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1.
Braz J Med Biol Res ; 52(9): e8525, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31411316

RESUMO

Many compounds of ginsenosides show anti-inflammatory properties. However, their anti-inflammatory effects in intervertebral chondrocytes in the presence of inflammatory factors have never been shown. Increased levels of pro-inflammatory cytokines are generally associated with the degradation and death of chondrocytes; therefore, finding an effective and nontoxic substance that attenuates the inflammation is worthwhile. In this study, chondrocytes were isolated from the nucleus pulposus tissues, and the cells were treated with ginsenoside compounds and IL-1ß, alone and in combination. Cell viability and death rate were assessed by CCK-8 and flow cytometry methods, respectively. PCR, western blot, and immunoprecipitation assays were performed to determine the mRNA and protein expression, and the interactions between proteins, respectively. Monomeric component of ginsenoside Rd had no toxicity at the tested range of concentrations. Furthermore, Rd suppressed the inflammatory response of chondrocytes to interleukin (IL)-1ß by suppressing the increase in IL-1ß, tumor necrosis factor (TNF)-α, IL-6, COX-2, and inducible nitric oxide synthase (iNOS) expression, and retarding IL-1ß-induced degradation of chondrocytes by improving cell proliferation characteristics and expression of aggrecan and COL2A1. These protective effects of Rd were associated with ubiquitination of IL-1 receptor accessory protein (IL1RAP), blocking the stimulation of IL-1ß to NF-κB. Bioinformatics analysis showed that NEDD4, CBL, CBLB, CBLC, and ITCH most likely target IL1RAP. Rd increased intracellular ITCH level and the amount of ITCH attaching to IL1RAP. Thus, IL1RAP ubiquitination promoted by Rd is likely to occur by up-regulation of ITCH. In summary, Rd inhibited IL-1ß-induced inflammation and degradation of intervertebral disc chondrocytes by increasing IL1RAP ubiquitination.


Assuntos
Condrócitos/efeitos dos fármacos , Ginsenosídeos/farmacologia , Proteína Acessória do Receptor de Interleucina-1/metabolismo , Interleucina-1beta/efeitos dos fármacos , Degeneração do Disco Intervertebral/metabolismo , Adulto , Idoso , Agrecanas/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/metabolismo , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Feminino , Ginsenosídeos/metabolismo , Humanos , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Dor Lombar/metabolismo , Masculino , Pessoa de Meia-Idade , Óxido Nítrico Sintase/metabolismo , Núcleo Pulposo/citologia , Núcleo Pulposo/efeitos dos fármacos , Núcleo Pulposo/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitinação
2.
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
3.
Nanoscale ; 11(16): 7921-7930, 2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-30964497

RESUMO

Poly(ethylene glycol) (PEG) based hydrogels are amongst the most studied synthetic hydrogels. However, reports on PEG-based hydrogels with high mechanical strength are limited. Herein, a class of novel, well-defined PEG-based nanocomposite hydrogels with tunable mechanical strength are synthesised via ring-opening reactions of diglycidyl ethers with carboxylate ions. The pH responsive crosslinked polyacid nanogels (NG) in the dispersed phase act as high functionality crosslinkers which covalently bond to the poly(ethylene glycol) diglycidyl ethers (PEGDGE) as the continuous matrix. A series of NG-x-PEG-y-z gels are prepared where x, y and z are concentrations of NGs, PEGDGE and the PEGDGE molecular weight, respectively. The hydrogel compositions and nano-structural homogeneity of the NGs have strong impact on the enhancement of mechanical properties which enables property tuning. Based on this design, a highly compressive PEG-based nanocomposite hydrogel (NG-13-PEG-20-6000) exhibits a compressive stress of 24.2 MPa, compressive fracture strain greater than 98% and a fracture energy density as high as 1.88 MJ m-3. The tensile fracture strain is 230%. This is amongst one of the most compressive PEG-based hydrogels reported to-date. Our chemically crosslinked gels are resilient and show highly recoverable dissipative energy. The cytotoxicity test shows that human nucleus pulposus (NP) cells remained viable after 8 days of culture time. The overall results highlight their potential for applications as replacements for intervertebral discs or articular cartilages.


Assuntos
Hidrogéis/química , Polietilenoglicóis/química , Polietilenoimina/química , Sobrevivência Celular/efeitos dos fármacos , Força Compressiva , Humanos , Concentração de Íons de Hidrogênio , Microscopia Eletrônica de Varredura , Núcleo Pulposo/citologia , Núcleo Pulposo/efeitos dos fármacos , Núcleo Pulposo/metabolismo , Polietilenoglicóis/síntese química , Polietilenoglicóis/toxicidade , Polietilenoimina/síntese química , Polietilenoimina/toxicidade , Espalhamento a Baixo Ângulo , Resistência à Tração , Difração de Raios X
4.
Int J Mol Med ; 43(4): 1679-1686, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30816449

RESUMO

Intervertebral disc degeneration (IDD) is widely considered to be one of the main causes of lower back pain, which is a chronic progressive disease closely related to inflammation, nucleus pulposus (NP) cell apoptosis and extracellular matrix (ECM) degradation. Berberine (BBR) is an alkaloid compound with an anti­inflammatory effect and has been reported to exert therapeutic action in several inflammatory diseases, including osteoarthritis. Therefore, it was hypothesized that BBR may have a therapeutic effect on IDD through inhibition of the inflammatory response. The aim of the present study was to evaluate the influence of BBR on IDD in interleukin (IL)­1ß­treated human NP cells in vitro. The results showed that BBR attenuated the upregulation of ECM­catabolic factors [matrix metalloproteinase (MMP)­3, MMP­13, a disintegrin and metalloproteinase with thrombospondin motif (ADAMTS)­4 and ADAMTS­5], and the downregulation of ECM­anabolic factors (type II collagen and aggrecan) following stimulation of the human NP cells with IL­1ß. Treatment with BBR also protected human NP cells from IL­1ß­induced apoptosis, as determined by western blotting and flow cytometry. Mechanistically, the IL­1ß­stimulated degradation of IκBα, and the phosphorylation and translocation of nuclear factor (NF)­κB p65 were found to be attenuated by BBR, indicating that NF­κB pathway activation was suppressed by BBR in the IL­1ß­treated human NP cells. The results of the experiments revealed a therapeutic potential of BBR for the prevention or treatment of IDD.


