RESUMEN
The in-situ osmolarity is an important physicochemical factor that regulates cell fate of nucleus pulposus cells (NPCs). Our previous studies demonstrated that reduced N-cadherin (NCDH) expression in nucleus pulposus cells is associated with cellular damage under hyper-osmolarity microenvironment. This study was aimed at exploring the impacts of NCDH on senescence and apoptosis of NPCs, as well as the potential molecular mechanism. By comparing NPCs from patients with lumbar fractures and lumbar disc herniation, we identified a correlation between decreased NCDH expression and increased endoplasmic reticulum stress (ERS), resulting in undesirable cell fate (senescence and apoptosis). After blocking Reactive oxygen species (ROS) or ERS, it was indicated that hyper-osmolarity microenvironment induced ERS was ROS-dependent. Further results demonstrated the correlation in rat NPCs. Upregulation of NCDH expression reduced ROS-dependent ERS, thus limiting undesirable cell fates in vitro. This was further confirmed through the rat tail acupuncture injection model. NCDH overexpression successfully mitigated ERS, preserved extracellular matrix production and alleviating intervertebral disc degeneration in vivo. Together, NCDH can alleviate senescence and apoptosis of NPCs by suppressing ROS-dependent ERS via the ATF4-CHOP signaling axis in the hyper-osmolarity microenvironment, thus highlighting the therapeutic potential of NCDH in combating degenerative disc diseases.
Asunto(s)
Degeneración del Disco Intervertebral , Núcleo Pulposo , Animales , Humanos , Ratas , Apoptosis/genética , Cadherinas/genética , Cadherinas/metabolismo , Senescencia Celular/genética , Estrés del Retículo Endoplásmico/genética , Degeneración del Disco Intervertebral/genética , Degeneración del Disco Intervertebral/terapia , Núcleo Pulposo/metabolismo , Concentración Osmolar , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Background: Nucleus pulposus (NP) degeneration is the core pathological change of intervertebral disc (IVD) degenerative diseases, but currently, no effective therapy is available. With the rapid development of biomaterials and tissue engineering in recent years, biomaterial-assisted cell transplantation becomes a promising therapy for IVD degeneration. However, the application is severely limited by the weak biological characteristics of NP cells (NPCs), such as a moderate proliferation ability, weak self-renewal capacity, and minimal extracellular matrix (ECM) synthesis capacity, caused by the current inappropriate cell seeding or grafting methods. Methods: Here, we developed a three-dimensional (3D) spheroidizing culture method to construct NPC spheroids and investigated repair and regeneration potential of these spheroids in vitro and in vivo. The in vitro biological characteristics (including cell viability and proliferation), and in vivo functions (including anti-degeneration potential and ability to induce tissue repair) of NPC spheroids and monolayer-cultured NPCs were compared. Furthermore, an RNA-seq-based transcriptome analysis and a series of function experiments were performed to elucidate the potential mechanisms of their differences that were involved in the tissue regeneration process. Results: NPC spheroids exhibited obviously superior self-renewal and ECM synthesis capacities compared to monolayers of NPCs in vitro. In vivo, NPC spheroids generated more functional ECM components, primarily aggrecan (ACAN) and collagen type II (Col2), and markedly promoted NP regeneration in the disc degeneration model induced by partial NP excision. Additionally, the biological characteristics and functions of NPC spheroids were to some extent regulated by the interaction of N-cadherin (N-CDH) and Integrinß1 (ITGß1), two key mechanosensing ECM-receptors expressed on NPCs. Conclusions: The NPC spheroidizing culture method is beneficial for cell renewal and the generation of functional ECM in NP tissue. The molecular mechanism involved in this regeneration process is closely associated with the regulation of the N-CDH and ITGß1 interaction-mediated ECM homeostesis. Moreover, the strategy of hydrogel-assisted NPC spheroids transplantation may potentially be used in the future treatment of IVD degeneration.
