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Low back pain (LBP) is a major physical and socioeconomic challenge worldwide. Nucleus pulposus (NP) is directly associated with LBP due to intervertebral disc (IVD) degeneration. IVD degeneration is mainly caused by structural and matrix-related changes within the IVD occurring during aging and degeneration. Mesenchymal stem cells (MSCs) can differentiate into multiple mesenchymal lineages under specific stimulatory conditions. This study is aimed at evaluating the effectiveness of the nucleus pulposus cell (NPC) conditioned medium for promoting the expression of MSCs and at confirming the expression of healthy NP phenotypic markers recently recommended by the Spine Research Interest Group. Expression was investigated using quantitative polymerase chain reaction (qPCR) and western blotting under normoxic and hypoxic conditions. qPCR and western blotting demonstrated significant upregulation of NP marker expression in MSCs cultured under hypoxic conditions and treated with the 50% or 100% NPC conditioned medium, compared with those cultured under normoxic conditions. Upregulation was highest in the presence of the 100% NPC conditioned medium compared with the control group (aggrecan, p < 0.01; brachyury, p < 0.05; collagen II, p < 0.001; KRT8, p < 0.01; KRT19, p < 0.001; and Shh, p < 0.01). The expression levels of genes in MSCs treated with the 50% NPC conditioned medium also showed upregulation compared with the control group (collagen II, p < 0.05; KRT8, p < 0.05; and KRT19, p < 0.01). These findings suggested that the NPC conditioned medium stimulated MSC differentiation into an NP-like phenotype with distinct characteristics. The results could inform strategies for IVD regeneration.
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PURPOSE: To elucidate the natural history of intervertebral disk (IVD) and characterize its embryonic beginnings and age-related degeneration. METHODS: Coronal sections of embryonic (E13.5-neonatal) and postnatal (4-60-week-old) Sprague-Dawley rat IVD were stained by a series of histological stainings (hematoxylin and eosin, Alcian blue, Picrosirius red, Masson, Periodic acid-Schiff). Growth kinetics within embryonic IVD were evaluated by immunohistochemical staining of Ki67 and proliferating cell nuclear antigen. Postnatal maturation and degeneration of IVD were visualized on radiology by X-ray, CT, and MR imaging. RESULTS: During the formation of rat IVD, inner annulus fibrosus (AF) and cartilaginous endplate (CEP) shared similar cell density, extracellular matrix, and potential of growth kinetics; notochord provided increased and enlarged cytoplasmic vacuoles to generate nucleus pulposus (NP), part of which was retained within CEP. Postnatally, vacuolated notochord cells were reduced by devacuolation, while chondrocytic NP cells increased; cartilaginous layers of CEP were narrowed by vertebrae growth and secondary ossification; fibrotic portion of AF decreased as cartilaginous matrix accumulated and infiltrated outward. In aged and degenerated IVD, large longitudinal fissures were detected near the boundaries between inner and outer AF, whereas both reduced cellularity and accumulated cell clusters were evident within the dehydrated NP; only part of these histocytological changes could be reported on radiology. CONCLUSIONS: By showing that the natural history of IVD is orchestrated by a dynamic histocytological regulation, our study may facilitate better understanding of the developmental defects, cellular heterogeneity, age-related degenerative mechanisms, and biological regeneration of IVD. These slides can be retrieved under Electronic Supplementary Material.
