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1.
Life Sci ; 243: 117244, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31891721

RESUMO

AIMS: Endplate chondrocyte apoptosis is an important contributor to the pathogenesis of cartilaginous endplate (CEP) degeneration that leads to the initiation and development of intervertebral disc degeneration (IDD). In this study, we hypothesized that Parkin-mediated mitophagy and nuclear factor E2-related factor 2 (Nrf2)-mediated antioxidant system played an important role in endplate chondrocyte survival under pathological conditions. MATERIALS AND METHODS: Human endplate chondrocytes were stimulated with H2O2 to mimic pathological conditions. Western blotting, immunofluorescence staining, and flow cytometry were applied to detect the indicators related to mitochondrial dynamics, mitophagy, Nrf2 signaling, and apoptosis. The puncture-induced rat models were established to evaluate the changes in vivo. KEY FINDINGS: Our results showed that H2O2 induced oxidative stress, mitochondrial dysfunction, and apoptosis in endplate chondrocytes. These H2O2-induced detrimental effects were inhibited by pretreatment with the mitochondria-targeted antioxidant Mito-TEMPO. In addition, mitochondrial dynamics, Parkin-mediated elimination of dysfunctional mitochondria, and Nrf2-mediated antioxidant system were promoted by H2O2. Knockdown of Parkin or Nrf2 increased H2O2-induced detrimental effects. Moreover, upregulation of Parkin and Nrf2 by polydatin protected endplate chondrocytes against H2O2-induced mitochondrial dysfunction, oxidative stress, and apoptosis. Finally, puncture-induced rat models showed that polydatin exerted a protective effect on CEP and disc degeneration. SIGNIFICANCE: Targeting Parkin and Nrf2 to improve mitochondrial homeostasis, redox balance and endplate chondrocyte survival may represent a potential therapeutic strategy for preventing IDD.


Assuntos
Apoptose/fisiologia , Condrócitos/patologia , Disco Intervertebral/patologia , Fator 2 Relacionado a NF-E2/fisiologia , Estresse Oxidativo/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Idoso , Animais , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Peróxido de Hidrogênio/farmacologia , Disco Intervertebral/efeitos dos fármacos , Disco Intervertebral/metabolismo , Masculino , Pessoa de Meia-Idade , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
2.
Cell Prolif ; 53(2): e12746, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31867863

RESUMO

Human high-temperature requirement protein 1 (HTRA1) is a member of serine proteases and consists of four well-defined domains-an IGFBP domain, a Kazal domain, a protease domain and a PDZ domain. HTRA1 is a secretory protein and also present intracellularly and associated with microtubules. HTRA1 regulates a broad range of physiological processes via its proteolytic activity. This review examines the role of HTRA1 in bone biology, osteoarthritis, intervertebral disc (IVD) degeneration and tumorigenesis. HTRA1 mediates diverse pathological processes via a variety of signalling pathways, such as TGF-ß and NF-κB. The expression of HTRA1 is increased in arthritis and IVD degeneration, suggesting that HTRA1 protein is attributed to cartilage degeneration and disease progression. Emerging evidence also suggests that HTRA1 has a role in tumorigenesis. Further understanding the mechanisms by which HTRA1 displays as an extrinsic and intrinsic regulator in a cell type-specific manner will be important for the development of HTRA1 as a therapeutic target.


Assuntos
Serina Peptidase 1 de Requerimento de Alta Temperatura A/metabolismo , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/patologia , Neoplasias/metabolismo , Neoplasias/patologia , Sequência de Aminoácidos , Animais , Humanos , Disco Intervertebral/metabolismo , Alinhamento de Sequência , Transdução de Sinais/fisiologia , Temperatura Ambiente
3.
Medicine (Baltimore) ; 98(52): e18465, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31876730

RESUMO

This study aimed to investigate the correlation of long noncoding RNA zinc finger antisense 1 (lncRNA ZFAS1) expression with disease risk, disease severity and inflammatory cytokines levels in lumbar disc degeneration (LDD) patients.83 LDD patients underwent surgery and 28 traumatized, non-LDD patients underwent lumbar disc surgery (controls) were consecutively enrolled in this case-control study. Lumbar disc tissue was obtained during surgery and herniated nucleus pulposus (HNP) was isolated to detect lncRNA ZFAS1 expression and inflammatory cytokines mRNA levels by RT-qPCR, and determine protein levels of inflammatory cytokines by western blot.HNP lncRNA ZFAS1 expression in LDD patients was up-regulated compared with controls (P < .001), and receiver operating characteristic (ROC) curve showed lncRNA ZFAS1 expression disclosed a good predictive value for LDD risk with area under curve (AUC) 0.753 (95% CI 0.646-0.859). And after adjustment by age, gender and body mass index (BMI), lncRNA ZFAS1 (P = .017) remained to be an independent predictive factor for higher LDD risk. In addition, lncRNA ZFAS1 expression was positively associated with Modified Pfirrmann Grade (P = .015). As to inflammatory cytokines, lncRNA ZFAS1 expression was observed to be positively correlated with TNF-α (P = .002), IL-1ß (P = .007) and IL-6 (P = .015) mRNAs expressions while reversely associated with IL-10 mRNA level (P = .014); and lncRNA ZFAS1 expression was also positively correlated with protein levels of TNF-α (P = .038) and IL-6 (P = .027) while reversely associated with IL-10 protein expression (P = .039).lncRNA ZFAS1 expression associates with increased risk, elevated disease severity and higher inflammatory cytokines levels in LDD patients.


