RESUMO
Cell transplantation is being actively explored as a regenerative therapy for discogenic back pain. This study explored the regenerative potential of Tie2+ nucleus pulposus progenitor cells (NPPCs) from intervertebral disc (IVD) tissues derived from young (<25 years of age) and old (>60 years of age) patient donors. We employed an optimized culture method to maintain Tie2 expression in NP cells from both donor categories. Our study revealed similar Tie2 positivity rates regardless of donor types following cell culture. Nevertheless, clear differences were also found, such as the emergence of significantly higher (3.6-fold) GD2 positivity and reduced (2.7-fold) proliferation potential for older donors compared to young sources. Our results suggest that, despite obtaining a high fraction of Tie2+ NP cells, cells from older donors were already committed to a more mature phenotype. These disparities translated into functional differences, influencing colony formation, extracellular matrix production, and in vivo regenerative potential. This study underscores the importance of considering age-related factors in NPPC-based therapies for disc degeneration. Further investigation into the genetic and epigenetic alterations of Tie2+ NP cells from older donors is crucial for refining regenerative strategies. These findings shed light on Tie2+ NPPCs as a promising cell source for IVD regeneration while emphasizing the need for comprehensive understanding and scalability considerations in culture methods for broader clinical applicability.
Assuntos
Núcleo Pulposo , Receptor TIE-2 , Humanos , Núcleo Pulposo/metabolismo , Núcleo Pulposo/citologia , Receptor TIE-2/metabolismo , Receptor TIE-2/genética , Adulto , Pessoa de Meia-Idade , Masculino , Feminino , Idoso , Fatores Etários , Adulto Jovem , Proliferação de Células , Células Cultivadas , Regeneração , Células-Tronco/citologia , Células-Tronco/metabolismo , Degeneração do Disco Intervertebral/terapia , Disco Intervertebral/metabolismo , Disco Intervertebral/citologia , Diferenciação Celular , Adolescente , Transplante de Células-Tronco/métodos , AnimaisRESUMO
Lower back pain (LBP) is a common condition closely associated with intervertebral disc degeneration (IDD), causing a significant socioeconomic burden. Inflammatory activation in degenerated discs involves pro-inflammatory cytokines, dysregulated regulatory cytokines, and increased levels of nerve growth factor (NGF), leading to further intervertebral disc destruction and pain sensitization. Macrophage polarization is closely related to autophagy. Based on these pathological features, a structured biomimetic nanoparticle coated with TrkA-overexpressing macrophage membranes (TMNP@SR) with a rapamycin-loaded mesoporous silica core is developed. TMNP@SR acted like sponges to adsorbe inflammatory cytokines and NGF and delivers the autophagy regulator rapamycin (RAPA) into macrophages through homologous targeting effects of the outer engineered cell membrane. By regulating autophagy activation, TMNP@SR promoted the M1-to-M2 switch of macrophages to avoid continuous activation of inflammation within the degenerated disc, which prevented the apoptosis of nucleus pulposus cells. In addition, TMNP@SR relieved mechanical and thermal hyperalgesia, reduced calcitonin gene-related peptide (CGRP) and substance P (SP) expression in the dorsal root ganglion, and downregulated GFAP and c-FOS signaling in the spinal cord in the rat IDD model. In summary, TMNP@SR spontaneously inhibits the aggravation of disc inflammation to alleviate disc degeneration and reduce the ingress of sensory nerves, presenting a promising treatment strategy for LBP induced by disc degeneration.
Assuntos
Autofagia , Degeneração do Disco Intervertebral , Nanopartículas , Ratos Sprague-Dawley , Degeneração do Disco Intervertebral/tratamento farmacológico , Degeneração do Disco Intervertebral/metabolismo , Animais , Autofagia/efeitos dos fármacos , Nanopartículas/química , Ratos , Masculino , Camundongos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Dor Lombar/tratamento farmacológico , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Sirolimo/farmacologia , Dióxido de Silício/química , Dióxido de Silício/farmacologia , Núcleo Pulposo/metabolismo , Inflamação/tratamento farmacológico , Citocinas/metabolismo , Biomimética/métodos , Modelos Animais de Doenças , Fator de Crescimento Neural/metabolismo , Células RAW 264.7RESUMO
Excessive exercise is an etiological factor of intervertebral disc degeneration (IVDD). Engineered extracellular vesicles (EVs) exhibit excellent therapeutic potential for disease-modifying treatments. Herein, we fabricate an exercise self-powered triboelectric-responsive microneedle (MN) assay with the sustainable release of optogenetically engineered EVs for IVDD repair. Mechanically, exercise promotes cytosolic DNA sensing-mediated inflammatory activation in senescent nucleus pulposus (NP) cells (the master cell population for IVD homeostasis maintenance), which accelerates IVDD. TREX1 serves as a crucial nuclease, and disassembly of TRAM1-TREX1 complex disrupts the subcellular localization of TREX1, triggering TREX1-dependent genomic DNA damage during NP cell senescence. Optogenetically engineered EVs deliver TRAM1 protein into senescent NP cells, which effectively reconstructs the elimination function of TREX1. Triboelectric nanogenerator (TENG) harvests mechanical energy and triggers the controllable release of engineered EVs. Notably, an optogenetically engineered EV-based targeting treatment strategy is used for the treatment of IVDD, showing promising clinical potential for the treatment of degeneration-associated disorders.
