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BACKGROUND: Intervertebral disc degeneration is caused by damage and degeneration of the nucleus pulposus and annulus fibrosus tissues inside the intervertebral disc, resulting in structural and functional changes of the intervertebral disc. However, there is yet no effective drug treatment for intervertebral disc degeneration. OBJECTIVE: To investigate the inhibitory effect of syringin on intervertebral disc degeneration. METHODS: A total of 10 male Sprague-Dawley rats were selected, and the coccygeal intervertebral disc (Co4/Co5) of each rat was set as model group, Co5/Co6intervertebral disc as syringin group, and Co6/Co7 intervertebral disc as control group. The control group did not receive any treatment. In the model group and syringin group, a miniature puncture needle was used to puncture the annulus fibrosus to establish an intervertebral disc degeneration model. Immediately after modeling, 2. 5μL of normal saline and syringin solution (5μmol/L) were given in the model and syringin groups, respectively. Four weeks after injection, the samples were taken. The degree of intervertebral disc degeneration in rats was observed by hematoxylin-eosin and safranine O-fast green staining. The expressions of type II collagen, aggrecan and matrix metalloproteinases 3 and 13 in intervertebral disc tissue were analyzed by immunohistochemical staining. RESULTS AND CONCLUSION: Hematoxylin-eosin staining showed that in the model group, the height of intervertebral disc decreased, the cartilage endplate became thinner and cracked, the fibrous ring structure was disordered and cracked, and the nucleus pulposus disappeared;in the syringin group, the height of intervertebral disc was normal or slightly lower than that in the control group, the degree of cartilage endplate degeneration was lighter than that in the model group, the fiber circle permutation was relatively regular with no cracks, and the nucleus pulposus was partially shrunk. Safranine O-fast green staining showed that in the model group, the cartilage endplate of the intervertebral disc was defective and the calcified layer of cartilage became thinner, showing obvious degeneration. The structure and morphology of intervertebral disc cartilage endplate in the syringin group recovered to some extent. Immunohistochemical staining showed that, compared with the control group, the expressions of type II collagen and aggrecan in the intervertebral disc cartilage were decreased in the model group (P＜0. 000 1), while the expressions of matrix metalloproteinases 3 and 13 increased (P＜0. 000 1). Compared with the model group, the expressions of type II collagen and aggrecan in the intervertebral disc cartilage tissue were increased in the syringin group (P＜0. 001, P＜0. 000 1), while the expressions of matrix metalloproteinases 3 and 13 decreased (P＜0. 001, P＜0. 000 1). These results showed that syringin could improve the structure and function of intervertebral disc by inhibiting the expression of matrix metalloproteinases 3 and 13 and increasing the expression of type II collagen and aggrecan, thus preventing and slowing down the procession of intervertebral disc degeneration [ABSTRACT FROM AUTHOR]

BACKGROUND: Intervertebral disc degeneration is a condition caused by the combined effects of various factors, such as cellular apoptosis, oxidative stress, and inflammatory responses. Currently, it is believed that the core of this condition lies in the degeneration and apoptosis of nucleus pulposus cells (NPCs). However, the specific pathological mechanisms remain unclear. OBJECTIVE: By investigating the expression of the phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt)/hypoxia-inducible factor 1-alpha (HIF-1α) signaling pathway and the apoptosis of NPCs under different oxygen concentrations, to clarify the biological characteristics of NPCs under varying oxygen levels, and to explore the mechanisms of intervertebral disc degeneration. METHODS: Normal and degenerated NPCs were collected. The second-generation cells were used for imaging identification. The third-generation cells were cultured in the following conditions: 37°C, air, 100% humidity. Leibovitz’s medium containing 100 μmol/L CoCl2 and 10% fetal bovine serum medium containing 100 μmol/L H2O2 were used to create distinct oxygen concentration environments for the third-generation cells, allowing for the investigation of cellular responses and behaviors under normal, low oxygen, and oxidative stress conditions. The third-generation cells were divided into six groups: normal NPCs+hypoxia, normal NPCs+normoxia, normal NPCs+oxidative stress, degenerated NPCs+hypoxia, degenerated NPCs+normoxia, and degenerated NPCs+oxidative stress groups. Cell viability and proliferation were assessed using the cell counting kit-8 method. Cell apoptosis was detected using flow cytometry. RT-PCR and western blot assay were utilized to examine the protein and mRNA expression levels of PI3K, AKT, and HIF-1α. RESULTS AND CONCLUSION: At different oxygen concentrations, the cell proliferation rate of both normal and degenerated NPCs decreased as the oxygen concentration increased. Conversely, the apoptosis rate increased as the oxygen concentration rose (P < 0.05). With the normal NPCs+hypoxia group as the control group, the effect of degenerated NPCs on the apoptosis rate was highly significant (P < 0.001). Oxygen concentration had a highly significant impact on both NPC proliferation rate and apoptosis rate (P < 0.001). The interaction between cell degeneration and oxygen concentration significantly affected both NPC proliferation and apoptosis rates (P < 0.05). At different oxygen concentrations, the protein and mRNA expression levels of PI3K, AKT, and HIF-1α in normal NPCs were highest under low oxygen concentration, followed by oxidative stress environment, and lowest under normoxia. In degenerated NPCs, the protein and mRNA expression levels of PI3K, AKT, and HIF-1α decreasd as the oxygen concentration increased. The protein and mRNA expression levels of PI3K and Akt in normal NPCs were significantly higher than those in degenerated NPCs (P < 0.05). With the normal NPCs+hypoxia group as the control group, the effects of normal or degenerated NPCs, as well as oxygen concentration, on the protein and mRNA expression of PI3K, AKT, and HIF-1α were highly significant. The interaction between cell degeneration and oxygen concentration also had an extremely significant effect on the protein and mRNA expression of PI3K, AKT, and HIF-1α (P < 0.001). To conclude, there is a close correlation between the proliferation and apoptosis of NPCs and both oxygen concentration and the cellular functional state. The PI3K/Akt/HIF-1α signaling pathway exhibits antagonistic regulatory effects under various cellular functional states and different oxygen concentration environments. The mechanism may be associated with the activation of the PI3K/Akt signaling pathway, which consequently transcribes HIF-1α, thus maintaining the balance of reactive oxygen species metabolism. This pathway plays a significant role in inhibiting oxidative stress, antagonizing NPCs apoptosis, and consequently delaying intervertebral disc degeneration. [ABSTRACT FROM AUTHOR]