Assuntos
Citocinas/metabolismo , Degeneração do Disco Intervertebral/metabolismo , Deslocamento do Disco Intervertebral/metabolismo , Disco Intervertebral/metabolismo , Vértebras Lombares , RNA Antissenso/metabolismo , RNA Longo não Codificante/metabolismo , Adulto , Biomarcadores/análise , Western Blotting , Estudos de Casos e Controles , Citocinas/análise , Feminino , Humanos , Interleucina-10/análise , Interleucina-10/metabolismo , Interleucina-1beta/análise , Interleucina-1beta/metabolismo , Interleucina-6/análise , Interleucina-6/metabolismo , Disco Intervertebral/química , Masculino , Pessoa de Meia-Idade , RNA Longo não Codificante/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Risco , Índice de Gravidade de Doença , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/metabolismo
4.
Spine (Phila Pa 1976) ; 44(22): E1290-E1297, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31689248

RESUMO

STUDY DESIGN: This is an in vitro study of bovine disc cells exposed to pulsed electromagnetic fields. OBJECTIVE: The purpose of the present study was to investigate whether pulsed electromagnetic fields (PEMF) effects on the expression of interleukin-6 (IL-6) expression is mediated by two known inflammation regulators, nuclear factor-κB (NF-κß) and phosphorylated mitogen-activated protein kinase p38 (p38-MAPK) signaling pathways SUMMARY OF BACKGROUND DATA.: Inflammatory cytokines play a dominant role in the pathogenesis of disc degeneration. Increasing evidence showed that PEMF, a noninvasive biophysical stimulation, can have physiologically beneficial effects on inflammation and tissue repair. Our previous research shows that PEMF treatment can reduce IL-6 expression by intervertebral disc cells. However, the underlying mechanisms of PEMF action are yet to be uncovered. METHODS: Intervertebral disc nuclear pulposus cells were challenged with interleukin-1α (IL-1α) (for mimicking inflammatory microenvironment) and treated with PEMF simultaneously up to 4 hours. Cells were then collected for NF-κß and phosphorylated p38-MAPK protein detection with Western blot. Additionally, the RelA (p65) subunit of NF-κß was examined with immunostaining for assessment of NF-κß activation. RESULTS: As expected, Western blot results showed that both NF-κß and phosphorylated p38 expression were significantly increased by IL-1α treatment. This induction was significantly inhibited to control condition levels by PEMF treatment. Immunostaining demonstrated similar trends, that PEMF treatment reduced the NF-κß activation induced by IL-1α exposure. CONCLUSION: Our data indicate that the previously-reported inhibitory effect of PEMF treatment on disc inflammation is mediated by NF-κß and phosphorylated p38-MAPK signaling pathways. These results further establish PEMFs anti-inflammatory activity, and may inform potential future clinical uses for management of inflammation associated with disc degeneration. LEVEL OF EVIDENCE: N/A.


Assuntos
Campos Eletromagnéticos , Interleucina-6/metabolismo , Disco Intervertebral , NF-kappa B/metabolismo , Transdução de Sinais/efeitos da radiação , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Bovinos , Disco Intervertebral/citologia , Disco Intervertebral/metabolismo , Disco Intervertebral/efeitos da radiação
5.
Spine (Phila Pa 1976) ; 44(23): 1613-1622, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31730570

RESUMO

STUDY DESIGN: Experimental study with human mesenchymal stem cells (MSCs) and intervertebral disc (IVD) tissue samples. OBJECTIVE: This study aimed to characterize the effect of MSC homing on the Tie2-positive IVD progenitor cell population, IVD cell survival, and proliferation. SUMMARY OF BACKGROUND DATA: Homing of human MSCs has been described as potential alternative to MSC injection, aiming to enhance the regenerative capacity of the IVD. IVD cells expressing Tie2 (also known as CD202b or Angiopoietin-1 receptor TEK tyrosine kinase) represent a progenitor cell population with discogenic differentiation potential. However, the fraction of Tie2-positive progenitor cells decreases with aging and degree of IVD degeneration, resulting in a potential loss of the IVD's regenerative capacity. METHODS: Human MSCs, isolated from vertebral bone marrow aspirates, were labeled and seeded onto the endplate of bovine IVDs and human IVD tissue. Following MSC migration for 5 days, IVD cells were isolated by tissue digestion. The fractions of Tie2-positive, dead, apoptotic, and proliferative IVD cells were evaluated by flow cytometry and compared to untreated IVDs. For human IVDs, 3 groups were investigated: nondegenerated (organ donors), IVDs of patients suffering from spinal trauma, and degenerative IVD tissue samples. RESULTS: MSC homing enhanced the fraction of Tie2-positive IVD cells in bovine and human IVD samples. Furthermore, a proliferative response and lower fraction of dead cells were observed after MSC homing in both bovine and human IVD tissues. CONCLUSION: Our findings indicate that MSC homing enhances the survival and regenerative capability of IVD cells, which may be mediated by intercellular communication. MSC homing could represent a potential treatment strategy to prevent the onset of the degenerative cascade in IVDs at risk such as IVDs adjacent to a fused segment or IVDs after herniation. LEVEL OF EVIDENCE: N/A.


