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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.
J Cell Physiol ; 235(3): 2195-2208, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31478571

RESUMO

Intervertebral disc degeneration (IDD) is a complex and chronic disease that involves disc cell senescence, death, and extracellular matrix (ECM) degradation. HOTAIR, a long non-coding RNA (lncRNA) is reportedly associated with autophagy, whereas autophagy is shown to promote IDD. However, how it affects nucleus pulposus (NP) cells, the primary component of intervertebral discs is still unclear. We hypothesized that HOTAIR promotes NP cell apoptosis and senescence through upregulating autophagy. Thus, silencing HOTAIR should inhibit autophagy and exert a therapeutic effect on IDD. Our in vitro experiments in human NP cells revealed that HOTAIR expression positively correlated with IDD grade, and overexpression enhanced autophagy. Autophagy inhibition via 3-methyladenine reversed HOTAIR stimulatory effects on apoptosis, senescence, and ECM catabolism, while the AMP-activated protein kinase (AMPK) inhibitor Compound C suppressed HOTAIR-induced autophagy through regulating AMPK/mTOR/ULK1 pathways. Our in vivo experiment then illustrated that silencing HOTAIR ameliorates IDD in rats. Collectively, we demonstrated that HOTAIR stimulates autophagy to promote NP cell apoptosis, senescence, and ECM catabolism. Therefore, silencing HOTAIR has the potential to become a treatment option for IDD.

3.
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.

4.
Theranostics ; 9(14): 4084-4100, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31281533

RESUMO

Objectives: Intervertebral disc degeneration (IDD) is widely accepted as a cause of low back pain and related degenerative musculoskeletal disorders. Nucleus pulposus (NP) cell apoptosis which is related to excessive endoplasmic reticulum (ER) stress in the intervertebral disc (IVD) could aggravate IDD progression. Many studies have shown the therapeutic potential of exosomes derived from bone marrow mesenchymal stem cells (MSC-exos) in degenerative diseases. We hypothesized that the delivery of MSC-exos could modulate ER stress and inhibit excessive NP cell apoptosis during IDD. Methods: The ER stress levels were measured in normal or degenerative NP tissues for contrast. The effects of MSC-exos were testified in advanced glycation end products (AGEs) -induced ER stress in human NP cells. The mechanism involving AKT and ERK signaling pathways was investigated using RNA interference or signaling inhibitors. Histological or immunohistochemical analysis and TUNEL staining were used for evaluating MSC-exos therapeutic effects in vivo. Results: The ER stress level and apoptotic rate was elevated in degenerative IVD tissues. MSC-exos could attenuate ER stress-induced apoptosis by activating AKT and ERK signaling. Moreover, delivery of MSC-exos in vivo modulated ER stress-related apoptosis and retarded IDD progression in a rat tail model. Conclusions: These results highlight the therapeutic effects of exosomes in preventing IDD progression. Our work is the first to demonstrate that MSC-exos could modulate ER stress-induced apoptosis during AGEs-associated IVD degeneration.

5.
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.

6.
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.

7.
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
8.
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
9.
Oxid Med Cell Longev ; 2019: 7810320, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31976028

RESUMO

Oxidative stress-induced mitochondrial dysfunction and nucleus pulposus (NP) cell apoptosis play crucial roles in the development of intervertebral disc degeneration (IDD). Increasing studies have shown that interventions targeting impaired autophagic flux can maintain cellular homeostasis by relieving oxidative damage. Here, we investigated the effect of curcumin (CUR), a known autophagy activator, on IDD in vitro and in vivo. CUR suppressed tert-butyl hydroperoxide- (TBHP-) induced oxidative stress and mitochondrial dysfunction and thereby inhibited human NP cell apoptosis, senescence, and ECM degradation. CUR treatment induced autophagy and enhanced autophagic flux in an AMPK/mTOR/ULK1-dependent manner. Notably, CUR alleviated TBHP-induced interruption of autophagosome-lysosome fusion and impairment of lysosomal function and thus contributed to the restoration of blocked autophagic clearance. These protective effects of CUR in TBHP-stimulated human NP cells resembled the effects produced by the autophagy inducer rapamycin, but the effects were partially eliminated by 3-methyladenine- and compound C-mediated inhibition of autophagy initiation or chloroquine-mediated obstruction of autophagic flux. Lastly, CUR also exerted a protective effect against puncture-induced IDD progression in vivo. Our results showed that suppression of excessive ROS production and mitochondrial dysfunction through enhancement of autophagy coupled with restoration of autophagic flux ameliorated TBHP-induced human NP cell apoptosis, senescence, and ECM degradation. Thus, maintenance of the proper functioning of autophagy represents a promising therapeutic strategy for IDD, and CUR might serve as an effective therapeutic agent for IDD.

