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1.
Cell Death Dis ; 11(2): 103, 2020 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-32029706

RESUMO

N6 methyladenosine (m6A) is one of the most prevalent epitranscriptomic modifications of mRNAs, and plays a critical role in various bioprocesses. Bone-derived mesenchymal stem cells (BMSCs) can attenuate apoptosis of nucleus pulposus cells (NPCs) under compression; however, the underlying mechanisms are poorly understood. This study showed that the level of m6A mRNA modifications was decreased, and the autophagic flux was increased in NPCs under compression when they were cocultured with BMSCs. We report that under coculture conditions, RNA demethylase ALKBH5-mediated FIP200 mRNA demethylation enhanced autophagic flux and attenuated the apoptosis of NPCs under compression. Specific silencing of ALKBH5 results in impaired autophagic flux and a higher proportion of apoptotic NPCs under compression, even when cocultured with BMSCs. Mechanistically, we further identify that the m6A "reader" YTHDF2 is likely to be involved in the regulation of autophagy, and lower m6A levels in the coding region of FIP200 lead to a reduction in YTHDF2-mediated mRNA degradation of FIP200, a core molecular component of the ULK1 complex that participates in the initiating process of autophagy. Taken together, our study reveals the roles of ALKBH5-mediated FIP200 mRNA demethylation in enhancing autophagy and reducing apoptosis in NPCs when cocultured with BMSCs.

2.
Arthritis Res Ther ; 21(1): 201, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31481088

RESUMO

BACKGROUND: Intervertebral disc degeneration (IDD) has a complicated and enigmatic pathogenic process. Accumulating evidence shows that long non-coding RNAs (LncRNAs) play a role in the pathogenesis of IDD. This study aimed to investigate the expression and role of the LncRNA HOTAIR in IDD pathogenesis. METHODS: Nucleus pulposus (NP) tissue samples from 10 patients with idiopathic scoliosis and 10 patients with lumbar disc herniation were collected. qRT-PCR was used to assess the expression of HOTAIR and ECM-related genes; western blotting was used to detect the expression of senescence biomarkers, apoptosis-related proteins, and Wnt/ß-catenin pathway; flow cytometry was used to detect apoptosis; and the MTT assay was used to determine cell proliferation. Moreover, a classic needle-punctured rat tail model was used to investigate the role of HOTAIR in IDD in vivo. RESULTS: The results showed that the expression of HOTAIR significantly increased during IDD progression. The overexpression of HOTAIR was found to induce nucleus pulposus (NP) cell senescence, apoptosis, and extracellular matrix (ECM) degradation. HOTAIR silencing by RNA interference in NP cells prevented interleukin-1ß-induced NP cell senescence, apoptosis, and ECM degradation. Furthermore, we found that the Wnt/ß-catenin pathway played a role in regulating HOTAIR to induce these changes in NP cells. Moreover, HOTAIR inhibition in a rat model effectively attenuated IDD symptoms in vivo. CONCLUSIONS: Our findings confirmed that HOTAIR promoted NP cell senescence, apoptosis, and ECM degradation via the activation of the Wnt/ß-catenin pathway, while silencing HOTAIR attenuated this degeneration process, indicating a potential therapeutic target against IDD.

3.
FEBS J ; 286(21): 4356-4373, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31230413

RESUMO

Previous studies identified advanced glycation end products (AGEs) accumulation in the intervertebral disc (IVD) as an essential risk factor associated with IVD degeneration via accelerated cell apoptosis and impeded extracellular-matrix metabolism; however, the underlying mechanisms have not been fully elucidated. Here, we investigated the effects and mechanisms of AGEs-mediated apoptosis in vitro and in vivo. We evaluated the effects of AGEs on endoplasmic reticulum (ER) stress, apoptosis, and subcellular calcium (Ca2+ ) redistribution. Our data indicated time- and concentration-dependent upregulation of ER-stress responses in AGEs-treated nucleus pulposus (NP) cells. Additionally, we observed marked suppression of AGEs-mediated apoptosis following the inhibition of ER stress using 4-phenylbutyric acid. Moreover, AGEs-induced sustained cytosolic Ca2+ ([Ca2+ ]c) elevation and ER luminal Ca2+ ([Ca2+ ]er) depletion in a concentration- and time-dependent manner in NP cells. Furthermore, we observed significant increases and decreases in levels of the ER-resident Ca2+ -release channels inositol 1,4,5-triphosphate receptor and ryanodine receptor and ER Ca2+ -reuptake pumps sarco/endoplasmic reticulum Ca2+ -ATPase, respectively. Pharmacologically blocking ER Ca2+ release using Ca2+ antagonists significantly ameliorated Ca2+ dyshomeostasis, ER stress, and subsequent apoptosis in NP cells and partially attenuated the progression of IVD degeneration in vivo. These results demonstrated that impaired Ca2+ homeostasis plays an essential role in AGEs-mediated ER stress and subsequent apoptosis in NP cells, with blockage of ER Ca2+ release partially ameliorating subcellular Ca2+ redistribution, ER stress, and apoptosis. Our findings provide novel mechanistic insight into the role of AGEs in the pathogenesis of IVD degeneration and a potential therapeutic strategy.