Assuntos
Proliferação de Células/fisiologia , Disco Intervertebral/metabolismo , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/metabolismo , Receptor TIE-2/biossíntese , Animais , Bovinos , Morte Celular/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Feminino , Humanos , Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/terapia , Técnicas de Cultura de Órgãos
6.
Biotech Histochem ; 94(7): 540-545, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31537133

RESUMO

Interleukin-23 (IL-23, IL-23p19) is a proinflammatory cytokine in the IL-12-related family. Although inflammatory cells in herniated discs have been shown to contain IL-23, little is known about the presence and role of IL-23 in human disc cells. We analyzed disc specimens for IL-23 localization using immunohistochemistry in control, herniated and non-herniated discs from which annulus fibrosus (annulus) cells were isolated and cultured to identify IL-23 gene expression and production. Microarray analysis was used to assess the expression of IL-23 in disc tissue and in cells exposed to two proinflammatory cytokines, IL-1ß and TNF-α. IL-23 was present in annulus cells at the protein level and its expression was up-regulated significantly in herniated compared to control disc tissue. Direct measurement of medium components confirmed production of IL-23 and its receptor, IL-23R, by annulus cells in vitro. Annulus cells in three-dimensional culture exposed to TNF-α, but not IL-1ß, resulted in significant up-regulation of IL-23 expression compared to control cells. Our findings are evidence for the constitutive presence of IL-23 in the human disc and that its expression in vitro is modified by exposure to TNF-α.


Assuntos
Subunidade p19 da Interleucina-23/metabolismo , Degeneração do Disco Intervertebral/metabolismo , Deslocamento do Disco Intervertebral/metabolismo , Disco Intervertebral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Anel Fibroso/metabolismo , Células Cultivadas , Citocinas/metabolismo , Expressão Gênica/fisiologia , Humanos , Degeneração do Disco Intervertebral/diagnóstico , Deslocamento do Disco Intervertebral/diagnóstico , Regulação para Cima
7.
Med Sci Monit ; 25: 4892-4900, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31263091

RESUMO

BACKGROUND Intervertebral disc degeneration (IDD) is associated with low back and neck pain, but the mechanisms underlying its pathogenesis are unclear. In this study, we explored the function of microRNA-149 (miR-149) in inflammatory response mediated by lipopolysaccharide (LPS) in nucleus pulposus (NP) cells. MATERIAL AND METHODS Quantitative real-time PCR was used to detect miRNA and mRNA levels, while Western blotting was utilized to determine protein levels. ELISA was used to examine chemokine production. The correlation between miR-149 and MyD88 was assessed by reporter assay. Apoptosis was examined by flow cytometry. RESULTS miR-149 expression was significantly decreased after LPS exposure in NP cells. Overexpression of miR-149 reversed LPS-induced inhibition in aggrecan and collagen II expression and attenuated LPS-mediated promotion in the levels of MMP3, ADAMTS4, and inflammatory cytokines. Moreover, we found that miR-149 exerted its function by targeting MyD88 in NP cells. CONCLUSIONS miR-149 can inhibit the inflammatory response mediated by LPS in NP cells, and might be a potential target for the treatment of IDD.


Assuntos
MicroRNAs/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Núcleo Pulposo/patologia , Animais , Apoptose/fisiologia , Citocinas/metabolismo , Matriz Extracelular/metabolismo , Disco Intervertebral/metabolismo , Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/genética , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/patologia , Lipopolissacarídeos/metabolismo , MicroRNAs/biossíntese , Fator 88 de Diferenciação Mieloide/genética , Núcleo Pulposo/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley
8.
Eur Radiol ; 29(12): 6443-6446, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31278582

RESUMO

KEY POINTS: • Molecular intervertebral disc damage was associated with LBP and radiculopathy.• Patients with radiculopathy and LBP demonstrated a depletion of gagCEST values compared with healthy controls.• GagCEST imaging may be a non-invasive tool for investigation of degeneration processes of lumbar intervertebral discs (IVDs). GagCEST imaging may be an imaging biomarker for biochemical IVD alterations.


