cells (NPCs) and to assess the role of ADR in inhibiting IDD.Methods: Hematoxylin-eosin (HE), toluidine blue, and immunofluorescence staining were used to identify NPCs. An NPC apoptosis model was constructed using a homemade cell pressurization device. The proliferation activity, reactive oxygen species (ROS) content, and apoptosis rate were detected using kits. The expression of related proteins was detected using Western blot. A rat tailbone IDD model was constructed using a homemade tailbone stress device. HE staining and safranine O-fast green FCF cartilage staining were used to observe the degeneration degree of the intervertebral disk.Results: ADR inhibits static mechanical pressure-induced apoptosis and ROS accumulation in NPCs and improves cell viability. ADR can promote the expression of Heme oxygenase-1 (HO-1), p-Nrf2, p-p38, p-Erk1/2, p-JNK, and other proteins, and its effects can be blocked by inhibitors of the above proteins.Conclusion: ADR can inhibit IDD by activating the MAPK/Nrf2/HO-1 signaling pathway and suppressing static mechanical pressure-induced ROS accumulation in the NPCs.Keywords: andrographolide, intervertebral disc degeneration, nucleus pulposus cells, apoptosis, reactive oxygen species, HO-1

Background: To clarify if a2-macroglobulin (a2M) has an antioxidative effect during the progression of the intervertebral disc degeneration (IVDD). Methods: The content of a2M and reactive oxygen species (ROS) were measured to compare mildly and severely degenerated human nucleus pulposus (NP) tissue by immunohistochemistry, mass spectrometry, and enzyme-linked immunosorbent assay (ELISA). Additionally, exogenic a2M was used to culture severely degenerated NP tissue in vitro. The effects of a2M on hypochlorite (HOCl)-treated NP cells were evaluated, containing antioxidative enzymes, ROS level, collagen II, and aggrecan expression, MMP3/13, and ADAMTS4/5. Results: ROS level increased in severely degenerated NP, accompanying with a decreased a2M content. Supplement of a2M could decrease the ROS level of cultured NP in vitro, meanwhile, the MMP13 and ADAMTS4 expression were also reduced. It was found that treatment of HOCl resulted in oxidative damage to NP cells and decreased a2M expression in a dose and time-dependent manner. Furthermore, exogenic a2M stimulation reversed the HOCl-triggered ROS accumulation. The promotion of SOD1/2, CAT, GPX1, collagen II, and aggrecan, and suppression of MMP3/13, ADAMTS4/5 expression caused by a2M were also observed. Conclusions: Our study indicates that a2M has an antioxidative ability in degenerated NP cells by promoting the antioxidative enzyme production.

Abstract Purpose Intervertebral disc degeneration (IDD) is one of the main causes of low back pain, which not only affects patients’ life quality, but also places a great burden on the public health system. Recently, ginsenoside Rg1 has been found to act in IDD; however, the mechanism is still unclear. The purpose of this study is to explore the function of ginsenoside Rg1 and its molecular mechanism in IDD. Methods The rat model of IDD and nucleus pulposus (NP) experimental groups treated with ginsenoside Rg1 was constructed for investing the role of ginsenoside Rg1 in IDD rats. In the in vitro and in vivo study, the histological morphological changes, motor threshold (MT), inflammatory factors, oxidative stress, apoptosis and expression of the YAP1/TAZ signaling pathway-related proteins of the intervertebral discs (IVD) were measured by histological staining, mechanical and thermal stimulation, ELISA, qRT-PCR, flow cytometry, and western blot, respectively. Results Ginsenoside Rg1 significantly increased the threshold for mechanical and thermal stimulation and alleviated histological changes in IDD rats. Ginsenoside Rg1 had a significant inhibitory effect on the secretion level of inflammatory factors, redox activity, extracellular matrix (ECM) degradation in IVD tissue and NP cells, and apoptosis in NP cells. Further investigation revealed that ginsenoside Rg1 significantly inhibited the expression of YAP1/TAZ signaling pathway-related proteins. Additionally, the above inhibitory effect of ginsenoside Rg1 on IDD progression was concentration-dependent, that is, the highest concentration of ginsenoside Rg1 was most effective. Conclusion Ginsenoside Rg1 inhibits IDD progression by suppressing the activation of YAP1/TAZ signaling pathway. This means that ginsenoside Rg1 has the potential to treat IDD.