Assuntos
Vesículas Extracelulares , Degeneração do Disco Intervertebral , Agulhas , Núcleo Pulposo , Optogenética , Degeneração do Disco Intervertebral/terapia , Degeneração do Disco Intervertebral/metabolismo , Vesículas Extracelulares/metabolismo , Animais , Núcleo Pulposo/metabolismo , Optogenética/métodos , Optogenética/instrumentação , Humanos , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Senescência Celular , Exodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/genética , Ratos , Dano ao DNA , Camundongos , Masculino , Modelos Animais de Doenças , Ratos Sprague-DawleyRESUMO
Objective: The mechanisms of intervertebral disc degeneration (IVDD) in low back pain (LBP) patients are multiples. In this study, we attempt to investigate whether melatonergic system plays a potential role in IVDD patients with LBP by analyzing their clinical specimens. The fucus will be given to the correlation between the melatonin receptor expression and intervertebral disc tissue apoptosis. Methods: In this clinical study, 107 lumbar intervertebral disc nucleus pulposus (NP) specimens from patients with LBP were collected with patients' consents. The disc height (DH) discrepancy ratio, range of motion and sagittal parameters of the pathological plane were measured and Pfirrmann grade was used to classified the grades of IVDD level. Discs at grades 1-3 were served as normal control and grades 4-5 were considered as IVDD. The expression levels of melatonin receptor 1A (MT1) and 1B (MT2) were measured by immunohistochemistry. The apoptosis of NP was assessed using TUNEL staining. Their potential associations among MT1/2, DH, apoptosis, sagittal parameters with IVDD and LBP were evaluated with statistical analysis. Results: The incidence of IVDD was positively associated with age and negatively related to VAS scores for LBP (p < 0.001). Patients with higher degree of IVDD also have higher DH discrepancy ratio (p < 0.001), higher prevalence of lumbar instability (p = 0.003) and higher cell apoptosis compared to the control. Nevertheless, no statistically significant correlation was identified between Pfirrmann grade and lumbar sagittal parameters. MT1 and MT2 both were highly expressed in the NP tissues. Importantly, MT1 expression but not MT2 was significantly increased in the intervertebral disc tissue of patients with IVDD and its level correlated well with cell apoptosis level and the severity of IVDD as well as lower VAS scores for LBP. Conclusion: The highly elevated MT1 expression was found in NP tissues of patients with IVDD and LBP compared to the control. This phenomenon probably reflects the compensating response of the body to the pathological alteration of the IVDD and LBP. Therefore, these findings provide the novel information to use selective agonists of MT1 to target IVDD and LBP clinically.
Assuntos
Apoptose , Degeneração do Disco Intervertebral , Dor Lombar , Humanos , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/patologia , Dor Lombar/patologia , Dor Lombar/metabolismo , Masculino , Feminino , Pessoa de Meia-Idade , Adulto , Núcleo Pulposo/metabolismo , Núcleo Pulposo/patologia , Vértebras Lombares/patologia , Receptor MT1 de Melatonina/metabolismo , Receptor MT2 de Melatonina/metabolismo , Idoso , Disco Intervertebral/patologia , Disco Intervertebral/metabolismoRESUMO
BACKGROUND: Human intervertebral disk degeneration (IVDD) is a sophisticated degenerative pathological process. A key cause of IVDD progression is nucleus pulposus cell (NPC) degeneration, which contributes to excessive endoplasmic reticulum stress in the intervertebral disk. However, the mechanisms underlying IVDD and NPC degeneration remain unclear. METHODS: We used interleukin (IL)-1ß stimulation to establish an NPC-degenerated IVDD model and investigated whether human urine-derived stem cell (USC) exosomes could prevent IL-1ß-induced NPC degeneration using western blotting, quantitative real-time polymerase chain reaction, flow cytometry, and transcriptome sequencing techniques. RESULTS: We successfully extracted and identified USCs and exosomes from human urine. IL-1ß substantially downregulated NPC viability and induced NPC degeneration while modulating the expression of SOX-9, collagen II, and aggrecan. Exosomes from USCs could rescue IL-1ß-induced NPC degeneration and restore the expression levels of SOX-9, collagen II, and aggrecan. CONCLUSIONS: USC-derived exosomes can prevent NPCs from degeneration following IL-1ß stimulation. This finding can aid the development of a potential treatment strategy for IVDD.