Assuntos
Degeneração do Disco Intervertebral/diagnóstico por imagem , Disco Intervertebral/diagnóstico por imagem , Dor Lombar/fisiopatologia , Imagem por Ressonância Magnética/métodos , Radiculopatia/fisiopatologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/análise , Feminino , Glicosaminoglicanos/metabolismo , Humanos , Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/complicações , Degeneração do Disco Intervertebral/metabolismo , Dor Lombar/etiologia , Dor Lombar/metabolismo , Vértebras Lombares/metabolismo , Vértebras Lombares/fisiopatologia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Radiculopatia/etiologia , Radiculopatia/metabolismo , Adulto Jovem
9.
PLoS One ; 14(6): e0218121, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31220091

RESUMO

The normal aging of the extracellular matrix and collagen content of the human lumbar intervertebral disc (IVD) remains relatively unknown despite vast amounts of basic science research, partly because of the use of inadequate surrogates for a truly normal, human IVD. Our objective in this study was to describe and compare the morphology and ultrastructure of lumbar IVDs in 2 groups of young (G1-<35 years) and elderly (G2->65 years). Thirty L4-5 and L5-S1 discs per group were obtained during autopsies of presumably-asymptomatic individuals and analyzed with magnetic resonance imaging (MRI), a morphological grading scale, light microscopy, scanning electron microscopy (SEM) and immunohistochemistry (IHC) for collagen types I, II, III, IV, V, VI, IX and X. As expected, a mild to moderate degree of degeneration was present in G1 discs and significantly more advanced in G2. The extracellular matrix of G2 discs was significantly more compact with an increase of cartilaginous features such as large chondrocyte clusters. Elastic fibers were abundant in G1 specimens and their presence correlated more with age than with degeneration grade, being very rare in G2. SEM demonstrated persistence of basic structural characteristics such as denser lamellae with Sharpey-type insertions into the endplates despite advanced age or degeneration grades. Immunohistochemistry revealed type II collagen to be the most abundant type followed by collagen IV. All collagen types were detected in every disc sector except for type X collagen. Statistical analysis demonstrated a general decrease in collagen expression from G1 to G2 with an annular- and another nuclear-specific pattern. These results suggest modifications of IVD morphology do not differ between the anterior or posterior annulus but are more advanced or happen earlier in the posterior areas of the disc. This study finally describes the process of extracellular matrix modification during disc degeneration in an unselected, general population and demonstrates it is similar to the same process in the cervical spine as published previously.


Assuntos
Disco Intervertebral/fisiologia , Disco Intervertebral/ultraestrutura , Vértebras Lombares , Adulto , Idoso , Colágeno Tipo II/metabolismo , Colágeno Tipo IV/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Disco Intervertebral/metabolismo , Imagem por Ressonância Magnética , Masculino , Microscopia Eletrônica de Varredura
10.
Life Sci ; 232: 116565, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31251999

RESUMO

HEADINGS AIMS: The present study determined whether nucleus pulposus (NP) cells express hypoxia-inducible factor-2alpha (HIF-2α) and assessed its role in regulating the expression of catabolic factors during intervertebral disc degeneration. MATERIALS AND METHODS: Human degenerated NP tissues were acquired to examine the HIF-2α expression levels using immunohistochemistry, western blotting, and Real-time PCR. Human NP cells were cultivated under normoxic or hypoxic conditions, and the HIF-2α expression was determined. Then, human NP cells were treated with HIF-2α plasmids, HIF-2α siRNA, and tumor necrosis factor-alpha (TNF-α) to evaluate the role of HIF-2α in regulating matrix metalloproteinase (MMP) and aggrecanase expression. An in vivo rabbit disc degeneration model was established to demonstrate that HIF-2α plays a critical role in disc degeneration. KEY FINDINGS: We found that HIF-2α had a markedly elevated expression in human degenerated discs in the Grade III stage. HIF-2α protein and gene transcript levels in vitro were relatively higher under hypoxic conditions. The expression of MMP-13, ADAMTS-4 was decreased significantly in HIF-2α silencing condition, while the over-expression resulted in significantly increased levels of MMP-13 and ADAMTS-4. When cytokine TNF-α was added, HIF-2α was induced by nuclear factor-κB (NF-κB). The in vivo experiments showed that the HIF-2α controlled the catabolic factors MMP-13 and ADAMTS-4 that regulated the collagen II and aggrecan metabolism in disc degeneration. SIGNIFICANCE: HIF-2α is a catabolic regulator in disc degeneration and directly controls the catabolic genes. The suppression of HIF-2α expression leads to decelerates extracellular matrix degradation that might represent a therapeutic target for the degenerative disc.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Degeneração do Disco Intervertebral/metabolismo , Adulto , Idoso , Agrecanas/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Citocinas/metabolismo , Endopeptidases/metabolismo , Feminino , Humanos , Hipóxia/metabolismo , Disco Intervertebral/metabolismo , Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/genética , Degeneração do Disco Intervertebral/patologia , Masculino , Metaloproteinases da Matriz/metabolismo , Pessoa de Meia-Idade , Núcleo Pulposo/metabolismo , Núcleo Pulposo/patologia , Coelhos , Transdução de Sinais/fisiologia , Transcriptoma , Fator de Necrose Tumoral alfa/metabolismo
11.
Mol Med Rep ; 20(1): 567-572, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31180546