Yuying Ge,1,&ast; Yuepeng Xie,2,&ast; Junlei Chai,3,&ast; Weifeng Ji,4,&ast; Xiulong Lou,1 Kun Tian,2 Ronghua Bao,3 Chengliang Wu,1 Hongfeng Ruan1 1Institute of Orthopaedics and Traumatology, The First Affiliated Hospital of Zhejiang Chinese Medical University (Zhejiang Provincial Hospital of Traditional Chinese Medicine), Hangzhou, People’s Republic of China; 2The Third Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, People’s Republic of China; 3Hangzhou Fuyang Hospital of TCM Orthopedics and Traumatology, Hangzhou, People’s Republic of China; 4Department of Orthopaedics, The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Hongfeng Ruan; Chengliang Wu, Institute of Orthopaedics and Traumatology, The First Affiliated Hospital of Zhejiang Chinese Medical University, 548 Binwen Road, Hangzhou, 310053, People’s Republic of China, Fax +86 571 86613684, Email rhf@zcmu.edu.cn; wcl@zcmu.edu.cnBackground: Intervertebral disc (IVD) degeneration (IVDD) is highly prevalent among the elderly population and stands as a leading cause of low back pain. Our prior studies have highlighted the therapeutic potential of Liuwei Dihuang decoction (LWDHD) and its component Cornus officinalis (CO)-derived compounds in alleviating IVDD and osteoarthritis, suggesting beneficial effects of CO in treating degenerative osteoarthropathies. However, uncertainty remains regarding the optimal CO dosage within LWDHD and its potential mechanism for effectively treating IVDD.Objective: To ascertain the optimal dosage of CO within LWDHD for enhancing its therapeutic efficacy in treating IVDD, through a comparison of its effects across varied dosages using a mouse IVDD model.Methods: Eight-week-old male C57BL/6J mice were subjected to a lumbar spine instability surgery to induce an IVDD model and received a modified LWDHD formulation containing varied dosages of CO (original dose of CO, or 5- or 10-time dose of CO (referred to as 1 × CO, 5 × CO, and 10 × CO)) for 8 weeks. The therapeutic efficacy on IVDD was evaluated through changes in lumbar spine function, histopathological morphology, extracellular matrix metabolism, nucleus pulposus cell viability, sensory nerve ingrowth, and nucleus pulposus (NP) cell pyroptosis.Results: Augmenting CO levels in LWDHD led to a dose-dependent increase in the levels of CO-sourced active compounds in the plasma of mice. The modified LWDHD formulations, particularly the 5 × CO, exhibited a favorable pharmacological effect on lumbar function, structural integrity, ECM composition, NP cell viability, and sensory nerve ingrowth. Importantly, all 3 formulations notably mitigated NP cell pyroptosis by activating NRF2/KEAP1 pathway, with the 5 × CO formulation exhibiting superior efficacy. Additionally, a comprehensive score analysis indicated that 5 × CO formulation achieved the highest score.Conclusion: These data underscore that elevating the dosage of CO to a specific threshold can enhance the effectiveness of LWDHD in treating IVDD.Keywords: intervertebral disc degeneration, liuwei dihuang decoction, nucleus pulposus cell, pyroptosis, Cornus officinalis

BACKGROUND: Nucleus pulposus cell apoptosis is the main pathological basis for intervertebral disc degeneration, and inflammation and peroxidation are important factors leading to apoptosis in the nucleus pulposus. Studies have shown that matrine has antioxidant, senescent, inflammatory and apoptotic effects, and may be a potential drug for the treatment of disc degeneration. OBJECTIVE: To investigate the effect of matrine on apoptosis of nucleus pulposus cells in rats with intervertebral disc degeneration by regulating the cyclic GMP-AMP synthase (cGAS) - stimulator of interferon genes (STING) signaling pathway. METHODS: (1) Nucleus pulposus cells of rats at a logarithmic phase were randomly separated into a control group, a model group, a low-dose matrine group, a high-dose matrine group, an empty group, and a high-dose matrine+cGAS overexpression group. Except for the control group, cell models of intervertebral disc degeneration were established in the other groups through oxygen-glucose deprivation. At the same time of modeling, the low-dose and high-dose groups were treated with 0.4 and 0.8 mmol/L matrine, respectively, and the empty group was transfected with the empty plasmid, while the high-dose+cGAS overexpression group was treated with 0.8 mmol/L matrine with the transfection of the cGAS overexpression plasmid. After 24 hours of treatment, cell activity and apoptosis, intracellular levels of reactive oxygen species, superoxide dismutase, tumor necrosis factor α and interleukin 1β, and intracellular expression of apoptotic proteins and cGAS-STING pathway proteins were detected. (2) Sixty Sprague-Dawley rats were randomized into six groups (n=10 per group): control group, model group, low-dose matrine group, high-dose matrine group, empty group, and high-dose+cGAS overexpression group. After 12 weeks of modeling, 60 and 120 mg/kg matrine were given by gavage in the low-dose and high-dose matrine groups, respectively (once a day), and the empty plasmid was injected into the tail vein in the empty group (2 times/week), while the high-dose+cGAS overexpression group was given 120 mg/kg matrine by gavage and injected with cGAS overexpression plasmid to the tail vein. Treatment in each group was given consecutively for 3 weeks. Samples were taken after drug administration and assayed for apoptosis, levels of reactive oxygen species, superoxide dismutase, tumor necrosis factor α and interleukin 1β, as well as apoptotic protein and cGAS-STING pathway protein expression. RESULTS AND CONCLUSION: Compared with the control group, in the model group, cell activity and superoxide dismutase levels were decreased (P < 0.05), and apoptosis rate, levels of reactive oxygen species, tumor necrosis factor α and interleukin 1β, and the expression of cGAS, STING, cleaved caspase-3 and Bax proteins were elevated (P < 0.05). Matrine dose-dependently ameliorated the above changes in each index due to cellular modeling (P < 0.05), whereas cGAS overexpression partially antagonized the ameliorative effect of high-dose matrine. Similar results to the in vitro cellular experiments were obtained in animal experiments. These results indicate that matrine could inhibit inflammation and oxidative stress by blocking the cGAS-STING signaling, which in turn attenuates apoptosis and elevates the activity of nucleus pulposus cells in rats with intervertebral disc degeneration. [ABSTRACT FROM AUTHOR]

BACKGROUND: Stem cell transplantation is a new way to prevent and cure intervertebral disc degeneration. However, whether the transplanted stem cells can survive, proliferate, differentiate, and restore the function of nucleus pulposus cells after transplantation, is the key and difficult point to overcome. OBJECTIVE: To explore the effects of Bushenhuoxue decoction on survival, proliferation, and nucleus pulposus-like differentiation of adipose-derived stem cells. METHODS: A Transwell chamber was used to construct a co-culture model of human adipose-derived stem cells and human degenerative nucleus pulposus cells. The experiment was divided into control group, model group, drug-containing serum group, and drug-free serum group. Except for the control group, the co-culture system of other groups was treated with 50 μmol/L tert-butyl hydrogen peroxide for 24 hours. The drug-containing serum group and drug-free serum group were treated with DMEM low-glucose complete culture medium containing drug-containing serum of Bushenhuoxue decoction or drug-free serum with 20% volume fraction for 48 hours. The sublayer adipose-derived stem cells were taken. Toluidine blue staining was used to detect proteoglycan synthesis levels. Real-time PCR method was used to detect mRNA expression of type II collagen, proteoglycan and SRY-box transcription factor 9. The protein expression of SOX9 was detected by western blot assay. Lactate dehydrogenase assay was used to detect cytotoxicity. Flow cytometry was used to detect reactive oxygen species, and β-galactosidase staining was used to detect cell senescence. RESULTS AND CONCLUSION: (1) Compared with the control group, the proportion of necrotic cells in the model group increased; toluidine blue staining became lighter, and the expression levels of type II collagen, proteoglycan, SOX9 mRNA and SOX9 protein decreased (P < 0.05). Compared with the model group, the drug-containing serum of Bushenhuoxue decoction could significantly reduce cell injury and promote the expression of type II collagen, proteoglycan, SOX9 mRNA, and SOX9 protein (P < 0.05), but the improvement in the drug-free serum group was not significant (P > 0.05). (2) Compared with the control group, the contents of cytotoxicity, reactive oxygen species, and cell senescence in the model group were significantly increased. Compared with the model group, the microenvironment of the coculture system was significantly improved by drug-containing serum of Bushenhuoxue decoction (P < 0.05), while drug-free serum had no significant effect on the microenvironment of the co-culture system (P > 0.05). (3) The results show that Bushenhuoxue decoction can promote the survival, proliferation, and nucleus pulposus-like differentiation of adipose-derived stem cells. [ABSTRACT FROM AUTHOR]