10.
Microsurgery ; 31(5): 388-94, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21503972

RESUMO

The acellular nerve graft that can provide internal structure and extracellular matrix components of the nerve is an alternative for repair of peripheral nerve defects. However, results of the acellular nerve grafting for nerve repair still remain inconsistent. This study aimed to investigate if supplementing bone marrow mesenchymal stromal cells (MSCs) could improve the results of nerve repair with the acellular nerve graft in a 10-mm sciatic nerve defect model in mice. Eighteen mice were divided into three groups (n = 6 for each group) for nerve repairs with the nerve autograft, the acellular nerve graft, and the acellular nerve graft by supplemented with MSCs (5 × 10(5)) fibrin glue around the graft. The mouse static sciatic index was evaluated by walking-track testing every 2 weeks. The weight preservation of the triceps surae muscles and histomorphometric assessment of triceps surae muscles and repaired nerves were examined at week 8. The results showed that the nerve repair by the nerve autografting obtained the best functional recovery of limb. The nerve repair with the acellular nerve graft supplemented with MSCs achieved better functional recovery and higher axon number than that with the acellular nerve graft alone at week 8 postoperatively. The results indicated that supplementing MSCs might help to improve nerve regeneration and functional recovery in repair of the nerve defect with the acellular nerve graft.


Assuntos
Transplante de Medula Óssea/métodos , Microcirurgia/métodos , Nervo Isquiático/cirurgia , Animais , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Regeneração Nervosa , Procedimentos Cirúrgicos Reconstrutivos/métodos , Recuperação de Função Fisiológica , Nervo Isquiático/lesões , Nervo Isquiático/fisiologia , Nervo Isquiático/transplante , Células Estromais/transplante , Transplante Autólogo
11.
Artigo em Chinês | MEDLINE | ID: mdl-22295505

RESUMO

OBJECTIVE: To investigate the phenotypic, molecular and biological characteristics of adipose tissue-derived stromal cells (ADSCs) differentiated alonely a Schwann cells (SCs) lineage and to provide a new cells' seed source for nerve tissue engineering or cell therapy. METHODS: Cultured ADSCs were isolated from SD rats and the undifferentiated ADSCs were confirmed by detection of MSC-specific cell-surface markers. The ADSCs were differentiated along a glial cell lineage using an established cocktail of growth factors. Following differention, we used immunofluorescene staining and RT-PCR to evaluate the characteristics of differentiated WJMSCs. RESULTS: ADSCs were successfully isolated from the rats' fat tissue. The isolated ADSCs expressed CD29, CD90 but not CD34, CD44 nor CD45. Osteogenic differentiation was detected by Alizarin red staining and adipogenic differentiation was comfirmed by Oil-red O staining. ADSCs treated with a mixture of glial growth factors adopted a spindle-like morphology similar to Schwann cells. Immunocytochemical staining and RT-PCR analysis revealed that the treated cells expressed the glial markers S100, P75 and glial fibrillary acidic protein indicative of differentiation. CONCLUSION: ADSCs can be differentiated into cells that are Schwann-like in terms of morphologic features and phenotype and could be suitable Schwann-cell substitutes for nerve repair in clinical applications.


Assuntos
Tecido Adiposo/citologia , Diferenciação Celular/fisiologia , Células-Tronco Mesenquimais/citologia , Células de Schwann/citologia , Animais , Células Cultivadas , Masculino , Ratos , Ratos Sprague-Dawley
12.
Artigo em Chinês | MEDLINE | ID: mdl-20632512

RESUMO

OBJECTIVE: Using chemically extracted acellular methods to treat extracranial section of the canine whole facial nerve, to evaluated its effects on nerve structure and the removal extent of Schwann cells and myelin. METHODS: Twenty whole facial nerves were exposed from 10 canines [weighing (18 +/- 3) kg]. The extracranial trunk of canine facial nerve and its branches (temporal branch, zygomatic branch, buccal branch, marginal mandibular branch, and cervical branch) were dissected under light microscope. Twenty facial nerves were divided into the experimental group (n = 12) and control group (n = 8) randomly. In experimental group, the nerve was extracted with the 3%TritonX-100 and 4% sodium deoxycholate. In control group, the nerve was not extracted. HE staining and immunofluorescence histological stainings for Hoechst33258, P75, Zero, and Laminin were performed. RESULTS: After histological staining, it was found that myelin and Schwann cells were removed from the facial nerve while the basal lamina tube remained intact. The whole canine facial nerves (one nerve trunk and multiple nerve branches) had the similar result. CONCLUSION: The canine whole facial nerve has natural structure (one nerve trunk and multiple nerve branches) by extracted with chemically extracted acellular methods, so it is an available graft for repairing the defect of the whole facial nerve.


Assuntos
Nervo Facial/anatomia & histologia , Células de Schwann/química , Células de Schwann/citologia , Animais , Cães , Fluorimunoensaio , Laminina/análise , Proteína P0 da Mielina/análise , Regeneração Nervosa
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