RESUMO

Cartilaginous endplate degeneration serves a key role in the process of intervertebral disc (IVD) degeneration, however, effective therapies are hindered by an incomplete understanding of the mechanisms that underlie cartilage endplate (CEP) homeostasis and degeneration. Wnt/ß­catenin signalling has been reported as a major factor in regulating biological processes. Whether Wnt/ß­catenin signalling engages in CEP homeostasis has not yet been investigated. The present study aimed to assess the function of CEP cells via the activation of Wnt/ß­catenin signalling to examine and promote the mechanism of degeneration of CEP in vitro. Rat CEP cells were confirmed to exhibit a chondrocytic phenotype by toluidine blue staining. The increased number of senescence­associated ß­galactosidase (SA­ß­gal)­positive cells and reduced cellular proliferation were investigated in the presence of a ß­catenin inhibitor, and the inhibitor improved the trend of senescence. An increased number of apoptotic cells was detected by lithium chloride treatment, and inhibiting Wnt/ß­catenin signalling protected the cells from apoptosis. Expression of the catabolic enzymes, metalloproteinase­13 and a disintegrin and metalloproteinase with thrombospondin motifs­5, and the decreased expression of aggrecan were also observed by Wnt/ß­catenin signalling activation, and a Wnt/ß­catenin signalling inhibitor decreased the expression of catabolic enzymes and increased the expression of aggrecan induced by Wnt/ß­catenin signalling activation. Wnt/ß­catenin signalling may provide potential strategies for preventing CEP degeneration.


Assuntos
Condrócitos/citologia , Disco Intervertebral/citologia , Via de Sinalização Wnt , Animais , Células Cultivadas , Condrócitos/metabolismo , Homeostase , Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/metabolismo , Masculino , Ratos Sprague-Dawley , beta Catenina/metabolismo
12.
Turk Neurosurg ; 29(4): 470-477, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31124572

RESUMO

AIM: To investigate the effect of dabigatran, a new oral anticoagulant, on human primary cell cultures isolated from intact intervertebral disc tissue. MATERIAL AND METHODS: Cell cultures were prepared from tissues obtained from six cases who had undergone surgery due to spinal trauma. Dabigatran, an active pharmacological agent, was applied to intact annulus fibrosus (AF)/nucleus pulposus (NP) primary cell cultures from the study group. After performing cell viability, toxicity, and proliferation tests on all cultures in the control and study groups, the surface morphologies of the samples were evaluated. Subsequently, chondroadherin (CHAD), cartilage oligomeric matrix protein (COMP), and matrix metalloproteinase (MMP)-13 and -19 expressions were measured via a real-time polymerase chain reaction (RT-PCR). Data were analyzed statistically. RESULTS: In the proliferation assays performed on the 20th day of the study, cells in the dabigatran-supplemented group were reported to have lost 46.37% more viability than those in the control group. Expressions of all genes examined except MMP-13 were evaluated in the control group by time, but in contrast to the control group results, COMP and MMP-19 gene expressions decreased in the dabigatran-treated group. No CHAD or MMP-13 expression was noted in these cultures. CONCLUSION: The potential for a systemically applied drug to accumulate in tissue and negatively affect surrounding tissues and microstructures must be emphasized.


Assuntos
Anticoagulantes/efeitos adversos , Dabigatrana/efeitos adversos , Disco Intervertebral/efeitos dos fármacos , Trombose/prevenção & controle , Administração Oral , Adolescente , Adulto , Anticoagulantes/administração & dosagem , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Dabigatrana/administração & dosagem , Proteínas da Matriz Extracelular/metabolismo , Feminino , Expressão Gênica , Humanos , Disco Intervertebral/metabolismo , Disco Intervertebral/cirurgia , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/cirurgia , Masculino , Pessoa de Meia-Idade , Cultura Primária de Células/métodos , Trombose/metabolismo , Adulto Jovem
13.
Acta Biochim Biophys Sin (Shanghai) ; 51(6): 571-579, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-31056633

RESUMO

The functional changes of nucleus pulposus (NP) cells are considered to be the initiating factors of intervertebral disc degeneration (IDD), and the differentially expressed circRNAs in NP cells may play an important role in the process of IDD. To identify circular RNAs (circRNAs) associated with human IDD, we isolated the NP cells from human degenerated and non-degenerated intervertebral disc and identified NP cells by microscopy and cell proliferation. CircRNA microarray expression profiles were obtained from NP cells of degenerated and non-degenerated intervertebral disc and further validated by quantitative reverse transcription PCR (qRT-PCR). The expression data were analyzed by bioinformatics. Microarray analysis identified 7294 circRNAs differentially expressed in degenerated human IDD NP cells. Among them, 3724 circRNAs were up-regulated and 3570 circRNAs were down-regulated by more than 2 folds. After validating by qRT-PCR, we predicted the possible miRNAs of the top dysregulated circRNAs using TargetScan, and miRanda. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the most modulated circRNAs regulate the viability, degradation, apoptosis and oxidative stress in NP cells, and the possible mechanism underlying IDD was discussed. These results revealed that circRNAs may play a role in IDD and might be a promising candidate molecular target for gene therapy.