4.
J Cell Mol Med ; 23(8): 5737-5750, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31211513

RESUMO

Intervertebral disc degeneration (IDD) is considered the primary culprit for low back pain. Although the underlying mechanisms remain unknown, hyperactive catabolism of the extracellular matrix (ECM) and inflammation are suggested to play critical roles in IDD progression. This study was designed to elucidate the role of angiopoietin-like protein 8 (ANGPTL8) in the progression of IDD, especially the relationship of ANGPTL8 with ECM metabolism and inflammation. A positive association between ANGPTL8 expression and degenerative grades of IDD was detected in the analysis of human nucleus pulposus tissue samples. Silencing of ANGPTL8 attenuated the degradation of the anabolic protein type collagen II, and reduced the expression of the catabolic proteins MMP3 and MMP9, and the inflammatory cytokine IL-6 through inhibition of NF-κB signalling activation. In addition, the effect of ANGPTL8 was evaluated in a rat model of puncture-induced IDD. Based on the imaging results and histological examination in animal study, knockdown of ANGPTL8 was demonstrated to ameliorate the IDD progression. These results demonstrate the detrimental role of ANGPTL8 expression in the pathogenesis of IDD and may provide a new therapeutic target for IDD treatment.

5.
Life Sci ; 228: 85-97, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31047897

RESUMO

AIM: Nucleus pulposus (NP) cell apoptosis induced by oxidative stress is known to be closely involved in the pathogenesis of intervertebral disc (IVD) degeneration. Berberine, a small molecule derived from Rhizoma coptidis, has been found to exert antioxidative activity and preserve cell viability. The present study aims to investigate whether berberine can prevent NP cell apoptosis under oxidative damage and the potential underlying mechanisms. METHODS AND MATERIALS: The effects of berberine on IVD degeneration were investigated both in vitro and in vivo. KEY FINDINGS: Our results showed that berberine significantly mitigated oxidative stress-decreased cell viability as well as apoptosis in human NP cells. Berberine treatment could attenuate oxidative stress-induced ER stress and autophagy in a concentration-dependent manner. With 4-PBA (ER stress specific inhibitor) and 3-MA (autophagy specific inhibitor) administration, we demonstrated that berberine inhibited oxidative stress-induced apoptosis by modulating the ER stress and autophagy pathway. We also found that the IRE1/JNK pathway was involved in the induction of ER stress-dependent autophagy. With Ca2+ chelator BAPTA-AM utilization, we revealed that oxidative stress-mediated ER stress and autophagy repressed by berberine could be restored by inducing intracellular Ca2+ dysregulation. Furthermore, in vivo study provided evidence that berberine treatment could retard the process of puncture-induced IVD degeneration in a rat model. SIGNIFICANCE: Our results indicate that berberine could prevent oxidative stress-induced apoptosis by modulating ER stress and autophagy, thus offering a novel potential pharmacological treatment strategy for IVD degeneration.


Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Berberina/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Degeneração do Disco Intervertebral/tratamento farmacológico , Núcleo Pulposo/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Antioxidantes/uso terapêutico , Autofagia/efeitos dos fármacos , Berberina/uso terapêutico , Células Cultivadas , Feminino , Humanos , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/patologia , Núcleo Pulposo/citologia , Núcleo Pulposo/metabolismo , Núcleo Pulposo/patologia , Ratos Sprague-Dawley
6.
World Neurosurg ; 125: 129-135, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30738941

RESUMO

BACKGROUND: Severe kyphoscoliosis associated with multiple giant spinal epidural arachnoid cysts (SEACs) is an extremely rare condition and remains a challenge in clinical practice. This study aimed to present a case of severe spinal deformity associated with multiple giant SEACs and to discuss strategies for the preoperative diagnosis and treatment. CASE DESCRIPTION: A 22-year-old man with severe thoracolumbar kyphoscoliosis associated with multiple giant SEACs presented with progressive scoliosis, spastic paralysis, numbness, and abnormal gait. X-ray and magnetic resonance imaging revealed severe rigid kyphoscoliosis, extensive diffuse cystic space-occupying lesions, and diffuse spinal cord compression. After multidisciplinary consultation and discussion, the patient underwent a cyst-peritoneal shunting surgery followed by posterior vertebral column resection (PVCR) correction. The postoperative course was uneventful. Both kyphosis and scoliosis were significantly corrected, and muscle weakness of the lower extremities and sensory disturbance partially improved. At the 2-year follow-up visit, the patient could freely walk without the aid of crutches, but there were some residual neurologic deficits in both legs. A plain radiograph showed that bony fusion was achieved, and the correction was well maintained. CONCLUSIONS: Cyst-peritoneal shunting surgery followed by PVCR, as in our case, could be an alternative surgical strategy for multiple giant SEACs associated with severe rigid kyphoscoliosis.


Assuntos
Cistos Aracnóideos/complicações , Cistos Aracnóideos/cirurgia , Cifose/complicações , Escoliose/complicações , Adolescente , Descompressão Cirúrgica/métodos , Humanos , Masculino , Procedimentos Neurocirúrgicos/métodos , Compressão da Medula Espinal/etiologia , Compressão da Medula Espinal/cirurgia , Fusão Vertebral/métodos
7.
J Orthop Surg Res ; 13(1): 312, 2018 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-30522509

RESUMO

BACKGROUND: Junction tuberculous spondylitis involves the stress transition zone of the spine and has a high risk of progression to kyphosis or paraplegia. Problems still exist with treatment for spinal junction tuberculosis. This study investigated the surgical approach and clinical outcomes of junction spinal tuberculosis. METHODS: From June 1998 to July 2014, 77 patients with tuberculous spondylitis were enrolled. All patients received 2-3 weeks of anti-tuberculous treatment preoperatively; treatment was prolonged for 2-3 months when active pulmonary tuberculosis was present. The patients underwent anterior debridement and were followed up for an average of 29.4 months clinically and radiologically. RESULTS: The cervicothoracic junction spine (C7-T3) was involved in 15 patients. The thoracolumbar junction spine (T11-L2) was involved in 39 patients. The lumbosacral junction spine (L4-S1) was involved in 23 patients. Two patients with recurrence underwent reoperation; the drugs were adjusted, and all patients achieved bone fusion. The preoperative cervicothoracic and thoracolumbar kyphosis angle and lumbosacral angle were 31.4 ± 10.9°, 32.9 ± 9.2°, and 19.3 ± 3.7°, respectively, and the corresponding postoperative angles were ameliorated significantly to 9.1 ± 3.2°, 8.5 ± 2.9°, and 30.3 ± 2.8°. The preoperative ESR and C-reactive protein level of all patients were 48.1 ± 11.3 mm/h and 65.5 ± 16.2 mg/L which decreased to 12.3 ± 4.3 mm/h and 8.6 ± 3.7 mg/L at the final follow-up, respectively. All patients that had neurological symptoms achieved function status improvement at different degrees. CONCLUSION: For spinal tuberculosis of spinal junctions, anterior debridement, internal fixation, and fusion can be preferred and achieved. If multiple segment lesions are too long or difficult for operation of anterior internal fixation, combining posterior pedicle screw fixation is appropriate.


Assuntos
Desbridamento/métodos , Gerenciamento Clínico , Fixadores Internos , Fusão Vertebral/métodos , Tuberculose da Coluna Vertebral/diagnóstico por imagem , Tuberculose da Coluna Vertebral/cirurgia , Adolescente , Adulto , Idoso , Vértebras Cervicais/diagnóstico por imagem , Vértebras Cervicais/cirurgia , Feminino , Seguimentos , Humanos , Vértebras Lombares/diagnóstico por imagem , Vértebras Lombares/cirurgia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Sacro/diagnóstico por imagem , Sacro/cirurgia , Fusão Vertebral/instrumentação , Vértebras Torácicas/diagnóstico por imagem , Vértebras Torácicas/cirurgia , Resultado do Tratamento , Adulto Jovem
8.
Exp Cell Res ; 372(2): 188-197, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30312603