Asunto(s)
Degeneración del Disco Intervertebral , Núcleo Pulposo , Cadherinas/metabolismo , Matriz Extracelular/metabolismo , Humanos , Degeneración del Disco Intervertebral/genética , Degeneración del Disco Intervertebral/metabolismo , Degeneración del Disco Intervertebral/terapia , Núcleo Pulposo/metabolismo , Ingeniería de TejidosRESUMEN
OBJECTIVE: To introduce a modified transverse process-pedicle puncture technique applied to unilateral extrapedicular percutaneous vertebroplasty (PVP) for the treatment of osteoporotic lumbar vertebral compression fractures. METHODS: A retrospective study was performed on 91 patients with osteoporotic vertebral compression fractures (OVCFs) who underwent unilateral extrapedicular PVP from June 2016 to September 2018. Lumbar and back pain was assessed through the visual analogue scale (VAS). Function recovery was assessed through the Oswestry disability index (ODI). Radiologic outcomes were assessed mainly on the basis of bone cement distribution and anterior vertebral height. RESULTS: A total of 101 fractured vertebrae were successfully treated using the extrapedicular technique without any recognized clinical complications. The postoperative VAS and ODI values were significantly lower than the corresponding preoperative values (P < 0.01). Radiologic outcomes in all fractured vertebrae showed that the diffusion of bone cement could exceed the midline of the vertebral body. There was no significant difference between preoperative and postoperative anterior vertebral heights (P < 0.05). CONCLUSION: The modified transverse process-pedicle approach applied to unilateral extrapedicular percutaneous vertebroplasty is a simple, safe, and effective surgical method.
Asunto(s)
Fracturas por Compresión , Fracturas Osteoporóticas , Fracturas de la Columna Vertebral , Vertebroplastia , Fracturas por Compresión/diagnóstico por imagen , Fracturas por Compresión/cirugía , Humanos , Vértebras Lumbares/diagnóstico por imagen , Vértebras Lumbares/cirugía , Fracturas Osteoporóticas/diagnóstico por imagen , Fracturas Osteoporóticas/cirugía , Estudios Retrospectivos , Fracturas de la Columna Vertebral/cirugía , Resultado del TratamientoRESUMEN
Mechanical overloading-induced nucleus pulposus cell (NPC) apoptosis plays a core role in the pathogenesis of intervertebral disc degeneration. In this study, we investigated the involvement of mammalian silent information regulator 2 homolog (SIRT1) in NPC apoptosis under high-magnitude compression. Our results showed that high-magnitude compression aggravated cellular apoptosis and attenuated the expression levels of SIRT1 and microtubule-associated protein-1 light chain-3B (LC3B) in rat NPCs in a three-dimensional (3D) cell culture model and an in vivo rat tail compression model, whereas SIRT1 overexpression in NPCs partially reversed these indicators. Moreover, SIRT1 overexpression increased the formation of the LC3B/Fas complex, alleviated activation of the NF-κB pathway, and reduced NPC apoptosis. Finally, downregulation of LC3B partially activated the NF-κB pathway and aggravated NPC apoptosis. Overall, upregulation of SIRT1 increases formation of the LC3B/Fas complex, which contributes to suppression of NPC apoptosis by inhibiting the NF-κB pathway under high compressive stress.
Asunto(s)
Proteínas Asociadas a Microtúbulos/metabolismo , Sirtuina 1/metabolismo , Receptor fas/metabolismo , Animales , Apoptosis , Humanos , Masculino , Núcleo Pulposo , Ratas , Ratas Sprague-Dawley , TransfecciónRESUMEN
Mechanical overloading-induced nucleus pulposus (NP) cells senescence plays an important role in the pathogenesis of intervertebral disc degeneration (IVDD). The silent mating type information regulator 2 homolog-1 (SIRT1)-mediated pathway preserves the normal NP cell phenotype and mitochondrial homeostasis under multiple stresses. We aimed to investigate the role of SIRT1 in IVDD by assessing the effects of SIRT1 overexpression on high-magnitude compression-induced senescence in NP cells. High-magnitude compression induced cellular senescence and mitochondrial dysfunction in human NP cells. Moreover, SIRT1 overexpression tended to alleviate NP cell senescence and mitochondrial dysfunction under compressive stress. Given the mitophagy-inducing property of SIRT1, activity of mitophagy was evaluated in NP cells to further demonstrate the underlying mechanism. The results showed that SIRT1-overexpression attenuated senescence and mitochondrial injury in NP cells subjected to high-magnitude compression. However, depletion of PINK1, a key mitophagic regulator, impaired mitophagy and blocked the protective role of SIRT1 against compression induced senescence in NP cells. In summary, these results suggest that SIRT1 plays a protective role in alleviating NP cell senescence and mitochondrial dysfunction under high-magnitude compression, the mechanism of which is associated with the regulation of PINK1-dependent mitophagy. Our findings may provide a potential therapeutic approach for IVDD treatment.