Assuntos
Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Degeneração do Disco Intervertebral/genética , Núcleo Pulposo/metabolismo , /genética , Adulto , Apoptose/genética , Sobrevivência Celular/genética , Células Cultivadas , Feminino , Ontologia Genética , Humanos , Disco Intervertebral/metabolismo , Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/patologia , Masculino , MicroRNAs/genética , Núcleo Pulposo/patologia
14.
PLoS One ; 14(4): e0215218, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30970007

RESUMO

Poor solute transport through the cartilage endplate (CEP) impairs disc nutrition and could be a key factor that limits the success of intradiscal biologic therapies. Here we demonstrate that treating the CEP with matrix metalloproteinase-8 (MMP-8) reduces the matrix constituents that impede solute uptake and thereby improves nutrient diffusion. Human CEP tissues harvested from four fresh cadaveric lumbar spines (age range: 38-66 years old) were treated with MMP-8. Treatment caused a dose-dependent reduction in sGAG, localized reductions to the amount of collagen, and alterations to collagen structure. These matrix modifications corresponded with 16-24% increases in the uptake of a small solute (376 Da). Interestingly, the effects of MMP-8 treatment depended on the extent of non-enzymatic glycation: treated CEPs with high concentrations of advanced glycation end products (AGEs) exhibited the lowest uptake compared to treated CEPs with low concentrations of AGEs. Moreover, AGE concentrations were donor-specific, and the donor tissues with the highest AGE concentrations appeared to have lower uptake than would be expected based on the initial amounts of collagen and sGAG. Finally, increasing solute uptake in the CEP improved cell viability inside diffusion chambers, which supports the nutritional relevance of enhancing the transport properties of the CEP. Taken together, our results provide new insights and in vitro proof-of-concept for a treatment approach that could improve disc nutrition for biologic therapy: specifically, matrix reduction by MMP-8 can enhance solute uptake and nutrient diffusion through the CEP, and AGE concentration appears to be an important, patient-specific factor that influences the efficacy of this approach.


Assuntos
Cartilagem/efeitos dos fármacos , Cartilagem/metabolismo , Disco Intervertebral/efeitos dos fármacos , Disco Intervertebral/metabolismo , Metaloproteinase 8 da Matriz/metabolismo , Metaloproteinase 8 da Matriz/farmacologia , Adulto , Idoso , Transporte Biológico/efeitos dos fármacos , Cartilagem/citologia , Sobrevivência Celular , Colágeno/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Humanos , Técnicas In Vitro , Degeneração do Disco Intervertebral/tratamento farmacológico , Degeneração do Disco Intervertebral/metabolismo , Pessoa de Meia-Idade , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia
15.
Mech Ageing Dev ; 180: 97-106, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31002926

RESUMO

Cellular senescence is a phenotype characterized by irreversible growth arrest, chronic elevated secretion of proinflammatory cytokines and matrix proteases, a phenomenon known as senescence-associated secretory phenotype (SASP). Biomarkers of cellular senescence have been shown to increase with age and degeneration of human disc tissue. Senescent disc cells in culture recapitulate features associated with age-related disc degeneration, including increased secretion of proinflammatory cytokines, matrix proteases, and fragmentation of matrix proteins. However, little is known of the metabolic changes that underlie the senescent phenotype of disc cells. To assess the metabolic changes, we performed a bioenergetic analysis of in vitro oxidative stress-induced senescent (SIS) human disc cells. SIS disc cells acquire SASP and exhibit significantly elevated mitochondrial content and mitochondrial ATP-linked respiration. The metabolic changes appear to be driven by the upregulated protein secretion in SIS cells as abrogation of protein synthesis using cycloheximide decreased mitochondrial ATP-linked respiration. Taken together, the results of the study suggest that the increased energy generation state supports the secretion of senescent associated proteins in SIS disc cells.


Assuntos
Senescência Celular , Metabolismo Energético , Disco Intervertebral/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo , Consumo de Oxigênio , Adulto , Feminino , Humanos , Disco Intervertebral/patologia , Masculino , Pessoa de Meia-Idade , Mitocôndrias/patologia
16.
J Mol Histol ; 50(3): 285-294, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30993430

RESUMO

The aim of this study was to ascertain whether, like many cell types in cartilaginous tissues if type XI collagen was a pericellular component of annulus fibrosus (AF) cells and chondrocytes. Fine fibrillar networks were visualised which were perlecan, HS (MAb 10E4) and type XI collagen positive. Heparitinase-III pre-digestion abolished the type XI collagen and 10E4 localisation in these fibrillar assemblies demonstrating a putative HS mediated interaction which localised the type XI collagen. Type XI collagen was confirmed to be present in the Heparitinase III treated AF monolayer media samples by immunoblotting. Heparitinase-III generated ΔHS stub epitopes throughout these fibrillar networks strongly visualised by MAb 3-G-10. Monolayers of murine hip articular chondrocytes from C57BL/6 and Hspg2 exon 3 null mice also displayed pericellular perlecan localisations, however type XI collagen was only evident in the Wild type mice. Perlecan was also immunolocalised in control and murine knee articular cartilage from the two mouse genotypes subjected to a medial meniscal destabilisation procedure which induces OA. This resulted in a severe depletion of perlecan levels particularly in the perlecan exon 3 null mice and was consistent with OA representing a disease of the pericellular matrix. A model was prepared to explain these observations between the NPP type XI collagen domain and HS chains of perlecan domain-I in the pericellular matrix of AF cells which likely contributed to cellular communication, tissue stabilization and the regulation of extracellular matrix homeostasis.