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Degeneración del Disco Intervertebral/patología , Disco Intervertebral/embriología , Envejecimiento/patología , Animales , Anillo Fibroso/citología , Anillo Fibroso/embriología , Anillo Fibroso/patología , Recuento de Células , Diferenciación Celular/fisiología , Condrocitos/patología , Matriz Extracelular , Femenino , Desarrollo Fetal/fisiología , Disco Intervertebral/diagnóstico por imagen , Disco Intervertebral/crecimiento & desarrollo , Disco Intervertebral/patología , Degeneración del Disco Intervertebral/diagnóstico por imagen , Imagen por Resonancia Magnética , Masculino , Notocorda/citología , Notocorda/embriología , Núcleo Pulposo/embriología , Núcleo Pulposo/patología , Radiografía , Ratas Sprague-Dawley , Tomografía Computarizada por Rayos XRESUMEN
Cellular loss induced by tumor necrosis factor alpha (TNF-α) contributes to the pathogenesis of intervertebral disc (IVD) degeneration. Cellular stress induced by TNF-α activates several processes to restore cell homeostasis. These processes include autophagy, endoplasmic reticulum stress, and related unfolded protein response (UPR). However, the effect and mechanism of UPR and autophagy regulated by TNF-α in IVD degeneration (IDD) remain unclear. The effect of autophagy on biological changes in nucleus pulposus cells (NPCs) also remains elusive. In this study, rat NPCs were cultured with TNF-α in the presence or absence of the UPR or autophagy pathway small-interfering RNAs. The associated genes and proteins were evaluated through immunofluorescence staining, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analyses to monitor UPR and autophagy signaling and identify the regulatory mechanism of autophagy by the UPR pathway. Trypan blue exclusion assay, cell flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, qRT-PCR, and western blot analyses were performed to examine the apoptosis of NPCs. The results showed that the acute exposure of TNF-α induced the apoptosis of rat NPCs and activated the protein kinase RNA-like ER kinase/eukaryotic translation initiation factor 2α (PERK/eIF2α) pathway of UPR and initiated autophagy. Silencing the PERK/eIF2α pathway or inhibiting autophagy enhanced the apoptosis of NPCs. Interference of the PERK/eIF2α pathway suppressed the autophagy of rat NPCs under TNF-α stimulation. Taken together, the PERK/eIF2α pathway reinforces the survival of NPCs under TNF-α stimulation by activating autophagy. Therefore, PERK/eIF2α-dependent autophagy could be a novel biological therapeutic target for IDD.
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Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Factor 2 Eucariótico de Iniciación/metabolismo , Núcleo Pulposo/patología , Transducción de Señal , Factor de Necrosis Tumoral alfa/farmacología , eIF-2 Quinasa/metabolismo , Animales , Proteína 5 Relacionada con la Autofagia/metabolismo , Beclina-1/metabolismo , Supervivencia Celular/efectos de los fármacos , Citoprotección/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Masculino , Modelos Biológicos , ARN Interferente Pequeño/metabolismo , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
OBJECTIVES: Tumour necrosis factor alpha (TNF-α) expressed by nucleus pulposus cells (NPCs) plays a critical role in intervertebral disc (IVD) degeneration. A key unfolded protein response (UPR) component, X-box binding protein 1 (XBP1) and nuclear factor-kappa B (NF-κB) are essential for cell survival and proliferation. The aim of our study was to elucidate the roles of XBP1 and NF-κB in IVD degeneration (IDD). MATERIALS AND METHODS: Rat NPCs were cultured with TNF-α in the presence or absence of XBP1 and NF-κB-p65 small interfering RNA. The associated genes and proteins were evaluated through quantitative real-time PCR, Western blot analyses and immunofluorescence staining to monitor UPR and NF-κB signalling and identify the regulatory mechanism of p65 by XBP1. Cell counting kit-8 assay, cell cycle analysis and related gene and protein expression were performed to examine the proliferation of NPCs. RESULTS: The acute exposure of TNF-α accelerated the proliferation of rat NPCs by activating the UPR/XBP1 pathway. XBP1 signalling favoured the phosphorylation and nuclear translocation of p65 subunit of NF-κB. The activation of NF-κB in the later phase also enhanced NPC proliferation. CONCLUSIONS: Unfolded protein response reinforces the survival and proliferation of NPCs under TNF-α stimulation by activating the XBP1 pathway, and NF-κB serves as a vital mediator in these events. The XBP1 signalling of UPR can be a novel therapeutic target in IDD.