Assuntos
Exossomos , Interleucina-1beta , Degeneração do Disco Intervertebral , Núcleo Pulposo , Fatores de Transcrição SOX9 , Humanos , Interleucina-1beta/metabolismo , Exossomos/metabolismo , Degeneração do Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/terapia , Núcleo Pulposo/metabolismo , Núcleo Pulposo/patologia , Núcleo Pulposo/citologia , Núcleo Pulposo/efeitos dos fármacos , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOX9/genética , Animais , Células-Tronco/metabolismo , Células Cultivadas , Agrecanas/metabolismo , Agrecanas/genética , Masculino , Urina/citologia , Urina/química , Feminino , Colágeno Tipo II/metabolismoRESUMO
The senescence of nucleus pulposus (NP) cells (NPCs), which is induced by the anomalous accumulation of reactive oxygen species (ROS), is a major cause of intervertebral disc degeneration (IVDD). In this research, glutathione-doped carbon dots (GSH-CDs), which are novel carbon dot antioxidant nanozymes, were successfully constructed to remove large amounts of ROS for the maintenance of NP tissue at the physical redox level. After significantly scavenging endogenous ROS via exerting antioxidant activities, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and total antioxidant capacity, GSH-CDs with good biocompatibility have been demonstrated to effectively improve mitochondrial dysfunction and rescue NPCs from senescence, catabolism, and inflammatory factors in vivo and in vitro. In vivo imaging data and histomorphological indicators, such as the disc height index (DHI) and Pfirrmann grade, demonstrated prominent improvements in the progression of IVDD after the topical application of GSH-CDs. In summary, this study investigated the GSH-CDs nanozyme, which possesses excellent potential to inhibit the senescence of NPCs with mitochondrial lesions induced by the excessive accumulation of ROS and improve the progression of IVDD, providing potential therapeutic options for clinical treatment.
Assuntos
Carbono , Glutationa , Degeneração do Disco Intervertebral , Núcleo Pulposo , Estresse Oxidativo , Espécies Reativas de Oxigênio , Degeneração do Disco Intervertebral/tratamento farmacológico , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/patologia , Núcleo Pulposo/metabolismo , Núcleo Pulposo/efeitos dos fármacos , Núcleo Pulposo/patologia , Animais , Estresse Oxidativo/efeitos dos fármacos , Carbono/química , Carbono/farmacologia , Glutationa/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Pontos Quânticos/química , Antioxidantes/farmacologia , Masculino , Senescência Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Microambiente Celular/efeitos dos fármacos , Catalase/metabolismo , Catalase/farmacologia , Superóxido Dismutase/metabolismoRESUMO
Intervertebral disc degeneration (IVDD) is characterized by the senescence and declining vitality of nucleus pulposus cells (NPCs), often driven by mitochondrial dysfunction. This study elucidates that mesenchymal stem cells (MSCs) play a crucial role in attenuating NPC senescence by secreting mitochondria-containing microvesicles (mitoMVs). Moreover, it demonstrates that static magnetic fields (SMF) enhance the secretion of mitoMVs by MSCs. By distinguishing mitoMV generation from exosomes, this study shifts focus to understanding the molecular mechanisms of SMF intervention, emphasizing cargo transport and plasma membrane budding processes, with RNA sequencing indicating the potential involvement of the microtubule-based transport protein Kif5b. The study further confirms the interaction between Rab22a and Kif5b, revealing Rab22a's role in sorting mitoMVs into microvesicles (MVs) and potentially mediating subsequent plasma membrane budding. Subsequent construction of a gelatin methacrylate (GelMA) hydrogel delivery system further addresses the challenges of in vivo application and verifies the substantial potential of mitoMVs in delaying IVDD. This research not only sheds light on the molecular intricacies of SMF-enhanced mitoMV secretion but also provides innovative perspectives for future IVDD therapeutic strategies.
Assuntos
Micropartículas Derivadas de Células , Degeneração do Disco Intervertebral , Campos Magnéticos , Células-Tronco Mesenquimais , Mitocôndrias , Núcleo Pulposo , Células-Tronco Mesenquimais/metabolismo , Degeneração do Disco Intervertebral/terapia , Degeneração do Disco Intervertebral/metabolismo , Mitocôndrias/metabolismo , Animais , Micropartículas Derivadas de Células/metabolismo , Núcleo Pulposo/metabolismo , Humanos , Ratos , Cinesinas/metabolismo , Células Cultivadas , Ratos Sprague-Dawley , Proteínas rab de Ligação ao GTP/metabolismo , MasculinoRESUMO
As a common disease, cervical spondylosis (CS) results from the degeneration of the cervical intervertebral disc. However, there are still no effective clinical strategies for the treatment of this disease. Needle-scalpel (Ns), a therapy guided by traditional Chinese medicine theory, alleviates intervertebral disc degradation and is widely used in the clinic to treat CS. Stromal cell-derived factor-1 (SDF-1) and its receptor CXC receptor 4 (CXCR4) in nucleus pulposus cells play an important role in CS onset and development. This study aimed to explore whether Ns can relieve pain and regulate the SDF-1/CXCR4 axis in nucleus pulposus cells to inhibit apoptosis, thereby delaying cervical intervertebral disc degradation in a rat model of CS. It was found that the Ns-treated groups exhibited higher mechanical allodynia scores than the model group, and H&E staining, MRI, and scanning electron microscopy revealed that Ns therapy inhibited intervertebral disc degeneration. Additionally, Ns therapy significantly inhibited increases in the RNA and protein expression levels of SDF-1 and CXCR4. Furthermore, these treatments alleviated the apoptosis of nucleus pulposus cells, which manifested as a decline in the proportion of apoptotic nucleus pulposus cells and inhibition of the decrease in the levels of Bcl-2/Bax. These findings indicated that Ns mitigated CS-induced pain, inhibited the apoptosis of nucleus pulposus cells, and alleviated intervertebral disc degeneration in CS rats. These effects may be mediated by specifically regulating the SDF-1/CXCR4 signaling axis. Based on these findings, we conclude that Ns might serve as a promising therapy for the treatment of CS.