Assuntos
Anel Fibroso/efeitos dos fármacos , Colágeno Tipo XI/genética , Proteoglicanas de Heparan Sulfato/genética , Animais , Anel Fibroso/metabolismo , Anel Fibroso/patologia , Cartilagem Articular/crescimento & desenvolvimento , Cartilagem Articular/metabolismo , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Éxons/genética , Matriz Extracelular/genética , Proteínas da Matriz Extracelular/genética , Homeostase/genética , Disco Intervertebral/crescimento & desenvolvimento , Disco Intervertebral/metabolismo , Camundongos , Polissacarídeo-Liase/farmacologia
17.
Spine (Phila Pa 1976) ; 44(18): 1257-1269, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30973506

RESUMO

STUDY DESIGN: A rat puncture injury intervertebral disc (IVD) degeneration model with structural, biomechanical, and histological analyses. OBJECTIVE: To determine if males and females have distinct responses in the IVD after injury. SUMMARY OF BACKGROUND DATA: Low back pain (LBP) and spinal impairments are more common in women than men. However, sex differences in IVD response to injury have been underexplored, particularly in animal models where sex differences can be measured without gender confounds. METHODS: Forty-eight male and female Sprague Dawley rats underwent sham, single annular puncture with tumor necrosis factor α (TNFα) injection (1×), or triple annular puncture with TNFα injection (3×) surgery. Six weeks after surgery, lumbar IVDs were assessed by radiologic IVD height, spinal motion segment biomechanical testing, histological degeneration grading, second harmonic generation (SHG) imaging, and immunofluorescence for fibronectin and α-smooth muscle actin. RESULTS: Annular puncture injuries significantly increased degenerative grade and IVD height loss for males and females, but females had increased degeneration grade particularly in the annulus fibrosus (AF). Despite IVD height loss, biomechanical properties were largely unaffected by injury at 6 weeks. However, biomechanical measures sensitive to outer AF differed by sex after 3× injury-male IVDs had greater torsional stiffness, torque range, and viscoelastic creep responses. SHG intensity of outer AF was reduced after injury only in female IVDs, suggesting sex differences in collagen remodeling. Both males and females exhibited decreased cellularity and increased fibronectin expression at injury sites. CONCLUSION: IVD injury results in distinct degeneration and functional healing responses between males and females. The subtle sex differences identified in this animal model suggest differences in response to IVD injury that might explain some of the variance observed in human LBP, and demonstrate the need to better understand differences in male and female IVD degeneration patterns and pain pathogenesis. LEVEL OF EVIDENCE: N/A.


Assuntos
Anel Fibroso/lesões , Degeneração do Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/fisiopatologia , Disco Intervertebral/lesões , Animais , Anel Fibroso/metabolismo , Anel Fibroso/patologia , Anel Fibroso/fisiopatologia , Colágeno/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Injeções , Disco Intervertebral/metabolismo , Disco Intervertebral/patologia , Disco Intervertebral/fisiopatologia , Dor Lombar/fisiopatologia , Masculino , Punções/efeitos adversos , Ratos , Ratos Sprague-Dawley , Fatores Sexuais , Fator de Necrose Tumoral alfa/metabolismo , Cicatrização
18.
Int J Mol Sci ; 20(7)2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-30974795

RESUMO

Transient receptor potential (TRP) channels have emerged as potential sensors and transducers of inflammatory pain. The aims of this study were to investigate (1) the expression of TRP channels in intervertebral disc (IVD) cells in normal and inflammatory conditions and (2) the function of Transient receptor potential ankyrin 1 (TRPA1) and Transient receptor potential vanilloid 1 (TRPV1) in IVD inflammation and matrix homeostasis. RT-qPCR was used to analyze human fetal, healthy, and degenerated IVD tissues for the gene expression of TRPA1 and TRPV1. The primary IVD cell cultures were stimulated with either interleukin-1 beta (IL-1ß) or tumor necrosis factor alpha (TNF-α) alone or in combination with TRPA1/V1 agonist allyl isothiocyanate (AITC, 3 and 10 µM), followed by analysis of calcium flux and the expression of inflammation mediators (RT-qPCR/ELISA) and matrix constituents (RT-qPCR). The matrix structure and composition in caudal motion segments from TRPA1 and TRPV1 wild-type (WT) and knock-out (KO) mice was visualized by FAST staining. Gene expression of other TRP channels (A1, C1, C3, C6, V1, V2, V4, V6, M2, M7, M8) was also tested in cytokine-treated cells. TRPA1 was expressed in fetal IVD cells, 20% of degenerated IVDs, but not in healthy mature IVDs. TRPA1 expression was not detectable in untreated cells and it increased upon cytokine treatment, while TRPV1 was expressed and concomitantly reduced. In inflamed IVD cells, 10 µM AITC activated calcium flux, induced gene expression of IL-8, and reduced disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) and collagen 1A1, possibly via upregulated TRPA1. TRPA1 KO in mice was associated with signs of degeneration in the nucleus pulposus and the vertebral growth plate, whereas TRPV1 KO did not show profound changes. Cytokine treatment also affected the gene expression of TRPV2 (increase), TRPV4 (increase), and TRPC6 (decrease). TRPA1 might be expressed in developing IVD, downregulated during its maturation, and upregulated again in degenerative disc disease, participating in matrix homeostasis. However, follow-up studies with larger sample sizes are needed to fully elucidate the role of TRPA1 and other TRP channels in degenerative disc disease.