Background: To clarify if a2-macroglobulin (a2M) has an antioxidative effect during the progression of the intervertebral disc degeneration (IVDD). Methods: The content of a2M and reactive oxygen species (ROS) were measured to compare mildly and severely degenerated human nucleus pulposus (NR) tissue by immunohistochemistry, mass spectrometry, and enzyme-linked immunosorbent assay (ELISA). Additionally, exogenic a2M was used to culture severely degenerated NP tissue in vitro. The effects of a2M on hypochlorite (HOCI)- treated NP cells were evaluated, containing antioxidative enzymes, ROS level, collagen II, and aggrecan expression, MMP3/13, and ADAMTS4/5. Results; ROS level increased in severely degenerated NP, accompanying with a decreased «2M content. Supplement of a2M could decrease the ROS level of cultured NP in vitro, meanwhile, the MMP13 and ADAMTS4 expression were also reduced. It was found that treatment of HOCI resulted in oxidative damage to NP cells and decreased «2M expression in a dose and time-dependent manner. Furthermore, exogenic a2M stimulation reversed the HOCI-triggered ROS accumulation. The promotion of SOD1/2, CAT, GPX1, collagen II, and aggrecan, and suppression of MMP3/13, ADAMTS4/5 expression caused by a2M were also observed. Conclusions: Our study indicates that «2M has an antiox-idative ability in degenerated NR cells by promoting the antioxidative enzyme production. [ABSTRACT FROM AUTHOR]

Background: Intervertebral disc degeneration (IDD) is a pathological process that occurs during the natural aging of intervertebral discs. Accumulating evidence suggests that noncoding RNAs (ncRNAs), including microRNAs and long ncRNAs (lncRNAs), participate in the pathogenesis and development of IDD. Herein, we examined the role of lncRNA MAGI2‐AS3 in the pathogenic mechanism of IDD. Material and Methods: To develop an IDD in vitro model, we treated human nucleus pulposus (NP) cells with lipopolysaccharide (LPS). Aberrant levels of lncRNA MAGI2‐AS3, miR‐374b‐5p, interleukin (IL)‐10 and extracellular matrix (ECM)‐related proteins in NP cells were examined using reverse transcription‐quantitative PCR and western blot analysis. LPS‐induced NP cell injury and inflammatory response were confirmed using the MTT assay, flow cytometry, Caspase3 activity, and enzyme‐linked immunosorbent assay. Dual‐luciferase reporter assay and rescue experiments were performed to confirm targets between lncRNA MAGI2‐AS3 and miR‐374b‐5p or miR‐374b‐5p and IL‐10. Results: LPS‐induced NP cells exhibited low levels of lncRNA MAGI2‐AS3 and IL‐10 expression, along with high miR‐374b‐5p expression. miR‐374b‐5p was a target of lncRNA MAGI2‐AS3 and IL‐10. LncRNA MAGI2‐AS3 ameliorated injury, inflammatory response, and ECM degradation in LPS‐treated NP cells by downregulating miR‐374b‐5p to upregulate IL‐10 expression. Conclusions: LncRNA MAGI2‐AS3 increased IL‐10 expression levels by sponging miR‐374b‐5p, which, in turn, alleviated LPS‐triggered decreased NP cell proliferation and increased apoptosis, inflammatory response, and ECM degradation. Therefore, lncRNA MAGI2‐AS3 may be a potential therapeutic target for IDD. [ABSTRACT FROM AUTHOR]