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Proliferación Celular , FN-kappa B/inmunología , Núcleo Pulposo/citología , Transducción de Señal , Factor de Necrosis Tumoral alfa/inmunología , Proteína 1 de Unión a la X-Box/inmunología , Animales , Células Cultivadas , Degeneración del Disco Intervertebral/inmunología , Núcleo Pulposo/inmunología , Ratas Sprague-DawleyRESUMEN
OBJECTIVES: Dysregulation of YAP by the Hippo signalling is associated with intervertebral disc degeneration (IDD). However, the relationship between the F-actin and Hippo pathway in IDD, and their effects on YAP remain poorly understood. METHODS: The characteristics of Hippo pathway and F-actin the in the NP (nucleus pulposus) and annulus fibrosus of immature, mature, ageing and disc degeneration model rats were observed by immunofluorescence, western blot and qPCR. Nucleus pulposus cells (NPCs) were transfected with lentivirus Sh-LATS A, Sh-LATS B and harvested for SA-ß-gal staining, qPCR, western blotting and immunofluorescence staining to investigate the mechanism of Hippo pathway and F-actin interact in NPCs. RESULTS: We observed moderate decreases in F-actin and YAP expression with age in healthy intervertebral discs (IVDs). F-actin stress fibres distributed throughout the cytoplasm disappeared following treatment with latrunculin B (Lat B), resulting in a punctate distribution. Depletion of large tumour suppressor homologues 1/2 (LATS1/2) did not decrease the rate of cellular senescence, and YAP remained in the cytoplasm following Lat B treatment. Furthermore, angiomotin 130 (AMOT130) was associated with F-actin through a conserved actin-binding domain to retain YAP in the cytoplasm. CONCLUSIONS: This study showed that a mechanism by which Hippo pathway and F-actin synergize to modulate YAP activation and localization in the context of IDD and help to control NPCs proliferation.
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Actinas/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Degeneración del Disco Intervertebral/metabolismo , Proteínas de la Membrana/metabolismo , Angiomotinas , Animales , Proliferación Celular/fisiología , Senescencia Celular/fisiología , Disco Intervertebral/patología , Masculino , Núcleo Pulposo/metabolismo , Ratas Sprague-Dawley , Proteínas Señalizadoras YAPRESUMEN
The Hippo pathway plays important roles in wound healing, tissue repair and regeneration, and in the treatment of degenerative diseases, by regulating cell proliferation and apoptosis in mammals. Intervertebral disc degeneration (IDD) is one of the major causes of low back pain, a widespread issue associated with a heavy economic burden. However, the mechanism underlying how the Hippo pathway regulates IDD is not well understood. Here, we demonstrate that the Hippo pathway is involved in natural IDD. Activation and dephosphorylation of yes-associated protein (YAP) were observed in younger rat discs, and decreased gradually with age. Surprisingly, Hippo pathway suppression was accompanied by overexpression of YAP, caused by acute disc injury, suggesting a limited ability for self-repair in IDD. We also demonstrated that YAP is inhibited by cell-to-cell contact via the Hippo pathway in vitro. Phosphorylation by large tumor suppressor kinases 1/2 (LATS1/2) led to cytoplasmic translocation and inactivation of YAP. YAP dephosphorylation was mainly localized in the nucleus and regulated by the Hippo pathway, whereas YAP dephosphorylation occurred in the cytoplasm and was associated with nucleus pulposus cell (NPC) senescence. Moreover, NPCs were transfected with shYAP and it accelerates the premature senescence of cells by interfered Hippo pathway through YAP. Therefore, our results indicate that the Hippo pathway plays an important role in maintaining the homeostasis of intervertebral discs and controlling NPC proliferation.
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Acid-sensing ion channel 1a (ASIC1a) participates in human intervertebral disc degeneration (IVDD) and regulates the destiny of nucleus pulposus cells (NPCs) in acid stimulus. However, the mechanism of ASIC1a activation and its downstream pathway remain unclear. Endoplasmic reticulum (ER) stress also participates in the acid-induced apoptosis of NPCs. The main purpose of this study was to investigate whether there is any connection between ASIC1a and ER stress in an acid-induced nucleus pulposus degeneration model. The IVDs of Sprague-Dawley rats were stained by immunohistochemical staining to evaluate the expression of ASIC1a in normal and degenerated rat nucleus pulposus. ASIC1a expression was also quantified by quantitative real-time-polymerase chain reaction and Western blotting analysis. NPCs were exposed to the culture media with acidity at pH 7.2 and 6.5 for 24 h, with or without 4-phenylbutyrate (4-PBA, a blocker of the ER stress pathway). Cell apoptosis was examined by Annexin V/Propidium Iodide (PI) staining and was quantified using flow cytometry analysis. ASIC1a-mediated intracellular calcium was determined by Ca2+ imaging using Fura-2-AM. Acidity-induced changes in ER stress markers were studied using Western blotting analysis. In vivo, ASIC1a expression was upregulated in natural degeneration. In vitro, acid stimulus increased intracellular calcium levels, but this effect was blocked by knockdown of ASIC1a, and this reversal was partly inhibited by 4-PBA. In addition, blockade of ASIC1a reduced expression of ER stress markers, especially the proapoptotic markers. ASIC1a partly regulates ER stress and promotes apoptosis of NPCs under acid stimulus and may be a novel therapeutic target in IVDD.