Assuntos
Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Degeneração do Disco Intervertebral/metabolismo , Disco Intervertebral/metabolismo , Núcleo Pulposo/metabolismo , Canal de Cátion TRPA1/biossíntese , Canais de Cátion TRPV/biossíntese , Animais , Sinalização do Cálcio , Matriz Extracelular/patologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/patologia , Camundongos , Camundongos Knockout , Núcleo Pulposo/patologia
19.
Spine (Phila Pa 1976) ; 44(15): E873-E881, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-30817728

RESUMO

STUDY DESIGN: In vivo and in vitro studies of the role of miR-2355-5p and its possible targets in intervertebral disc degeneration (IVDD). OBJECTIVE: To elucidate the regulatory role of miR-2355-5p in IVDD and the underlying mechanisms. SUMMARY OF BACKGROUND DATA: IVDD, which is caused by multiple factors, is the main cause of lower back pain with or without extremity pain. However, the underlying cellular mechanisms of IVDD pathogenesis are not well elucidated. Cell hyper-proliferation, inflammation, and epidermal growth factor receptor activation have been implicated in IVDD. Up-regulated miR-2355-5p level was identified to associate with IVDD. ERRFI1 (the product of mitogen-inducible gene 6 [MIG6]) was known to inhibit epidermal growth factor receptor activation. METHODS: We monitored the expression of miR-2355-5p and ERRFI1 in IVDD tissues and lipopolysaccharides (LPS)-treated nucleus pulposus (NP) cells. We explored the effects of ERFFI1 on NP cells proliferation and LPS-induced pro-inflammatory cytokines production. We searched the targets of miR-2355-5p and explored the effects of miR-2355-5p on NP cells proliferation and cytokines production. RESULTS: We identified the up-regulation of miR-2355-5p and down-regulation of ERFFI1 in IVDD samples and LPS-treated NP cells. ERFFI1 inhibited NP cells proliferation and LPS-induced pro-inflammatory cytokine production. MiR-2355-5p targeted ERFFI1 and negatively regulated ERFFI1 expression. MiR-2355-5p regulated IVDD by targeting ERFFI1. CONCLUSION: MiR-2355-5p negatively regulated ERFFI1 and prevented the effects of ERRFI1 on inhibiting NP cells proliferation and inflammation. LEVEL OF EVIDENCE: N/A.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Degeneração do Disco Intervertebral/genética , Degeneração do Disco Intervertebral/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Núcleo Pulposo/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proliferação de Células/efeitos dos fármacos , Citocinas/biossíntese , Regulação para Baixo , Humanos , Inflamação/genética , Inflamação/metabolismo , Disco Intervertebral/metabolismo , Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/patologia , Lipopolissacarídeos/farmacologia , MicroRNAs/biossíntese , Núcleo Pulposo/efeitos dos fármacos , Núcleo Pulposo/patologia , Proteínas Supressoras de Tumor/biossíntese , Regulação para Cima
20.
Spine (Phila Pa 1976) ; 44(17): E992-E999, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-30896588

RESUMO

STUDY DESIGN: Simulation of antibiotics transport into human intervertebral disc with intravenous infusion. OBJECTIVE: The objective of this study was to quantitatively investigate antibiotic concentrations in the disc. SUMMARY OF BACKGROUND DATA: Intravenous infusion of antibiotics is typically used to treat intervertebral disc infection in clinics. However, it is difficult to evaluate the drug concentrations within discs in vivo. METHODS: A computational model was used in this study. The variation of drug charge with pH was considered in the model. Thirty-minute infusions of two commonly used antibiotics in clinic-vancomycin and cefepime-were numerically investigated. Spatial and temporal concentration distributions of these drugs in both nondegenerated and moderately degenerated discs were calculated. RESULTS: For intravenous infusion of 1 g vancomycin and 2 g cefepime in 30 minutes repeated every 12 hours, it was predicted that vancomycin concentration in the disc fluctuated between 17.0 and 31.0 times of its minimum inhibitory concentration (1 ug/mL) and cefepime concentration fluctuated between 1.1 and 4.2 times of its minimum inhibitory concentration (i.e., 8 ug/mL) in about 2 days. It was also found that vancomycin concentration in moderately degenerated disc was lower than that in the nondegenerated disc. CONCLUSION: This study provides quantitative guidance on selecting proper dosage for treating disc infection. The method used in this study could be used to provide quantitative information on transport of other antibiotics and drugs in discs as well. LEVEL OF EVIDENCE: N/A.


Assuntos
Antibacterianos/farmacocinética , Cefepima/farmacocinética , Disco Intervertebral/metabolismo , Vancomicina/farmacocinética , Antibacterianos/administração & dosagem , Cefepima/administração & dosagem , Humanos , Infusões Intravenosas , Disco Intervertebral/química , Degeneração do Disco Intervertebral/metabolismo , Modelos Biológicos , Vancomicina/administração & dosagem
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