Asunto(s)
Senescencia Celular , Degeneración del Disco Intervertebral/enzimología , Mitocondrias/enzimología , Mitofagia , Núcleo Pulposo/enzimología , Proteínas Quinasas/metabolismo , Sirtuina 1/metabolismo , Adulto , Reactores Biológicos , Células Cultivadas , Femenino , Humanos , Degeneración del Disco Intervertebral/genética , Degeneración del Disco Intervertebral/patología , Masculino , Persona de Mediana Edad , Mitocondrias/ultraestructura , Núcleo Pulposo/ultraestructura , Estrés Oxidativo , Presión , Proteínas Quinasas/genética , Transducción de Señal , Sirtuina 1/genética , Estrés MecánicoRESUMEN
Bone loss is one of the major complications of advanced cancers such as breast cancer, prostate cancer, and lung cancer. Extensive research has revealed that the receptor activator of NF-κB ligand (RANKL), which is considered to be a key factor in osteoclast differentiation, plays an important role in cancer-associated bone resorption. Therefore, agents that can suppress this bone loss have therapeutic potential. In this study, we detected whether quetiapine (QUE), a commonly used atypical antipsychotic drug, can inhibit RANKL-induced osteoclast differentiation in vitro and prevent human breast cancer-induced bone loss in vivo. RAW 264.7 cells and bone marrow-derived macrophages (BMMs) were used to detect inhibitory effect of QUE on osteoclastogenesis in vitro. Mouse model of breast cancer metastasis to bone was used to test suppressive effect of QUE on breast cancer-induced bone loss in vivo. Our results show that QUE can inhibit RANKL-induced osteoclast differentiation from RAW 264.7 cells and BMMs without signs of cytotoxicity. Moreover, QUE reduced the occurrence of MDA-MB-231 cell-induced osteolytic bone loss by suppressing the differentiation of osteoclasts. Finally, molecular analysis revealed that it is by inhibiting RANKL-mediated MAPK and NF-κB signaling pathways that QUE suppressed the osteoclast differentiation. We demonstrate, for the first time, the novel suppressive effects of QUE on RANKL-induced osteoclast differentiation in vitro and human breast cancer-induced bone loss in vivo, suggesting that QUE may be a potential therapeutic drug for osteolysis treatment.
Asunto(s)
Resorción Ósea/genética , Neoplasias de la Mama/tratamiento farmacológico , Dibenzotiazepinas/administración & dosificación , Ligando RANK/genética , Animales , Resorción Ósea/complicaciones , Resorción Ósea/patología , Neoplasias de la Mama/complicaciones , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Femenino , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/patología , Ratones , FN-kappa B/genética , Osteoclastos/efectos de los fármacos , Osteoclastos/patología , Fumarato de Quetiapina , Ligando RANK/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacosRESUMEN
Bone is a frequent target of lung cancer metastasis, which is associated with significant morbidity and poor prognosis; however, the molecular basis of this process is still unknown. This study investigated the role of extracellular matrix metalloproteinase inducer (also known as cluster of differentiation (CD)147) in osteoclastogenesis resulting from bone metastasis, based on the enrichment of this glycoprotein on the surface of many malignant bone tumors. RNA interference was used to silence CD147 expression in A549 human lung cancer cells. Compared with conditioned medium (CM) from control cells (A549-CM), CM from CD147-deficient cells (A549-si-CM) suppressed receptor activator of nuclear factor κB ligand-stimulated osteoclastogenesis in RAW 264.7 cells and bone marrow-derived macrophages. The mRNA levels of osteoclast-specific genes such as tartrate-resistant acid phosphatase, calcitonin receptor, and cathepsin K were also reduced in the presence of A549-si-CM. CD147 knockdown in A549 cells decreased interleukin (IL)-8mRNA and protein expression. IL-8 is present in large amounts in A549-CM and mimicked its inductive effect on osteoclastogenesis; this was reversed by depletion of IL-8 from the medium. Taken together, these results indicate that CD147 promotes lung cancer-induced osteoclastogenesis by modulating IL-8 secretion, and suggest that CD147 is a potential therapeutic target for cancer-associated bone resorption in lung cancer patients.