Intervertebral disc degenerative diseases are one of the main causes of lower back pain, with significant limitations in both traditional conservative and surgical treatments. In recent years, biopolymeric nanomaterials have become a hotspot in research due to their excellent biodegradability, biocompatibility, highly precise drug delivery, prolonged drug release time, and enhanced drug efficacy. This paper systematically reviews the relevant research progress of biopolymeric nanomaterials in the regenerative therapy of degenerative intervertebral discs, providing useful insights for achieving precise treatment of degenerative intervertebral disc diseases. [ABSTRACT FROM AUTHOR]

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

Shang Gao,1 Chenmoji Wang,1 Lijie Qi,2 Songlin Liang,1 Xintian Qu,1 Wei Liu,3,&ast; Nianhu Li4,&ast; 1The First Clinical Medical College of Shandong University of Traditional Chinese Medicine, Jinan, Shandong Province, People’s Republic of China; 2Qilu Hospital of Shandong University, Jinan, Shandong Province, People’s Republic of China; 3Shandong University of Traditional Chinese Medicine, Jinan, Shandong Province, People’s Republic of China; 4Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, Shandong Province, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Nianhu Li; Wei Liu, Email tigerlee073@126.com; weiliu800109@163.comBackground: The method of action of Bushen Formula (BSHXF) in the treatment of intervertebral disc degeneration (IVDD) was uncovered in this work using in vivo and in vitro tests. To clarify the mechanism of action of BSHXF, we validated the rat intervertebral disc degeneration model and the nucleus pulposus cell degeneration model.Methods: In an in vivo model of IVDD the study explores the impact of BSHXF on mitochondrial function, pro-inflammatory cytokines, pro-apoptotic factors, and matrix metalloproteinases. Additionally, it evaluates the induced degeneration of nucleus pulposus (NP) cells in an in vitro model stimulated by interleukin-1 β (IL-1β). The study measures the effects of BSHXF on both the inflammatory response and mitochondrial function.Results: The MRI results showed that BSHXF reduced intervertebral disc volume reduction and degradation of NP tissue. HE, SO-FG and immunofluorescence further confirmed the protective effect of BSHXF on degenerative intervertebral discs. BSHXF reduced the inflammatory levels of IL-6 IL-1β and TNF-α in degenerative intervertebral disc tissue. Meanwhile, JC-1, mPTP and ROS detection revealed that BSHXF can restore mitochondrial function by regulating the expression of antioxidant proteins, playing a protective role in NP cells. Finally, the WB results showed that BSHXF can alleviate IL-1β mediate the degeneration of NP cells. BSHXF can alleviate NP cell apoptosis by inhibiting the expression of bax, cleaved caspase-3, caspase-3, and cyt-c, and increasing the expression of Bcl-2.Conclusion: This study reveals that BSHXF inhibits the development of inflammatory factors, which may play a significant role in intervertebral disc degeneration. This implies that BSHXF is a suitable herbal medication for future research into inflammatory cytokine treatment.Keywords: intervertebral disc degeneration, nucleus pulposus cell, BSHXF, apoptosis, inflammatory

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

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

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

Vitamin D affects calcium homeostasis and is a crucial factor in cytoplasmic and mitochondrial interactions implicated in muscle energy metabolism. This study probed the effect of vitamin D on lumbar disk degeneration based on the PINK1/Parkin signaling pathway. Primary nucleus pulposus cells from Sprague-Dawley rats were cultured to construct a model of oxidative stress in vitro, and the expression and activity of target proteins in related genes and signaling pathways were detected via real-time quantitative polymerase chain reaction and western blotting. We simulated disk degeneration by inducing high levels of oxidative stress in nucleus pulposus cells with 100 μM hydrogen peroxide. Vitamin D treatment could effectively increase the expression of genes related to the nucleus pulposus matrix as well as decrease disk degeneration factors’ expression. Further mechanism results displayed that vitamin D effectively reduced oxidative stress indexes in nucleus pulposus cell s and alleviated hydrogen peroxide-mediated degeneration of nucleus pulposus cells by activating the PINK1/Parkin pathway. We confirm that vitamin D activates the PINK1/Parkin pathway to play an antioxidative stress role and delay the aging and degeneration of nucleus pulposus cells. [ABSTRACT FROM AUTHOR]

This study aimed to explore the specific function of M2 macrophages in intervertebral disc degeneration (IDD). Intervertebral disc (IVD) samples from normal (n = 4) and IDD (n = 6) patients were collected, and the expression of M2-polarized macrophage marker, CD206, was investigated using immunohistochemical staining. Nucleus pulposus cells (NPCs) in a TNF-α environment were obtained, and a mouse caudal IVD puncture model was established. Mice with Rheb deletions, specifically in the myeloid lineage, were generated and subjected to surgery-induced IDD. IDD-induced damage and cell apoptosis were measured using histological scoring, X-ray imaging, immunohistochemical staining, and TdT-mediated dUTP nick end labeling (TUNEL) assay. Finally, mice and NPCs were treated with R-spondin-2 (Rspo2) or anti-Rspo2 to investigate the role of Rspo2 in IDD. Accumulation of CD206 in human and mouse IDD tissues was detected. Rheb deletion in the myeloid lineage (RheBcKO) increased the number of CD206+ M2-like macrophages (mean difference 18.6% [15.7–21.6%], P < 0.001), decreased cell apoptosis (mean difference −15.6% [−8.9 to 22.2%], P = 0.001) and attenuated the IDD process in the mouse IDD model. NPCs treated with Rspo2 displayed increased extracellular matrix catabolism and apoptosis; co-culture with a conditioned medium derived from RheBcKO mice inhibited these changes. Anti-Rspo2 treatment in the mouse caudal IVD puncture model exerted protective effects against IDD. Promoting CD206+ M2-like macrophages could reduce Rspo2 secretion, thereby alleviating experimental IDD. Rheb deletion may help M2-polarized macrophages accumulate and attenuate experimental IDD partially by inhibiting Rspo2 production. Hence, M2-polarized macrophages and Rspo2 may serve as therapeutic targets for IDD. [ABSTRACT FROM AUTHOR]