Background: Intervertebral disk degeneration (IDD) is a common orthopedic disorder, and nucleus pulposus cells (NPCs) serve to stabilize the intervertebral disk. Objective: This study was implemented to explore the protective effect of endplate stem cells (EPSCs) on IDD. NPCs and EPSCs were isolated from rats and tert-Butyl peroxide (TBHP) was used to treat NPCs and simulate IDD. EPSC-derived conditioned medium (CM) was collected and mixed with a culture medium at different proportions, which was then used to treat NPCs. SOD assay was employed to examine the oxidative stress level in the NPCs. To assess apoptosis and caspase-3 activity, flow cytometry and western blots for cleaved PARP1 and BAX were carried out. The mRNA expression levels of different genes were detected by quantitative real-time polymerase chain reaction. Furthermore, the concentrations of amphiregulin (Areg) and inflammatory factors were measured with ELISA kits and cell signaling pathways were evaluated using western blotting. Results: Following TBHP treatment, caspase-3 activity in the NPCs increased. However, exposing TBHP-induced NPCs to the EPSC-derived CM reduced oxidative stress, caspase-3 activity, cell apoptosis, fibrotic response, and cytokine production. Additionally, the EPSC-derived CM contained a higher concentration of Areg compared to that in the NPCs. Furthermore, Areg decreased oxidative stress, cell apoptosis, fibrosis, and inflammation via both PI3K/AKT and ERK1/2 signaling pathways. Conclusion: ECSCs reduce NPC apoptosis, oxidative stress, production of inflammatory factors, and fibrotic response via the release of Areg. Moreover, Areg can exert these protective effects on NPCs via PI3K/AKT and ERK1/2 signaling pathways. [ABSTRACT FROM AUTHOR]

Background: Excessive oxidative stress plays a critical role in the progression of various diseases, including intervertebral disk degeneration (IVDD). Recent studies have found that anemonin (ANE) possesses antioxidant and anti-inflammatory effects. However, the role of ANE in IVDD is still unclear. Therefore, this study investigated the effect and mechanism of ANE on H2O2 induced degeneration of nucleus pulposus cells (NPCs). Methods: NPCs were pretreated with ANE, and then treated with H2O2. NOX4 was upregulated by transfection of pcDNA-NOX4 into NPCs. Cytotoxicity was detected by MTT, oxidative stress-related indicators and inflammatory factors were measured by ELISA, mRNA expression was assessed by RT-PCR, and protein expression was tested by western blot. Results: ANE attenuated H2O2-induced inhibition of NPCs activity. H2O2 enhanced oxidative stress, namely, increased ROS and MDA levels and decreased SOD level. However, these were suppressed and pretreated by ANE. ANE treatment repressed the expression of inflammatory factors (IL-6, IL-1β and TNF-α) in H2O2-induced NPCs. ANE treatment also prevented the degradation of extracellular matrix induced by H2O2, showing the downregulation of MMP-3, 13 and ADAMTS-4, 5 and the upregulation of collagen II. NOX4 is a key factor regulating oxidative stress. Our study confirmed that ANE could restrain NOX4 and p-NF-κB. In addition, overexpression of NOX4 counteracted the antioxidant and anti-inflammatory activities of ANE in H2O2-induced NPCs, and the inhibition of the degradation of extracellular matrix induced by ANE was also reversed by overexpression of NOX4. Conclusion: ANE repressed oxidative stress, inflammation and extracellular matrix degradation in H2O2-induced NPCs by inhibiting NOX4/NF-κB pathway. Our study indicated that ANE might be a candidate drug for the treatment of IVDD. [ABSTRACT FROM AUTHOR]