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Intervertebral disc (IVD) degeneration is closely related to inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α). The endoplasmic reticulum (ER) serves several important cell functions, which are essential for normal cell metabolism and survival. This study aims to clarify the role of ER stress and unfolded protein response (UPR) in TNF-α-induced biological changes in rat nucleus pulposus cells (NPCs) and IVD degeneration. In our research, rat NPCs were cultured with different concentrations of TNF-α in the presence or absence of ER stress inhibitors. Related genes and proteins were measured by immunofluorescence staining, quantitative real-time PCR, and Western blot analyses to monitor ER stress. Cell proliferation was evaluated by CCK-8 assay and cyclin D1 expression. Apoptosis was detected by flow cytometry and Western blot analyses. Our results showed that TNF-α induced the apoptosis of some NPCs in the early stage and then accelerated the proliferation of surviving cells. In addition, TNF-α stimulus upregulated ER stress markers and initiated UPR. However, these effects could be reversed by inhibitors, thereby reducing cell proliferation and enhancing apoptosis. In conclusion, ER stress reinforces the survival and proliferation of NPCs in TNF-α stimulus by activating UPR signaling, which could be an important therapeutic target in the future.
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Estrés del Retículo Endoplásmico/fisiología , Núcleo Pulposo/metabolismo , Respuesta de Proteína Desplegada/fisiología , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular , Supervivencia Celular , Retículo Endoplásmico/fisiología , Disco Intervertebral , Degeneración del Disco Intervertebral/patología , Masculino , Núcleo Pulposo/fisiología , Cultivo Primario de Células/métodos , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/efectos de los fármacos , Estrés Fisiológico/fisiología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Notochord nucleus pulposus cells are characteristic of containing abundant and giant cytoplasmic vacuoles. This review explores the embryonic formation, biological function, and postnatal exhaustion of notochord vacuoles, aiming to characterize the signal network transforming the vacuolated nucleus pulposus cells into the vacuole-less chondrocytic cells. Embryonically, the cytoplasmic vacuoles within vertebrate notochord originate from an evolutionarily conserved vacuolation process during neurulation, which may continue to provide mechanical and signal support in constructing a mammalian intervertebral disc. For full vacuolation, a vacuolating specification from dorsal organizer cells, synchronized convergent extension, well-structured notochord sheath, and sufficient post-Golgi trafficking in notochord cells are required. Postnatally, age-related and species-specific exhaustion of vacuolated nucleus pulposus cells could be potentiated by Fas- and Fas ligand-induced apoptosis, intolerance to mechanical stress and nutrient deficiency, vacuole-mediated proliferation check, and gradual de-vacuolation within the avascular and compression-loaded intervertebral disc. These results suggest that the notochord vacuoles are active and versatile organelles for both embryonic notochord and postnatal nucleus pulposus, and may provide novel information on intervertebral disc degeneration to guide cell-based regeneration.
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STUDY DESIGN: Retrospective, case control evaluation of 86 patients who underwent microendoscopic discectomy (MED) and percutaneous transforaminal endoscopic discectomy (PTED) for the treatment of lumbar disc herniation (LDH). PURPOSE: To evaluate the safety and the outcomes of MED and PTED for the treatment of LDH. OVERVIEW OF LITERATURE: MED and PTED are minimally invasive surgical techniques for lower back pain. Studies to date have shown that MED and PTED are safe and effective treatment modalities for LDH. METHODS: A retrospective study was performed in patients with LDH treated with MED (n=50) and transforaminal endoscopic discectomy (PTED; n=36) in our hospital. All patients were followed-up with self-evaluation questionnaires, Oswestry disability index (ODI), medical outcomes study 36-item short form health survey and MacNab criteria. All the patients in both groups were followed up to 12 months after the operation. RESULTS: ODI questionnaire responses were not statistically different between the MED and PTED groups (53.00 vs. 48.72) before treatment. Average scores and minimal disability after 5 days to 12 months of follow-up were 4.96 in the MED group and 3.61 in the PTED group. According to MacNab criteria, 92.0% of the MED group and 94.4% of the PTED group had excellent or good results with no significant difference. CONCLUSIONS: There was no significant difference between MED and PTED outcomes. Further large-scale, randomized studies with long-term follow-up are needed.