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

Objective·To investigate the effect of hydrogel stiffness on nucleus pulposus cell phenotype and its function in repairing intervertebral disc degeneration in rats.Methods·Methacrylate gelatin (GelMA) hydrogels with different concentrations were constructed. The stiffness of the hydrogels was investigated by using rheological analysis and uniaxial compression test. The microstructure and morphology of the hydrogels were observed by scanning electron microscopy (SEM). Nucleus pulposus cells with normal phenotype were inoculated on the surface of GelMA hydrogels. The biocompatibility of the hydrogel was evaluated by live-dead cell staining and the growth pattern of nucleus pulposus cells on hydrogels with different stiffness was observed with phalloidin staining under microscope. Immunofluorescence staining was performed to examine the nuclear localization of Yes-associated protein (YAP) and real-time quantitative reverse transcription PCR (qRT-PCR) was used to detect the expression levels of nucleus pulposus cell-associated genes [neural cell adhesion molecule 1 (Ncam-1), aggrecan (Acan), sex-determing region of Y chromosome (SRY)-box transcription factor 9 (Sox9)]. A rat caudal acupuncture intervertebral disc degeneration model was established. Nucleus pulposus cells cultured on different hydrogels were harvested and injected into the degenerated discs separately. Four weeks after surgery, magnetic resonance imaging (MRI) was performed to analyze the water content of the intervertebral discs in each group. Histological tests were performed to examine the disc structure and proteoglycan levels.Results·The elastic modulus of the hydrogels was 1 kPa and 200 kPa when the concentration of GelMA prepolymerisation solution was at 4% and 15% respectively. SEM observation revealed that the hydrogels showed a loose and porous microstructure, and the porosity of hydrogels decreased significantly with the decrease of their stiffness. In vitro experiments demonstrated that both GelMA hydrogel mediums showed good biocompatibility and the ability to support cell proliferation. Nucleus pulposus cells cultured on the soft matrix (4%GelMA) had a lower elongation and spreading area than those cultured on the stiff matrix (15%GelMA), showing a tendency of YAP concentration in the cytoplasm. The gene expression of nucleus pulposus cells was examined and the levels of Sox9, Acan and Ncam-1 in the soft matrix hydrogel group were 23.7, 6.6 and 12.7 times of those in the control group respectively. In vivo experiments on rat disc degeneration showed that the soft hydrogel matrix group had higher disc water content and structural integrity than the stiff hydrogel matrix group.Conclusion·Compared to stiff GelMA hydrogels, hydrogels with low stiffness better maintain the growth phenotypes in the nucleus pulposus cells and have better therapeutic effect on disc degeneration in vivo.

Abstract Background Low back pain (LBP) has become the second leading cause of disability worldwide, which has brought great economic burden to people. It is generally believed that intervertebral disc degeneration (IDD) is the main cause of LBP. This study aimed to explore the role of circ-STC2 in the pathogenesis of IDD. Methods Nucleus pulposus cells (NPCs) were treated with T-Butyl Hydrogen Peroxide (TBHP) to establish IDD model in vitro. RT-qPCR was performed to detect mRNA expressions. The cell viability was detected with CCK-8 assay. The levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), Fe2+ and glutathione (GSH) of NPCs were measured by corresponding kits. The protein expressions were determined by western blot. Dual-luciferase reporter and RNA pull-down assays were conducted to verify the relationship between circ-STC2 or transferrin recepto 2 (TFR2) and miR-486-3p. Results Circ-STC2 and TFR2 expressions were up-regulated in IDD tissues, and miR-486-3p expression was down-regulated. Knockdown of circ-STC2 promoted the cell viability and inhibited the ferroptosis of the NPCs. The GSH levels, and glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) protein expressions were increased, the LDH, MDA and Fe2+ levels and achaete-scute complexlike 4 (ASCL4) protein expressions were decreased after circ-STC2 knockdown. Knockdown of miR-486-3p abrogated the si-circ-STC2 effects and overexpression of TFR2 reversed the miR-486-3p mimic effects. Conclusions Circ-STC2 inhibits the cell viability, induced the ferroptosis of the TBHP treated NPCs via targeting miR-486-3p/TFR2 axis.

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

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

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

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

Low back pain (LBP) is a major public health problem worldwide and a significant health and economic burden. Intervertebral disc degeneration (IDD) is the reason for LBP. However, we have not identified effective therapeutic strategies to address this challenge. With accumulating knowledge on the role of circular RNAs in the pathogenesis of IDD, we realised that circular RNAs (circRNAs) may have tremendous therapeutic potential and clinical application prospects in this field. This review presents an overview of the current understanding of characteristics, classification, biogenesis, and function of circRNAs and summarises the protective and detrimental circRNAs involved in the intervertebral disc that have been studied thus far. This review is aimed to help researchers better understand the regulatory role of circRNAs in the progression of IDD, reveal their clinical therapeutic potential, and provide a theoretical basis for the prevention and targeted treatment of IDD.

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

Background: Low back pain (LBP) has become the second leading cause of disability worldwide, which has brought great economic burden to people. It is generally believed that intervertebral disc degeneration (IDD) is the main cause of LBP. This study aimed to explore the role of circ-STC2 in the pathogenesis of IDD. Methods: Nucleus pulposus cells (NPCs) were treated with T-Butyl Hydrogen Peroxide (TBHP) to establish IDD model in vitro. RT-qPCR was performed to detect mRNA expressions. The cell viability was detected with CCK-8 assay. The levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), Fe2+ and glutathione (GSH) of NPCs were measured by corresponding kits. The protein expressions were determined by western blot. Dual-luciferase reporter and RNA pull-down assays were conducted to verify the relationship between circ-STC2 or transferrin recepto 2 (TFR2) and miR-486-3p. Results: Circ-STC2 and TFR2 expressions were up-regulated in IDD tissues, and miR-486-3p expression was down-regulated. Knockdown of circ-STC2 promoted the cell viability and inhibited the ferroptosis of the NPCs. The GSH levels, and glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) protein expressions were increased, the LDH, MDA and Fe2+ levels and achaete-scute complexlike 4 (ASCL4) protein expressions were decreased after circ-STC2 knockdown. Knockdown of miR-486-3p abrogated the si-circ-STC2 effects and overexpression of TFR2 reversed the miR-486-3p mimic effects. Conclusions: Circ-STC2 inhibits the cell viability, induced the ferroptosis of the TBHP treated NPCs via targeting miR-486-3p/TFR2 axis. [ABSTRACT FROM AUTHOR]