Hypoxia-inducible factor (HIF) plays a crucial role in regulating the hypoxia-inducible state of nucleus pulposus cells in the intervertebral disc. In addition, the oxygen-dependent conversion of HIF-1α in nucleus pulposus cells is controlled by the protein proline 4-hydroxylase domain (PHD) family. To explore whether HIF-1α can be regulated by modulating PHD homologs to inhibit nucleus pulposus degeneration, PHD2-shRNAs were designed and a PHD2 interference vector was constructed. The expression of HIF-1α and PHD2 genes in the nucleus pulposus cells in the experimental group was detected by RT-PCR, and the expression of HIF-1α, MMP-2, Aggrecan, and Col II proteins in the P0-P3 cells in the experimental group and the control group was detected by Western blotting. The apoptosis of P0-P3 nucleus pulposus cells was detected by flow cytometry. After lentivirus infection, the interference efficiency of the PHD2 gene decreased with cell passage. The apoptosis of P1-P3 cells in the experimental group was significantly lower than that in the control group or degeneration group. Compared to the control group, the expression of HIF-1α, Aggrecan, and Col II proteins increased significantly, and the expression of MMP-2 protein decreased significantly. In conclusion, interference with PHD2 can upregulate the expression of HIF-1α, accelerate anabolism, reduce catabolism, inhibit apoptosis of nucleus pulposus cells, and then these can inhibit degeneration of nucleus pulposus cells. Our results can provide an effective therapeutic target in intervertebral discs during intervertebral disc degeneration, and this may have important clinical significance. [ABSTRACT FROM AUTHOR]

Intervertebral disc degeneration (IVDD) is a frequent and intractable chronic condition in orthopedics that causes enormous discomfort in patients' lives and thoughts, as well as a significant economic burden on society and the nation. As a result, understanding the pathophysiology of IVDD is critical. The pathophysiology of IVDD has been linked to numerous variables, including oxidative stress, apoptosis, matrix metalloproteinases, and inflammatory factors. Cellular senescence has recently attracted a lot of attention in the study of age-related diseases. It has been discovered that IVDD is intimately linked to human senescence, in which nucleus pulposus cell senescence may play a significant role. Previously, our group did a comprehensive and systematic clarification of the pathogenesis of IVDD from an immune perspective and discovered that the fundamental pathogenesis of IVDD is inflammatory upregulation and nucleus pulposus cell death caused by an imbalance in the immune microenvironment. In this review, we will treat nucleus pulposus cell senescence as a novelty point to clarify the pathophysiology of IVDD and further explore the probable relationship between senescence and immunity along with the dysregulation of the immunological microenvironment to propose new therapeutic approaches for IVDD.

Objective: The nucleus pulposus (NP) comprises notochordal NP cells (NCs) and chondrocyte-like NP cells (CLCs). Although morphological similarities between CLCs and chondrocytes have been reported, interactions between CLCs and NCs remain unclear. In this study, we aimed to clarify regulatory mechanisms of cells in the NP and chondrocytes.Design: We performed single-cell RNA sequencing (scRNA-seq) analysis of the articular cartilage (AC) and NP of three-year-old cynomolgus monkeys in which NCs were present. We then performed immunohistochemical analysis of NP and distal femur. We added sonic hedgehog (SHH) to primary chondrocyte culture.Results: The scRNA-seq analysis revealed that CLCs and some articular chondrocytes had similar gene expression profiles, particularly related to GLI1, the nuclear mediator of the hedgehog pathway. In the NP, cell–cell interaction analysis revealed SHH expression in NCs, resulting in hedgehog signaling to CLCs. In contrast, no hedgehog ligands were expressed by chondrocytes in AC samples. Immunohistochemical analysis of the distal end of femur indicated that SHH and Indian hedgehog (IHH) were expressed around the subchondral bone that was excluded from our scRNA-seq sample. scRNA-seq data analysis and treatment of primary chondrocytes with SHH revealed that hedgehog proteins mediated an increase in hypoxia-inducible factor 1-alpha (HIF-1α) levels.Conclusion: CLCs and some articular chondrocytes have similar transcriptional profiles, regulated by paracrine hedgehog proteins secreted from NCs in the NP and from the subchondral bone in the AC to promote the HIF-1α pathway.