Assuntos
Apoptose , Quimiocina CXCL12 , Modelos Animais de Doenças , Degeneração do Disco Intervertebral , Núcleo Pulposo , Ratos Sprague-Dawley , Receptores CXCR4 , Animais , Receptores CXCR4/metabolismo , Receptores CXCR4/genética , Quimiocina CXCL12/metabolismo , Apoptose/efeitos dos fármacos , Núcleo Pulposo/metabolismo , Núcleo Pulposo/efeitos dos fármacos , Núcleo Pulposo/patologia , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/tratamento farmacológico , Ratos , Masculino , Vértebras Cervicais , Transdução de Sinais/efeitos dos fármacos , Espondilose/metabolismo , Espondilose/patologiaRESUMO
BACKGROUND: Cellular senescence features irreversible growth arrest and secretion of multiple proinflammatory cytokines. Cyclic GMP-AMP synthase (cGAS) detects DNA damage and activates the DNA-sensing pathway, resulting in the upregulation of inflammatory genes and induction of cellular senescence. This study aimed to investigate the effect of cGAS in regulating senescence of nucleus pulposus (NP) cells under inflammatory microenvironment. METHODS: The expression of cGAS was evaluated by immunohistochemical staining in rat intervertebral disc (IVD) degeneration model induced by annulus stabbing. NP cells were harvested from rat lumbar IVD and cultured with 10ng/ml IL-1ß for 48 h to induce premature senescence. cGAS was silenced by cGAS specific siRNA in NP cells and cultured with IL-1ß. Cellular senescence was evaluated by senescence-associated beta-galactosidase (SA-ß-gal) staining and flow cytometry. The expression of senescence-associated secretory phenotype including IL-6, IL-8, and TNF-a was evaluated by ELISA and western blotting. RESULTS: cGAS was detected in rat NP cells in cytoplasm and the expression was significantly increased in degenerated IVD. Culturing in 10ng/ml IL-1ß for 48 h induced cellular senescence in NP cells with attenuation of G1-S phase transition. In senescent NP cells the expression of cGAS, p53, p16, NF-kB, IL-6, IL-8, TNF-α was significantly increased while aggrecan and collagen type II was reduced than in normal NP cells. In NP cells with silenced cGAS, the expression of p53, p16, NF-kB, IL-6, IL-8, and TNF-α was reduced in inflammatory culturing with IL-1ß. CONCLUSION: cGAS was increased by NP cells in degenerated IVD promoting cellular senescence and senescent inflammatory phenotypes. Targeting cGAS may alleviate IVD degeneration by reducing NP cell senescence.
Assuntos
Senescência Celular , Degeneração do Disco Intervertebral , Nucleotidiltransferases , Núcleo Pulposo , Ratos Sprague-Dawley , Senescência Celular/fisiologia , Animais , Núcleo Pulposo/metabolismo , Núcleo Pulposo/patologia , Degeneração do Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/metabolismo , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Células Cultivadas , Ratos , Masculino , Inflamação/metabolismo , Inflamação/patologia , Interleucina-1beta/metabolismoRESUMO
Metformin has been the goto medical treatment for addressing type 2 diabetes mellitus (T2DM) as a frontline oral antidiabetic. Obesity, cancer and bone deterioration are linked to T2DM, which is considered a metabolic illness. Numerous diseases associated with T2DM, such as tumours, cardiovascular disease and bone deterioration, may be treated with metformin. Intervertebral disc degeneration (IVDD) is distinguished by degeneration of the spinal disc, accompanied by the gradual depletion of proteoglycans and water in the nucleus pulposus (NP) of the IVD, resulting in lower back pain. The therapeutic effect of metformin on IVDD has also attracted much attention. By stimulating AMPactivated kinase, metformin could enhance autophagy and suppress cell senescence, apoptosis and inflammation, thus effectively delaying IVDD. The present review aimed to systematically explain the development of IVDD and mechanism of metformin in the treatment and prevention of IVDD to provide a reference for the clinical application of metformin as adjuvant therapy in the treatment of IVDD.