Objective·To investigate the effect of hydrogel stiffness on nucleus pulposus cell phenotype and its function in repairing intervertebral disc degeneration in rats. Methods·Methacrylate gelatin (GelMA) hydrogels with different concentrations were constructed. The stiffness of the hydrogels was investigated by using rheological analysis and uniaxial compression test. The microstructure and morphology of the hydrogels were observed by scanning electron microscopy (SEM). Nucleus pulposus cells with normal phenotype were inoculated on the surface of GelMA hydrogels. The biocompatibility of the hydrogel was evaluated by livedead cell staining and the growth pattern of nucleus pulposus cells on hydrogels with different stiffness was observed with phalloidin staining under microscope. Immunofluorescence staining was performed to examine the nuclear localization of Yes-associated protein (YAP) and real-time quantitative reverse transcription PCR (qRT-PCR) was used to detect the expression levels of nucleus pulposuscell-associated genes [neural cell adhesion molecule 1 (Ncam-1), aggrecan (Acan), sex-determing region of Y chromosome (SRY)-box transcription factor 9 (Sox9)]. A rat caudal acupuncture intervertebral disc degeneration model was established. Nucleus pulposus cells cultured on different hydrogels were harvested and injected into the degenerated discs separately. Four weeks after surgery, magnetic resonance imaging (MRI) was performed to analyze the water content of the intervertebral discs in each group. Histological tests were performed to examine the disc structure and proteoglycan levels. Results·The elastic modulus of the hydrogels was 1 kPa and 200 kPa when the concentration of GelMA prepolymerisation solution was at 4% and 15% respectively. SEM observation revealed that the hydrogels showed a loose and porous microstructure, and the porosity of hydrogels decreased significantly with the decrease of their stiffness. In vitro experiments demonstrated that both GelMA hydrogel mediums showed good biocompatibility and the ability to support cell proliferation. Nucleus pulposus cells cultured on the soft matrix (4%GelMA) had a lower elongation and spreading area than those cultured on the stiff matrix (15%GelMA), showing a tendency of YAP concentration in the cytoplasm. The gene expression of nucleus pulposus cells was examined and the levels of Sox9, Acan and Ncam-1 in the soft matrix hydrogel group were 23.7, 6.6 and 12.7 times of those in the control group respectively. In vivo experiments on rat disc degeneration showed that the soft hydrogel matrix group had higher disc water content and structural integrity than the stiff hydrogel matrix group. Conclusion·Compared to stiff GelMA hydrogels, hydrogels with low stiffness better maintain the growth phenotypes in the nucleus pulposus cells and have better therapeutic effect on disc degeneration in vivo. [ABSTRACT FROM AUTHOR]

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

Background: Intervertebral disk degeneration (IVDD) is a major cause of low back pain and one of the most common health problems all over the world. However, the early diagnosis of IVDD is still restricted. The purpose of this study is to identify and validate the key characteristic gene of IVDD and analyze its correlation with immune cell infiltration. Methods: 3 IVDD-related gene expression profiles were downloaded from the Gene Expression Omnibus database to screen for differentially expressed genes (DEGs). Gene Ontology (GO) and gene set enrichment analysis (GSEA) were conducted to explore the biological functions. Two machine learning algorithms were used to identify characteristic genes, which were tested to further find the key characteristic gene. The receiver operating characteristic curve was performed to estimate the clinical diagnostic value of the key characteristic gene. The excised human intervertebral disks were obtained, and the normal nucleus pulposus (NP) and degenerative NP were carefully separated and cultured in vitro. The expression of the key characteristic gene was validated by real-time quantitative PCR (qRT-PCR). The related protein expression in NP cells was detected by Western blot. Finally, the correlation was investigated between the key characteristic gene and immune cell infiltration. Results: A total of 5 DEGs, including 3 upregulated genes and 2 downregulated genes, were screened between IVDD and control samples. GO enrichment analysis showed that DEGs were enriched to 4 items in BP, 6 items in CC, and 13 items in MF. They mainly included the regulation of ion transmembrane transport, transporter complex, and channel activity. GSEA suggested that the cell cycle, DNA replication, graft versus host disease, and nucleotide excision repair were enriched in control samples, while complement and coagulation cascades, Fc γ R-mediated phagocytosis, neuroactive ligand-receptor interaction, the NOD-like receptor signaling pathway, gap junctions, etc., were enriched in IVDD samples. Furthermore, ZNF542P was identified and tested as key characteristic gene in IVDD samples through machine learning algorithms and showed a good diagnostic value. The results of qRT-PCR showed that compared with normal NP cells, the expression of ZNF542P gene was decreasedin degenerated NP cells. The results of Western blot suggested that compared with normal NP cells, the expression of NLRP3 and pro Caspase-1 was increased in degenerated NP cells. Finally, we found that the expression of ZNF542P was positively related to the proportions of T cells gamma delta (γδT cells). Conclusion: ZNF542P is a potential biomarker in the early diagnosis of IVDD and may be associated with the NOD-like receptor signaling pathway and the infiltration of γδT cells. [ABSTRACT FROM AUTHOR]

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

Study Design.: Isolated human nucleus pulposus (hNP) cells from the degenerated intervertebral disc (IVD) were incubated under hydrostatic pressure (HP) and evaluated for regenerative potential. Objectives.: To characterize metabolic turnover in hNP cells isolated from degenerated IVDs classified by Pfirrmann grade under physiologically relevant HP at high osmolality in vitro. Summary of Background Data.: We demonstrated that bovine caudal nucleus pulposus cells isolated from healthy cows produced more extracellular matrix under cyclic HP followed by constant pressure (mimicking physiological intradiscal pressure in humans) than under no pressure in vitro. We assessed the effects of pressure on human degenerated cells isolated under the same regimen of pressure used for bovine cells. Materials and Methods.: hNP cells isolated from discarded tissue classified as Pfirrmann grade 2 to 3 (n = 13: age, 46.7 ± 14.0) and grade 4 (n = 13: age, 53.0 ± 11.5) were incubated under cyclic HP at 0.2 to 0.7 MPa, 0.5 Hz for 2 days followed by constant pressure at 0.3 MPa for 1 day, repeated twice over 6 days. The gene expression and immunohistology of matrix molecules and catabolic and anticatabolic proteins were evaluated. Results.: Aggrecan and collagen type II expression were significantly more upregulated under HP in grades 2 to 3 than in grade 4 tissues (both, P < 0.01). Linear regression analysis showed a positive correlation between matrix metalloproteinase 13 and tissue inhibitor for metalloproteinase 2 expression in grades 2 to 3, whereas a negative correlation was found in grade 4 (P < 0.05). Immunohistological staining revealed the activation of a mechanoreceptor, transient receptor potential vanilloid 4, under HP. Conclusions.: Resident cells in mild-moderate degenerated discs classified as Pfirrmann grade 2 to 3 have the potential to promote extracellular matrix production and maintain adequate cell viability under physiological spinal loading. Relevance.: This study explored the potential of degenerated remnant nucleus pulposus cells under a physiological environment, possibly leading to establishing strategies for IVD regeneration. [ABSTRACT FROM AUTHOR]