RESUMO

Regulated in development and DNA damage response 1 (REDD1) is an evolutionarily conserved, ubiquitous protein that responds to various cell stresses. Studies have proved REDD1 is involved in many diseases, such as osteoarthritis and cancer. The present study aimed to investigate the potential role of REDD1 in the pathogenesis of intervertebral disc degeneration (IDD). Analysis of clinical tissue samples showed REDD1 expression was up-regulated during IDD and was correlated with the grade of disc degeneration. Overexpression of REDD1 in normal human nucleus pulposus (NP) cells resulted in extracellular matrix (ECM) degeneration. Further, we investigated the function of REDD1 using a serum deprivation-induced IDD vitro model and found that REDD1 was up-regulated in a temporal manner. However, hypoxia abolished this increase through down-regulation of NF-κB. Knockdown of REDD1 or NF-κB by si-RNA significantly rescued ECM from degeneration both in normoxia and hypoxia. In addition, NF-κB/REDD1 mediated the protection of hypoxia from serum deprivation-induced apoptosis and autophagy in NP cells. These results suggest that REDD1 might play a pivotal role in IDD pathogenesis, thereby potentially providing a new therapeutic target for IDD treatment.


Assuntos
Matriz Extracelular/genética , Degeneração do Disco Intervertebral/genética , Núcleo Pulposo/metabolismo , Fatores de Transcrição/genética , Adulto , Apoptose/genética , Autofagia/genética , Hipóxia Celular/genética , Matriz Extracelular/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Degeneração do Disco Intervertebral/fisiopatologia , Masculino , Pessoa de Meia-Idade , NF-kappa B/genética , Núcleo Pulposo/patologia
9.
Redox Biol ; 19: 339-353, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30216853

RESUMO

Intervertebral disc (IVD) degeneration contributes largely to pathoanatomical and degenerative changes of spinal structure that increase the risk of low back pain. Apoptosis in nucleus pulposus (NP) can aggravate IVD degeneration, and increasing studies have shown that interventions targeting NP cell apoptosis can ameliorate IVD degeneration, exhibiting their potential for use as therapeutic strategies. Recent data have shown that advanced glycation end products (AGEs) accumulate in NP tissues in parallel with the progression of IVD degeneration and form a microenvironment of oxidative stress. This study examined whether AGEs accumulation aggravates NP cell apoptosis and IVD degeneration, and explored the mechanisms underlying these effects. We observed that the viability and proliferation of human NP cells were significantly suppressed by AGEs treatment, mainly due to apoptosis. Furthermore, activation of the mitochondrial apoptosis pathway was detected after AGEs treatment. In addition, the molecular data showed that AGEs could significantly aggravate the generation of mitochondrial reactive oxygen species and prolonged activation of the mitochondrial permeability transition pore, as well as the increased level of Bax protein and decreased level of Bcl-2 protein in mitochondria. These effects could be reduced by antioxidant (2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl) triphenylphosphonium chloride (MitoTEMPO) and Visomitin (SKQ1). Importantly, we identified that impairment of Sirtuin3 (SIRT3) function and the mitochondrial antioxidant network were vital mechanisms in AGEs-induced oxidative stress and secondary human NP cell apoptosis. Finally, based on findings that nicotinamide mononucleotide (NMN) could restore SIRT3 function and rescue human NP cell apoptosis through adenosine monophosphate-activated protein kinase and peroxisome proliferator-activated receptor-γ coactivator 1α (AMPK-PGC-1α) pathway in vitro, we confirmed its protective effect on AGEs-induced IVD degeneration in vivo. In conclusion, our data demonstrate that SIRT3 protects against AGEs-induced human NP cell apoptosis and IVD degeneration. Targeting SIRT3 to improve mitochondrial redox homeostasis may represent a potential therapeutic strategy for attenuating AGEs-associated IVD degeneration.