Assuntos
Degeneração do Disco Intervertebral , Metformina , Metformina/uso terapêutico , Metformina/farmacologia , Degeneração do Disco Intervertebral/tratamento farmacológico , Degeneração do Disco Intervertebral/prevenção & controle , Degeneração do Disco Intervertebral/metabolismo , Humanos , Animais , Progressão da Doença , Núcleo Pulposo/efeitos dos fármacos , Núcleo Pulposo/metabolismo , Núcleo Pulposo/patologia , Hipoglicemiantes/uso terapêutico , Hipoglicemiantes/farmacologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Autofagia/efeitos dos fármacosRESUMO
Intervertebral disc degeneration (IVDD) is a prevalent chronic condition causing spinal pain and functional impairment. This study investigates the role of extracellular vesicles (EVs) derived from human umbilical cord mesenchymal stem cells (hUCMSCs) in regulating IVDD. Using RNA-seq, we analyzed differential expressions of lncRNA and miRNA in nucleus pulposus tissues from various mouse groups. We identified key regulatory molecules, MALAT1 and miRNA-138-5p, which contribute to IVDD. Further experiments demonstrated that MALAT1 can up-regulate SLC7A11 expression by competitively binding to miR-138-5p, forming a MALAT1/miR-138-5p/SLC7A11 coexpression regulatory network. This study elucidates the molecular mechanism by which hUCMSC-derived EVs regulate IVDD and could help develop novel therapeutic strategies for treating this condition. Our findings demonstrate that hUCMSCs-EVs inhibit ferroptosis in nucleus pulposus cells, thereby improving IVDD. These results highlight the therapeutic potential of hUCMSCs-EVs in ameliorating the development of IVDD, offering significant scientific and clinical implications for new treatments.
Assuntos
Vesículas Extracelulares , Degeneração do Disco Intervertebral , Células-Tronco Mesenquimais , MicroRNAs , RNA Longo não Codificante , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Degeneração do Disco Intervertebral/terapia , Degeneração do Disco Intervertebral/genética , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/patologia , Humanos , Células-Tronco Mesenquimais/metabolismo , Animais , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/genética , Camundongos , Núcleo Pulposo/metabolismo , Núcleo Pulposo/patologia , Cordão Umbilical/citologia , Cordão Umbilical/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Regulação da Expressão Gênica , Ferroptose/genéticaRESUMO
Intervertebral disc degeneration (IVDD), a common degenerative disc disease, is a major etiological factor for back pain, affecting a significant number of middle-aged and elderly individuals worldwide. Thus, IVDD is a major socio-economic burden. The factors contributing to the complex IVDD etiology, which has not been elucidated, include inflammation, oxidative stress, and natural aging. In particular, inflammation and aging of nucleus pulposus cells are considered primary pathogenic factors. Isorhapontigenin (ISO) is a polyphenolic compound commonly found in traditional Chinese herbs and grapes. We have demonstrated that ISO exerts anti-inflammatory and anti-aging effects and mitigates extracellular matrix (ECM) degradation. In this study, in vitro experiments revealed that, ISO delays aging and ECM degradation by promoting PI3K/AKT/mTOR-mediated autophagy. Meanwhile, in vivo experiments affirmed that ISO delays the progression of IVDD.
Assuntos
Autofagia , Senescência Celular , Matriz Extracelular , Degeneração do Disco Intervertebral , Núcleo Pulposo , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Serina-Treonina Quinases TOR , Degeneração do Disco Intervertebral/tratamento farmacológico , Degeneração do Disco Intervertebral/metabolismo , Núcleo Pulposo/efeitos dos fármacos , Núcleo Pulposo/metabolismo , Núcleo Pulposo/patologia , Serina-Treonina Quinases TOR/metabolismo , Autofagia/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Transdução de Sinais/efeitos dos fármacos , Matriz Extracelular/metabolismo , Matriz Extracelular/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Senescência Celular/efeitos dos fármacos , Masculino , Ratos Sprague-Dawley , Humanos , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Ratos , Células Cultivadas , EstilbenosRESUMO
BACKGROUND: Intervertebral disc degeneration (IDD) is a common musculoskeletal degenerative disease, which often leads to low back pain and even disability, resulting in loss of labor ability and decreased quality of life. Although many progresses have been made in the current research, the underlying mechanism of IDD remains unclear. The apoptosis of nucleus pulposus (NP) cells (NPCs) is an important pathological mechanism in intervertebral disc degeneration (IDD). This study evaluated the relationship between S100A6 and NPCs and its underlying mechanism. METHODS: Mass spectrometry, bioinformatics, and quantitative real-time polymerase chain reaction (qRT-PCR) analyses were used to screen and verify hub genes for IDD in human IVD specimens with different degeneration degrees. Western blotting, immunohistochemistry (IHC), and/or immunofluorescence (IF) were used to detect the expression level of S100A6 in human NP tissues and NPCs. The apoptotic phenotype of NPCs and Wnt/ß-catenin signaling pathway were evaluated using flow cytometry, western blotting, and IF. S100A6 was overexpressed or knocked down in NPCs to determine its impact on apoptosis and Wnt/ß-catenin signaling pathway activity. Moreover, we used the XAV-939 to inhibit and SKL2001 to activate the Wnt/ß-catenin signaling pathway. The therapeutic effect of S100A6 inhibition on IDD was also evaluated. RESULTS: S100A6 expression increased in IDD. In vitro, increased S100A6 expression promoted apoptosis in interleukin (IL)-1ß-induced NPCs. In contrast, the inhibition of S100A6 expression partially alleviated the progression of annulus fibrosus (AF) puncture-induced IDD in rats. Mechanistic studies revealed that S100A6 regulates NPC apoptosis via Wnt/ß-catenin signaling pathway. CONCLUSIONS: This study showed that S100A6 expression increased during IDD and promoted NPCs apoptosis by regulating the Wnt/ß-catenin signaling pathway, suggesting that S100A6 is a promising new therapeutic target for IDD.