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

BACKGROUND: Platelet-rich plasma therapy for disc degeneration has received increasing attention. Platelet-rich plasma preparation methods for intervertebral disc repair include manual preparation using one-spin centrifugation and kit preparation using two-spin centrifugation. There was insufficient evidence of which preparation method is better. OBJECTIVE: To compare the effects of intervention of porine nucleus pulposus cells with manually prepared platelet-rich plasma, kit-prepared platelet-rich plasma and whole blood and to explore the better preparation method of platelet-rich plasma for intervertebral disc repair. METHODS: The porcine venous blood was extracted to prepare platelet-rich plasma using the manual preparation method of one-spin centrifugation and OSP kit preparation method of two-spin centrifugation. Some whole blood was reserved as control. And PBS was used to be blank control. The concentration of platelets and leukocytes in platelet-rich plasma was detected to calculate fold enrichment. ELISA was used to detect the concentration of transforming growth factor-β1, platelet-derived growth factor-BB, tumor necrosis factor-α and interleukin-1β in platelet-rich plasma lysate. Porcine nucleus pulposus cells were cultivated to P3 and intervened with prepared platelet-rich plasma and whole blood lysate. Cell counting kit-8 was used to detect the proliferation of nucleus pulposus cells after intervention with platelet-rich plasma and whole blood lysate. Western blot was used to detect the anabolic effects of platelet-rich plasma on type II collagen and aggrecan in nucleus pulposus cells. RESULTS AND CONCLUSION: Compared with the manual group, the OSP group showed significantly longer preparation time and higher platelet and leukocyte concentrations in the preparation of platelet-rich plasma. The fold enrichment of platelet-rich plasma was 5.6 in the OSP group and 3.0 in the manual group, while the fold enrichment of leukocytes was 2.21 and 1.36 respectively. The concentrations of transforming growth factor-β1, platelet-derived growth factor-BB, tumor necrosis factor-α and interleukin-1β of the OSP group were significantly higher than those of the manual group (P < 0.05). In the cell counting kit-8 detection of cell proliferation, the absorbance value at 450 nm of the OSP group was significantly higher than that of the other groups (P < 0.000 1); there was no significant difference in the absorbance value at 450 nm of the manual group compared with the whole blood and control groups (P=0.192 7). The relative expression of type II collagen in the OSP group was significantly higher than that in the other groups (P < 0.000 1). As for the synthesis of aggrecan, the relative expression of aggrecan in the manual group was lower than that in the remaining groups (P=0.040 5). These findings indicate that compared with the manual preparation method, the kit preparation method represented by OSP consumes more time and can acquire significantly higher concentration of leukocytes and proinflammatory cytokines. However, the kit preparation method can obtain higher concentrations of platelets and growth factors and has better cell proliferation effect and synthesis effect of extracellular matrix. For a better therapeutic effect of platelet-rich plasma, it is suggested to enhance the isolation of leukocytes when using the kit preparation method. [ABSTRACT FROM AUTHOR]

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

BACKGROUND: Endplate chondrocytes are regulated by many factors in the microenvironment of the intervertebral disc and the influence of microenvironmental changes inside the intervertebral disc caused by abnormal stress on endplate chondrocytes still needs to be explored. OBJECTIVE: To observe the exosome secretion of nucleus pulposus cells under cyclic mechanical tension conditions and the effect of exosomes on endplate chondrocytes under this condition. METHODS: Nucleus pulposus cells were isolated and cultured from the nucleus pulposus of patients undergoing lumbar surgery in vitro. The cells were loaded with cyclic mechanical tension by FX-5000T system. Exosomes secreted from nucleus pulposus cells under stress and non-stress conditions were extracted by magnetic bead method. Purity and concentration of nucleus pulposus cells-derived exosomes were identified by flow cytometry. The exosomes were labeled with PKH67 fluorescent dye and incubated with endplate chondrocytes for 24 hours. Endplate chondrocytes cultured alone were used as control. The uptake of exosomes by endplate chondrocytes was observed under inverted fluorescence microscope. Then the apoptosis and activity of endplate chondrocytes were observed. Type II collagen, depolymerized protein like metalloproteinase 5, matrix metalloproteinase 3, bone morphogenetic protein 2, and β-catenin mRNA expression were detected. RESULTS AND CONCLUSION: Under certain cyclic mechanical tension conditions, nucleus pulposus cells could be promoted to secrete exosomes, and the particle concentration of nucleus pulposus cells-derived exosomes was higher than that under non-stress conditions. Under the inverted fluorescence microscope, both stress and non-stress nucleus pulposus cells-derived exosomes could be ingested by endplate chondrocytes. Compared with the control group, the viability of endplate chondrocytes decreased significantly (P < 0.05) and the apoptosis rate increased in the stress group (P < 0.05). Compared with the control group, the mRNA expression of matrix metalloproteinase 3 and bone morphogenetic protein 2 in endplate chondrocytes decreased significantly in the non-stress group (P < 0.05). Compared with the control group, the mRNA expression of type I collagen decreased significantly in the stress group (P < 0.05), while the mRNA expression of depolymerized protein like metalloproteinase 5, matrix metalloproteinase 3 and β-catenin increased significantly in the stress group (P < 0.05). Compared with the non-stress group, the mRNA expression of type II collagen decreased significantly in the stress group, while the mRNA expression of depolymerized protein like metalloproteinase 5, matrix metalloproteinase 3 and bone morphogenetic protein 2 increased significantly in the stress group (P < 0.05). Therefore, abnormal stress could promote the exosome secretion of nucleus pulposus cells, and the exosomes under this condition could inhibit the viability of endplate chondrocytes and cause the disorder of cell matrix synthesis and catabolism. [ABSTRACT FROM AUTHOR]

Abstract Functionalized self-assembling peptides, which display functional growth-factor bioactivity, can be designed by connecting the C-terminus of a pure self-assembling peptide with a short functional motif. In this study, we designed a novel functionalized peptide (RADA16-SNVI) in which an SNVI motif with hBMP-7 activity was conjugated onto the C-terminus of the RADA16 peptide via solid-phase synthesis. A mix of RADA16-SNVI and RADA16 solutions was used to create a functionalized peptide nanofiber scaffold (SNVI-RADA16). The hydrogels were analyzed by atomic force microscopy, circular dichroism, and scanning electron microscopy. The results showed that the SNVI-RADA16 solution effectively formed hydrogel. Next, we seeded the SNVI-RADA16 scaffold with adipose-derived stem cells (ADSCs) and investigated whether it displayed biological properties of nucleus pulposus tissue. SNVI-RADA16 displayed good biocompatibility with the ADSCs and induced their expression. Cells in SNVI-RADA16 gel had a greater secretion of the extracellular matrix marker collagen type II and aggrecan compared to ADSCs grown in monolayer and control gel (p