Assuntos
Produtos Finais de Glicação Avançada/metabolismo , Degeneração do Disco Intervertebral/metabolismo , Mitocôndrias/metabolismo , Sirtuína 3/metabolismo , Adolescente , Adulto , Animais , Apoptose , Células Cultivadas , Feminino , Humanos , Degeneração do Disco Intervertebral/patologia , Masculino , Pessoa de Meia-Idade , Mitocôndrias/patologia , Oxirredução , Estresse Oxidativo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Adulto Jovem
10.
Oncotarget ; 8(17): 27868-27881, 2017 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-28427186

RESUMO

PURPOSE: This study investigated the expression and function of the microRNA-494 in intervertebral disc degeneration (IDD). RESULTS: MicroRNA-494 expression was upregulated during IDD progression; its overexpression increased the expression of ECM catabolic factors such as matrix metalloproteinase and A disintegrin and metalloproteinase with thrombospondin motif in NP cells while decreasing that of anabolic genes such as type II collagen and aggrecan; it also induced the apoptosis of NP cells, as determined by flow cytometry. These effects were reversed by microRNA-494 inhibitor treatment. SOX9 was identified as a target of negative regulation by microRNA-494. Promoter hypomethylation and NF-κB activation were associated with microRNA-494 upregulation in IDD. MATERIALS AND METHODS: MicroRNA-494 expression in degenerative nucleus pulposus (NP) tissue was assessed by quantitative real-time PCR. The effect of microRNA-494 on extracellular matrix (ECM) metabolism and NP cell apoptosis was evaluated by transfection of microRNA-494 mimic or inhibitor. The regulation of SRY-related high mobility group box (SOX)9 expression by microRNA-494 was assessed with the luciferase reporter assay, and the methylation status of the microRNA-494 promoter was evaluated by methylation-specific PCR and bisulfite sequencing PCR. The role of activated nuclear factor (NF)-κB in the regulation of microRNA-494 expression was evaluated using specific inhibitors. CONCLUSIONS: MicroRNA-494 promotes ECM degradation and apoptosis of degenerative human NP cells by directly targeting SOX9.


Assuntos
Matriz Extracelular/metabolismo , Degeneração do Disco Intervertebral/genética , MicroRNAs/metabolismo , Núcleo Pulposo/patologia , Fatores de Transcrição SOX9/genética , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteínas ADAMTS/genética , Proteínas ADAMTS/metabolismo , Adulto , Agrecanas/genética , Agrecanas/metabolismo , Apoptose , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Metilação de DNA , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Imunofluorescência , Humanos , Imuno-Histoquímica , Degeneração do Disco Intervertebral/patologia , Masculino , MicroRNAs/antagonistas & inibidores , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Núcleo Pulposo/citologia , Cultura Primária de Células , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição SOX9/metabolismo , Regulação para Cima
11.
Biotechnol Lett ; 39(4): 623-632, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28039556

RESUMO

OBJECTIVES: To determine the role of microRNA-15b (miR-15b) in interleukin-1 beta (IL-1ß)-induced extracellular matrix (ECM) degradation in the nucleus pulposus (NP). RESULTS: MiR-15b was up-regulated in degenerative NP tissues and in IL-1ß-stimulated NP cells, as compared to the levels in normal controls (normal tissue specimens from patients with idiopathic scoliosis). Bioinformatics and luciferase activity analyses showed that mothers against decapentaplegic homolog 3 (SMAD3), a key mediator of the transforming growth factor-ß signaling pathway, was directly targeted by miR-15b. Functional analysis demonstrated that miR-15b overexpression aggravated IL-1ß-induced ECM degradation in NP cells, while miR-15b inhibition had the opposite effects. Prevention of IL-1ß-induced NP ECM degeneration by the miR-15b inhibitor was attenuated by small-interfering-RNA-mediated knockdown of SMAD3. In addition, activation of MAP kinase and nuclear factor-κB up-regulated miR-15b expression and down-regulated SMAD3 expression in IL-1ß-stimulated NP cells. CONCLUSIONS: MiR-15b contributes to ECM degradation in intervertebral disc degeneration (IDD) via targeting of SMAD3, thus providing a novel therapeutic target for IDD treatment.


Assuntos
Matriz Extracelular/metabolismo , Inativação Gênica , Interleucina-1beta/farmacologia , MicroRNAs/metabolismo , Núcleo Pulposo/patologia , Proteína Smad3/metabolismo , Adulto , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Degeneração do Disco Intervertebral/genética , Degeneração do Disco Intervertebral/patologia , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Núcleo Pulposo/citologia , Proteólise , Transdução de Sinais , Proteína Smad3/genética , Transfecção
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