Assuntos
Apoptose , Degeneração do Disco Intervertebral , Núcleo Pulposo , Proteína A6 Ligante de Cálcio S100 , Via de Sinalização Wnt , Núcleo Pulposo/metabolismo , Núcleo Pulposo/patologia , Apoptose/genética , Humanos , Proteína A6 Ligante de Cálcio S100/metabolismo , Proteína A6 Ligante de Cálcio S100/genética , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/genética , Degeneração do Disco Intervertebral/patologia , Animais , Masculino , Feminino , Ratos , Adulto , Pessoa de Meia-Idade , beta Catenina/metabolismo , beta Catenina/genética , Ratos Sprague-Dawley , Modelos Animais de Doenças , Proteínas de Ciclo CelularRESUMO
The nucleus pulposus (NP) in the intervertebral disc (IVD) arises from embryonic notochord. Loss of notochordal-like cells in humans correlates with onset of IVD degeneration, suggesting that they are critical for healthy NP homeostasis and function. Comparative transcriptomic analyses identified expression of progenitor-associated genes (GREM1, KRT18, and TAGLN) in the young mouse and non-degenerated human NP, with TAGLN expression reducing with aging. Lineage tracing using Tagln-CreERt2 mice identified peripherally located proliferative NP (PeriNP) cells in developing and postnatal NP that provide a continuous supply of cells to the entire NP. PeriNP cells were diminished in aged mice and absent in puncture-induced degenerated discs. Single-cell transcriptomes of postnatal Tagln-CreERt2 IVD cells indicate enrichment for TGF-ß signaling in Tagln descendant NP sub-populations. Notochord-specific removal of TGF-ß/BMP mediator Smad4 results in loss of Tagln+ cells and abnormal NP morphologies. We propose Tagln+ PeriNP cells are potential progenitors crucial for NP homeostasis.
Assuntos
Degeneração do Disco Intervertebral , Núcleo Pulposo , Células-Tronco , Núcleo Pulposo/metabolismo , Núcleo Pulposo/patologia , Degeneração do Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/genética , Animais , Humanos , Camundongos , Células-Tronco/metabolismo , Disco Intervertebral/metabolismo , Disco Intervertebral/patologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
OBJECTIVE: The continuously increasing extracellular matrix stiffness during intervertebral disc degeneration promotes disease progression. In an attempt to obtain novel treatment methods, this study aims to investigate the changes in nucleus pulposus cells under the stimulation of a stiff microenvironment. DESIGN: RNA sequencing and metabolomics experiments were combined to evaluate the primary nucleus pulposus and screen key targets under mechanical biological stimulation. Additionally, small molecules work in vitro were used to confirm the target regulatory effect and investigate the mechanism. In vivo, treatment effects were validated using a rat caudal vertebrae compression model. RESULTS: Our research results revealed that by activating TRPC6, hyperforin, a herbaceous extract can rescue the inflammatory phenotype caused by the stiff microenvironment, hence reducing intervertebral disc degeneration (IDD). Mechanically, it activates mitochondrial fission to inhibit PFKFB3. CONCLUSION: In summary, this study reveals the important bridging role of TRPC6 between mechanical stiffness, metabolism, and inflammation in the context of nucleus pulposus degeneration. TRPC6 activation with hyperforin may become a promising treatment for IDD.