Background: Intervertebral disc degeneration (IVDD) is the leading cause of low back pain, and apoptosis plays a key role in its pathogenesis. Distal-less homeobox 5 (Dlx5) has been reported to induce cell apoptosis. Melatonin, as a powerful antiapoptotic agent, has been widely reported., Aim: This study aimed to investigate the role of DLX5 in the pathogenesis of IVDD and the potential therapeutic role of melatonin in targeting DLX5 in IVDD., Materials & Methods: Western blotting, RT-qPCR, immunohistochemistry, si-DLX5, Ex-DLX5, flow cytometry, and immunofluorescence were used to examine the regulatory effect of DLX5 on apoptosis. Therapeutic efficacy was assessed by the intraperitoneal injection of melatonin into IVDD mice., Results: The expression level of DLX5 is significantly increased in IVDD, and the expression levels were positively correlated with the grade of IVDD. DLX5 was significantly upregulated in TNF-α-induced degenerative NP cells. Degenerative NP cells transfected with si-DLX5 exhibited significantly less apoptosis than control cells. Melatonin significantly alleviated IVDD in surgically induced IVDD model mice., Discussion: The results revealed that the expression of DLX5 was positively correlated with the severity of IVDD and that melatonin ameliorated DLX5-induced apoptosis and extracellular matrix imbalance by inhibiting the TGF-β/Smad signaling pathway. This study may provide therapeutic strategies to alleviate inflammation-induced apoptosis IVDD-associated inflammation-induced apoptosis., Conclusion: DLX5 plays an important role in IVDD progression by promoting apoptosis, and melatonin represents a promising therapeutic strategy for alleviating IVDD-associated inflammation and apoptosis., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Author(s). JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Background: Erastin has been found to induce ferroptosis; however, whether erastin may have roles other than ferroptosis inducer in cells is unknown. Nutrient deficiency is one of the major causes of many diseases including intervertebral disc (IVD) degeneration., Purpose: The current study investigates the effect of erastin in nucleus pulposus cells under nutrient deprivation condition., Study Design: Experiment in vitro and ex vivo., Methods: The effect of erastin on the cell survival of nucleus pulposus cells was evaluated in fetal bovine serum (FBS) and glucose deprivation condition. RSL3 and ferrostatin-1 were applied to illustrate whether the effect of erastin is ferroptosis dependent. The involvement of solute carrier family 7, membrane 11(SLC7A11), autophagy as well as mechanistic target of rapamycin kinase complex 1(mTORC1) and transcription factor EB (TFEB) were assessed to demonstrate the working mechanism of erastin., Results: Erastin may induce cell death at the concentration of ≥ 5μM; however, it may protect nucleus pulposus cells against nutrient deprivation induced cell death at lower concentration (0.25-1μM) and the effect of erastin is ferroptosis independent. The mechanism study showed that the effect of erastin may relate to its SCL7A11 regulation, as SCL7A11 knock-down may have the similar effect as erastin. Furthermore, it was also demonstrated that mTORC1-TFEB mediated autophagy was involved in protective effect of erastin., Conclusions: Low dose erastin may promote cell survival under nutrient deprivation condition, and its effect is ferroptosis independent; erastin may exert its protective effect through mTORC1-TFEB mediated autophagy regulation., Clinical Significance: Nutrient deprivation is a major contributor to intervertebral disc degeneration. Our in vitro and ex vivo study showed that low dose of erastin may suppress nutrient deprivation induced cell death in IVD degeneration. Although it was not validated in vivo model due to lack of in vivo nutrient deprivation induced IVD degeneration model currently, this study may still provide a potential therapeutic option for IVD degeneration, which of cause need further validation., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier Inc.)

Low back pain is thought to be mainly caused by intervertebral disc degeneration (IVDD), and there is a lack of effective treatments. Cellular senescence and matrix degradation are important factors that cause disc degeneration. Mitochondrial dysfunction induced by oxidative stress is an important mechanism of cellular senescence and matrix degradation in the nucleus pulposus (NP), and mitophagy can effectively remove damaged mitochondria, restore mitochondrial homeostasis, and mitigate the damage caused by oxidative stress. Optineurin (OPTN) is a selective mitophagy receptor, and its role in intervertebral disc degeneration remains unclear. Here, we aimed to explore the effect of OPTN on H2O2-induced nucleus pulposus cell (NPCs) senescence and matrix degradation in a rat model of disc degeneration. Western blot analysis showed that OPTN expression was reduced in degenerative human and rat nucleus pulposus tissues and increased in H2O2-induced senescent NPCs. OPTN overexpression significantly inhibited H2O2-induced senescence and increased matrix-associated protein expression in NPCs, but OPTN knockdown showed the opposite effect. As previous reports have suggested that mitophagy significantly reduces mitochondrial damage and reactive oxygen species (ROS) caused by oxidative stress, and we used the mitophagy agonist CCCP, the mitophagy inhibitor cyclosporin A (CsA), and the mitochondrial ROS (mtROS) scavenger mitoTEMPO and confirmed that OPTN attenuated NPCs senescence and matrix degeneration caused by oxidative stress by promoting mitophagy to scavenge damaged mitochondria and excess reactive oxygen species, thereby slowing the progression of IVDD. In conclusion, our research suggests that OPTN is involved in IVDD and exerts beneficial effects against IVDD. [ABSTRACT FROM AUTHOR]

Abstract Background Nucleus pulposus cell (NPC) degeneration is widely accepted as one of the major causes of intervertebral disc (IVD) degeneration (IVDD). The pathogenesis of IVDD is complex and consists of inflammation, oxidative stress, and the loss of extracellular matrix (ECM). Cannabinoid type 2 receptor (CB2) has been shown to be involved in the pathological mechanism of a variety of diseases due to its anti-inflammatory effects and antioxidative stress capacity. Method In Vitro, H2O2 was used to induce degeneration of nucleus pulposus cells, mRNA and protein expression level was determined by RT-PCR and Western Blot, and Immunocytochemical staining were used to detect expression of collagen II, aggrecan, MMP3/13, superoxide dismutase 2 (SOD2) and inducible nitric oxide synthase (iNOS). In vivo, the potential therapeutic effect of CB2 was detected in the rat acupuncture model. Result In vitro, we found that the CB2 agonist (JWH133) treatment reduced the oxidative stress level in NPCs induced by hydrogen peroxide (H2O2) treatment. Furthermore, the expression of inflammatory cytokines was also decreased by JWH133 treatment. We found that collagen II and aggrecan expression was preserved, whereas matrix metalloproteinase levels were reduced. In vivo, we established a rat model by needle puncture. Imaging assessment revealed that the disc height index (DHI) and morphology of IVD were significantly improved, and the disc degeneration process was delayed by treatment of JWH133. Furthermore, immunohistochemical (IHC) staining revealed that JWH133 could inhibit the degradation of collagen II and decrease the expression of MMP3. Conclusions The experiment indicates the oxidative stress and inflammatory response of rat NPCs induced by H2O2 could be inhibited by activating CB2. This study reveals that CB2 activation can effectively delay the development of IVDD, providing an effective therapeutic target for IVDD.

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

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

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

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

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

Overactivated inflammation and catabolism induced by proinflammatory macrophages are involved in the pathological processes of intervertebral disc (IVD) degeneration (IVDD). Our previous study suggested the protective role of inhibiting heat shock protein 90 (HSP90) in IVDD, while the underlying mechanisms need advanced research. The current study investigated the effects of HSP90 inhibitor 17-AAG on nucleus pulposus (NP) inflammation and catabolism induced by M1-polarized macrophages. Immunohistochemical staining of degenerated human IVD samples showed massive infiltration of macrophages, especially M1 phenotype, as well as elevated levels of interleukin (IL)-1β, tumor necrosis factor (TNF)-α and matrix metalloproteinase (MMP)13. The conditioned medium (CM) of inflamed NP cells (NPCs) enhanced M1 polarization of macrophages, while the CM of M1 macrophages but not M2 macrophages promoted the expression of inflammatory factors and matrix proteases in NPCs. Additionally, we found that 17-AAG could represent anti-inflammatory and anti-catabolic effects by modulating both macrophages and NPCs. On the one hand, 17-AAG attenuated the pro-inflammatory activity of M1 macrophages via inhibiting nuclear factor-κB (NF-κB) pathway and mitogen-activated protein kinase (MAPK) pathways. On the other hand, 17-AAG dampened M1-CM-induced inflammation and catabolism in NPCs by upregulating HSP70 and suppressing the Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) pathway. Moreover, both in vitro IVD culture models and murine disc puncture models supported that 17-AAG treatment decreased the levels of inflammatory factors and matrix proteases in IVD tissues. In conclusion, HSP90 inhibitor 17-AAG attenuates NP inflammation and catabolism induced by M1 macrophages, suggesting 17-AAG as a promising candidate for IVDD treatment.

Intervertebral disc degeneration (IDD) is closely associated with low back pain, but its underlying mechanism remains unclear. Cholesterol is an essential nutrient in mammalian cells. Alterations in cholesterol levels lead to impairments in cell physiology, such as cell proliferation and signal transduction. Previous clinical studies demonstrated that hypercholesterolemia could be a potential risk factor for IDD, but how cholesterol induces IDD remains unknown. The current study aimed to explore the regulatory role of cholesterol in IDD development and the potential underlying mechanisms. It was found that different forms of cholesterol levels were elevated in degenerative nucleus pulposus (NP) tissues in both humans and Sprague–Dawley rats. Rats fed a high cholesterol diet (HCD) exhibited degenerative features in the lumbar intervertebral disc compared with those fed a standard diet. Interestingly, this effect could be abolished by cholesterol-lowering drug atorvastatin. In NP cells treated with TNF-α and IL-1β, a significantly higher level of cholesterol was observed. These results suggested a pivotal role of cholesterol in the progression of IDD. We also observed accelerated pyroptosis in NP cells and extracellular matrix (ECM) degradation in the rat NP cells treated with exogenous cholesterol. We further demonstrated that endoplasmic reticulum stress was responsible for cholesterol-induced pyroptosis and ECM degradation. Moreover, RNA-seq analysis revealed that the mature form of SREBP1 (mSREBP1), an important regulator of lipid metabolism, is involved in regulating endoplasmic reticulum stress in knockdown experiments. In conclusion, this study demonstrated that cholesterol could induce pyroptosis in NP cells and ECM degradation by activating endoplasmic reticulum stress through stimulating mSREBP1 in IDD.

Intervertebral disc (IVD) degeneration is the leading cause of low back pain, which has a striking impact on numerous patients. Therefore, comprehensively illuminating the regulatory mechanisms of IVD degeneration is of great significance. Here, we discuss the latest advances in understanding the main epigenetic mechanisms regulating IVD degeneration. [ABSTRACT FROM AUTHOR]

Bergenin is a C‐glucoside of 4‐O‐methyl gallic acid with a variety of biological activities, such as antioxidant and anti‐inflammatory. Herein, we investigated the involvement of bergenin in the protective effect against H2O2‐induced oxidative stress and apoptosis in human nucleus pulposus cells (HNPCs) and the underlying mechanisms. HNPCs were cotreated with various concentrations of bergenin and 200 μM H2O2 for 24 h. Cell viability was detected by Cell Counting Kit‐8 and lactate dehydrogenase release assays. Reactive oxygen species (ROS) was evaluated utilizing 2′,7′‐dichlorofluorescein‐diacetate. Superoxide dismutase (SOD) and catalase (CAT) activities and glutathione (GSH) levels were measured to assess oxidative stress. Apoptosis was evaluated using terminal deoxynucleotidyl transferase dUTP nick end labeling and caspase‐3/7 activity assays. Expression of protein was determined by western blotting. Results indicated that treatment with bergenin significantly alleviated H2O2‐induced viability reduction and ROS overproduction in HNPCs in a dose‐dependent manner. Bergenin alleviated H2O2‐induced oxidative stress in HNPCs by increased activity of superoxide dismutase and level of glutathione peroxidase. H2O2‐induced apoptosis and activity of caspase‐3/7 were also suppressed by bergenin treatment in HNPCs. Western blotting showed that H2O2‐induced decrease in expression of peroxisome proliferator‐activated receptor γ (PPAR‐γ) and increase in nuclear factor κB (NF‐κB) were inhibited by bergenin. However, the inhibitory effect of bergenin on H2O2‐induced viability reduction, oxidative stress and apoptosis were noticeably abrogated in PPAR‐γ knockdown HNPCs. In conclusion, our results indicated that bergenin alleviates H2O2‐induced oxidative stress and apoptosis in HNPCs by activating PPAR‐γ and suppressing NF‐κB pathway. [ABSTRACT FROM AUTHOR]

Background: Nucleus pulposus cell (NPC) degeneration is widely accepted as one of the major causes of intervertebral disc (IVD) degeneration (IVDD). The pathogenesis of IVDD is complex and consists of inflammation, oxidative stress, and the loss of extracellular matrix (ECM). Cannabinoid type 2 receptor (CB2) has been shown to be involved in the pathological mechanism of a variety of diseases due to its anti-inflammatory effects and antioxidative stress capacity. Method: In Vitro, H2O2 was used to induce degeneration of nucleus pulposus cells, mRNA and protein expression level was determined by RT-PCR and Western Blot, and Immunocytochemical staining were used to detect expression of collagen II, aggrecan, MMP3/13, superoxide dismutase 2 (SOD2) and inducible nitric oxide synthase (iNOS). In vivo, the potential therapeutic effect of CB2 was detected in the rat acupuncture model. Result: In vitro, we found that the CB2 agonist (JWH133) treatment reduced the oxidative stress level in NPCs induced by hydrogen peroxide (H2O2) treatment. Furthermore, the expression of inflammatory cytokines was also decreased by JWH133 treatment. We found that collagen II and aggrecan expression was preserved, whereas matrix metalloproteinase levels were reduced. In vivo, we established a rat model by needle puncture. Imaging assessment revealed that the disc height index (DHI) and morphology of IVD were significantly improved, and the disc degeneration process was delayed by treatment of JWH133. Furthermore, immunohistochemical (IHC) staining revealed that JWH133 could inhibit the degradation of collagen II and decrease the expression of MMP3. Conclusions: The experiment indicates the oxidative stress and inflammatory response of rat NPCs induced by H2O2 could be inhibited by activating CB2. This study reveals that CB2 activation can effectively delay the development of IVDD, providing an effective therapeutic target for IVDD. [ABSTRACT FROM AUTHOR]

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

Nucleus pulposus cells (NPCs) apoptosis and inflammation are the extremely critical factors of intervertebral disc degeneration (IVDD). Nevertheless, the underlying procedure remains mysterious. Macrophage migration inhibitory factor (MIF) is a cytokine that promotes inflammation and has been demonstrated to have a significant impact on apoptosis and inflammation. For this research, we employed a model of NPCs degeneration stimulated by lipopolysaccharides (LPS) and a rat acupuncture IVDD model to examine the role of MIF in vitro and in vivo, respectively. Initially, we verified that there was a significant rise of MIF expression in the NP tissues of individuals with IVDD, as well as in rat models of IVDD. Furthermore, this augmented expression of MIF was similarly evident in degenerated NPCs. Afterwards, it was discovered that ISO-1, a MIF inhibitor, effectively decreased the quantity of cells undergoing apoptosis and inhibited the release of inflammatory molecules (TNF-α, IL-1β, IL-6). Furthermore, it has been shown that the PI3K/Akt pathway plays a vital part in the regulation of NPCs degeneration by MIF. Ultimately, we showcased that the IVDD process was impacted by the MIF inhibitor in the rat model. In summary, our experimental results substantiate the significant involvement of MIF in the degeneration of NPCs, and inhibiting MIF activity can effectively mitigate IVDD. [ABSTRACT FROM AUTHOR]