Assuntos
Matriz Extracelular , Degeneração do Disco Intervertebral , Dinâmica Mitocondrial , Núcleo Pulposo , Floroglucinol , Ratos Sprague-Dawley , Animais , Núcleo Pulposo/metabolismo , Núcleo Pulposo/patologia , Núcleo Pulposo/efeitos dos fármacos , Degeneração do Disco Intervertebral/tratamento farmacológico , Degeneração do Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/metabolismo , Ratos , Floroglucinol/farmacologia , Floroglucinol/análogos & derivados , Floroglucinol/uso terapêutico , Dinâmica Mitocondrial/efeitos dos fármacos , Matriz Extracelular/metabolismo , Matriz Extracelular/efeitos dos fármacos , Masculino , Células Cultivadas , Humanos , Terpenos/farmacologia , Terpenos/uso terapêutico , Canais de Cátion TRPC/metabolismo , Modelos Animais de Doenças , Inflamação/tratamento farmacológicoRESUMO
BACKGROUND: Nucleus pulposus cell (NPC) senescence in intervertebral disc (IVD) tissue is the major pathological cause of intervertebral disc degeneration (IDD). N6-methyladenosine (m6A) methylation and gut microbiota play important roles in the progression of IDD. This study investigated whether methyltransferase-like 3 (METTL3) regulates TLR2 m6A modification and gut microbiota to influence NPC senescence. METHODS: An IDD rat model was established by lumbar IVD puncture and NPCs were challenged with IL-1ß to mimic IVD injury. IDD rats and IL-1ß-exposed NPCs were treated with METTL3-interfering lentivirus and the TLR2 agonist Pam3CSK4. Compositional changes in the rat gut microbiota were analyzed and fecal microbiota transplantation procedures were used. NPC senescence, cell cycle, and the expression of senescence-associated secretory phenotype (SASP) factors were assessed. The m6A enrichment of TLR2 and the binding of IGF2BP1 to TLR2 mRNA were examined. RESULTS: METTL3 and TLR2 were highly expressed in IDD rats. METTL3 silencing attenuated senescent phenotypes and reduced secretion of SASP factors. Pam3CSK4 reversed the beneficial effects of METTL3 silencing on NPC senescence and IVD injury. METTL3 stabilized TLR2 mRNA in an IGF2BP1-dependent manner. METTL3 silencing restored specific gut microbiota levels in IDD rats, which was further reversed by administration of Pam3CSK4. Fecal microbiota from METTL3 silenced IDD rats altered the pathological phenotypes of IDD rats. CONCLUSIONS: These results demonstrate the beneficial effects of METTL3 silencing on NPC senescence and amelioration of IVD injury, involving modulation of TLR2 m6A modification and gut microbiota. These findings support METTL3 silencing as a potential therapeutic target for IDD.
Assuntos
Senescência Celular , Microbioma Gastrointestinal , Degeneração do Disco Intervertebral , Metiltransferases , Núcleo Pulposo , Ratos Sprague-Dawley , Receptor 2 Toll-Like , Animais , Receptor 2 Toll-Like/metabolismo , Receptor 2 Toll-Like/genética , Ratos , Metiltransferases/metabolismo , Metiltransferases/genética , Núcleo Pulposo/metabolismo , Núcleo Pulposo/patologia , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/microbiologia , Masculino , Modelos Animais de Doenças , Metilação , Adenosina/análogos & derivados , Adenosina/metabolismoRESUMO
Intervertebral disc degeneration (IVDD) is a common degenerative disease which is closely related to low back pain (LBP) and brings huge economic and social burdens. In this study, we explored the therapeutic effects of Homoplantaginin (Hom) for IVDD due to its convincing anti-inflammatory and antioxidant functions. TNF-α was used to simulate the inflammatory environment for nucleus pulposus (NP) cells in vitro. We verified that Hom could alleviate the TNF-α-induced inflammation and disturbance of ECM homeostasis through blocking the NF-κB/MAPK signaling pathways. Subsequently, we screened the binding targets of Hom and confirmed that Hom could directly bind to TAK1 and inhibit its phosphorylation to down-regulate the inflammation-related pathways. The therapeutic effects of Hom on IVDD were further validated through a needle puncture rat model in vivo. Overall, Hom was a promising small molecule for IVDD early intervention, possessing huge clinical translational value.
Assuntos
Degeneração do Disco Intervertebral , MAP Quinase Quinase Quinases , NF-kappa B , Animais , Humanos , Masculino , Ratos , Células Cultivadas , Degeneração do Disco Intervertebral/tratamento farmacológico , Degeneração do Disco Intervertebral/metabolismo , MAP Quinase Quinase Quinases/metabolismo , MAP Quinase Quinase Quinases/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , NF-kappa B/metabolismo , NF-kappa B/antagonistas & inibidores , Núcleo Pulposo/metabolismo , Núcleo Pulposo/efeitos dos fármacos , Núcleo Pulposo/patologia , Ligação Proteica/fisiologia , Ligação Proteica/efeitos dos fármacos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/metabolismoRESUMO
This study aimed to reveal the specific role of early growth response protein 1 (EGR1) and nuclear receptor 4A3 (NR4A3) in nucleus pulposus cells (NPCs) and the related molecular mechanism and to identify a new strategy for treating intervertebral disc degeneration (IVDD). Bioinformatics analysis was used to explore and predict IVDD-related differentially expressed genes, and chromatin immunoprecipitation sequencing (ChIP-seq) revealed NR4A3 as the EGR1 target gene. An in vitro NPC model induced by tributyl hydrogen peroxide (TBHP) and a rat model induced by fibrous ring acupuncture were established. Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemical staining, immunofluorescence staining, and flow cytometry were used to detect the effects of EGR1 and NR4A3 knockdown and overexpression on NPC apoptosis and the expression of extracellular matrix (ECM) anabolism-related proteins. Interactions between EGR1 and NR4A3 were analyzed via ChIP-qPCR and dual luciferase assays. EGR1 and NR4A3 expression levels were significantly higher in severely degenerated discs (SDD) than in mildly degenerated discs (MDD), indicating that these genes are important risk factors in IVDD progression. ChIP-seq and RNA-seq revealed NR4A3 as a direct downstream target of EGR1, and this finding was verified by ChIP-qPCR and dual luciferase reporter experiments. Remarkably, the rescue experiments showed that EGR1 promotes TBHP-induced NPC apoptosis and impairs ECM anabolism, dependent on elevated NR4A3 expression. In summary, the EGR1-NR4A3 axis mediates the progression of NPC apoptosis and ECM impairment and is a potential therapeutic target in IVDD.
Assuntos
Apoptose , Proteína 1 de Resposta de Crescimento Precoce , Degeneração do Disco Intervertebral , Núcleo Pulposo , Estresse Oxidativo , Receptores dos Hormônios Tireóideos , Adulto , Animais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ratos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/genética , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/genética , Degeneração do Disco Intervertebral/patologia , Proteínas do Tecido Nervoso , Núcleo Pulposo/metabolismo , Núcleo Pulposo/patologia , Ratos Sprague-Dawley , Receptores de Esteroides/metabolismo , Receptores de Esteroides/genética , Receptores dos Hormônios Tireóideos/metabolismo , Receptores dos Hormônios Tireóideos/genética , Regulação para CimaRESUMO
BACKGROUND: Intervertebral disc (IVD) degeneration (IVDD) is a prevalent condition contributing to back pain and disability. Periostin (POSTN) has emerged as a potential molecular marker and therapeutic target in IVDD, prompting further investigation into its role and mechanisms. METHODS: This study employs bioinformatics analysis combined with experimental validation to explore the role of POSTN in IVDD. Gene expression datasets from the GEO database were analyzed to identify genes associated with IVDD, and the effects of POSTN on rat nucleus pulposus (NP) cells senescence and extracellular matrix (ECM) metabolism were assessed both in vitro and in vivo. RESULTS: Elevated POSTN expression was observed in degenerated discs from IVDD patients, correlating with disease severity. In vitro experiments demonstrated that POSTN promotes NP cells senescence and ECM metabolism in a dose- and time-dependent manner. In vivo studies confirmed that POSTN inhibition can ameliorate the progression of IVDD. Further mechanistic insights revealed that POSTN may exert its effects by activating the NF-κB and Wnt/ß-catenin signaling pathways. CONCLUSION: POSTN plays a significant role in the pathogenesis of IVDD, with its upregulated expression closely linked to NP cells senescence and ECM metabolism. Targeting POSTN could offer a novel therapeutic strategy for IVDD. Additionally, the study predicts small molecules that may inhibit POSTN expression, providing potential candidates for the development of new drug treatments.
Assuntos
Moléculas de Adesão Celular , Senescência Celular , Matriz Extracelular , Degeneração do Disco Intervertebral , NF-kappa B , Núcleo Pulposo , Ratos Sprague-Dawley , Via de Sinalização Wnt , Núcleo Pulposo/metabolismo , Núcleo Pulposo/patologia , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/patologia , Moléculas de Adesão Celular/metabolismo , Moléculas de Adesão Celular/genética , Animais , Matriz Extracelular/metabolismo , Humanos , NF-kappa B/metabolismo , Ratos , Masculino , Feminino , Pessoa de Meia-Idade , Adulto , beta Catenina/metabolismoRESUMO
Intervertebral disc degeneration (IVDD) severely affects the work and the quality of life of people. We previously demonstrated that silencing activation transcription factor 3 (ATF3) blocked the IVDD pathological process by regulating nucleus pulposus cell (NPC) ferroptosis, apoptosis, inflammation, and extracellular matrix (ECM) metabolism. Nevertheless, whether miR-874-3p mediated the IVDD pathological process by targeting ATF3 remains unclear. We performed single-cell RNA sequencing (scRNA-seq) and bioinformatics analysis to identify ATF3 as a key ferroptosis gene in IVDD. Then, Western blotting, flow cytometry, ELISA, and animal experiments were performed to validate the roles and regulatory mechanisms of miR-874-3p/ATF3 signalling axis in IVDD. ATF3 was highly expressed in IVDD patients and multiple cell types of IVDD rat, as revealed by scRNA-seq and bioinformatics analysis. GO analysis unveiled the involvement of ATF3 in regulating cell apoptosis and ECM metabolism. Furthermore, we verified that miR-874-3p might protect against IVDD by inhibiting NPC ferroptosis, apoptosis, ECM degradation, and inflammatory response by targeting ATF3. In vivo experiments displayed the protective effect of miR-874-3p/ATF3 axis on IVDD. These findings propose the potential of miR-874-3p and ATF3 as biomarkers of IVDD and suggest that targeting the miR-874-3p/ATF3 axis may be a therapeutic target for IVDD.