Assuntos
Apoptose/efeitos dos fármacos , Berberina/farmacologia , Matriz Extracelular/metabolismo , Interleucina-1beta/efeitos adversos , NF-kappa B/metabolismo , Núcleo Pulposo/citologia , Transdução de Sinais/efeitos dos fármacos , Adolescente , Adulto , Sobrevivência Celular/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Humanos , Modelos Biológicos , Adulto Jovem
5.
J Neurol Surg A Cent Eur Neurosurg ; 80(3): 174-179, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30818407

RESUMO

PURPOSE: To investigate the effects of methylene blue (MB) on the viability and secretory phenotype of rat nucleus pulposus (NP) cells in vitro. METHODS: Rat NP cells were isolated, cultured, and treated with different MB concentrations (0-6.25 ng/mL) for different lengths of time. We evaluated the changes in cell morphology and cell viability. We also examined the cells for expression of collagen II, aggrecan, matrix metalloproteinase (MMP)-3, MMP-9, and inducible nitric oxide synthase (iNOS). RESULTS: After 2.5 to 6.25 ng/mL MB induced for 6 hours, numerous NP cells were dyed blue and rounded up. The adherent cell number was reduced by MB treatment. The viability of rat NP cells was significantly inhibited by MB in a dose- and time-dependent manner. Treatment with a very low dose of MB (1.5625 ng/mL) resulted in lower expression of collagen II and aggrecan and higher expression of MMP-3, MMP-9, and iNOS in rat NP cells. CONCLUSIONS: Rat NP cells exposed in vitro to MB significantly reduced their viability. Moreover, MB upregulated catabolism gene expression and downregulated anabolism gene expression in rat NP cells. These results suggest MB may be harmful to NP cells. The dose of intradiskal injected MB should be as low as possible to prevent or limit the damage to intervertebral disks.


Assuntos
Sobrevivência Celular/efeitos dos fármacos , Azul de Metileno/farmacologia , Núcleo Pulposo/efeitos dos fármacos , Agrecanas/metabolismo , Animais , Células Cultivadas , Colágeno Tipo II/metabolismo , Matriz Extracelular/metabolismo , Masculino , Metaloproteinase 3 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Núcleo Pulposo/citologia , Núcleo Pulposo/metabolismo , Ratos , Ratos Sprague-Dawley
6.
Mol Med Rep ; 19(3): 2164-2172, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30747227

RESUMO

Nucleus pulposus cell (NPC) apoptosis serves an important role in intervertebral disc degeneration (IDD); however, the roles of long noncoding RNAs (lncRNAs) in this process remain unknown. The present study aimed to determine the effects of the lncRNA growth arrest­specific transcript 5 (GAS5) on the apoptosis of primary human NPCs derived from the intervertebral disc, and to investigate the underlying mechanisms. TargetScan was used to predict the lncRNAs targeted by microRNA­155 (miR­155). Then, NPCs were subjected to lentivirus­mediated transduction of miR­155 or GAS5. A human lncRNA and mRNA array was used to screen differentially expressed lncRNAs following miR­155 overexpression. GAS5 and miR­155 expression levels were determined by reverse transcription­quantitative polymerase chain reaction. After GAS5 overexpression, apoptosis was assessed by flow cytometry via Annexin V/propidium iodide staining. Western blotting was employed to determine the expression of apoptosis­associated proteins, including caspase­3 and B cell lymphoma 2 (Bcl­2). TargetScan indicated GAS5 had one binding site for miR­155. Following exogenous transfection of miR­155 mimics, GAS5 expression levels in NPCs were significantly decreased (P<0.05). Interestingly, miR­155 overexpression in NPCs resulted in 721 differentially expressed lncRNAs compared with the negative control group (P<0.05), including 492 and 229 upregulated and downregulated lncRNAs respectively. In addition, 18 transcripts of GAS5 exhibited a downregulated expression profile. GAS5 overexpression in NPCs resulted in enhanced caspase­3 decreased Bcl­2 expression levels; the apoptosis of NPCs was significantly increased (P<0.05). The results of the present study revealed that overexpression of lncRNA GAS5 may promotes NPC apoptosis via Bcl­2 downregulation and caspase­3 upregulation, which may be associated with miR­155. The results of the present study suggest that lncRNA GAS5­silenced NPCs, or lentivirus­mediated lncRNA GAS5 knockdown may be precise and effective therapeutic strategies in the treatment of IDD.


Assuntos
Apoptose , Caspase 3/genética , Disco Intervertebral/citologia , Núcleo Pulposo/citologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Longo não Codificante/genética , Células Cultivadas , Regulação para Baixo , Humanos , Disco Intervertebral/metabolismo , MicroRNAs/genética , Núcleo Pulposo/metabolismo , Regulação para Cima
7.
Virus Res ; 259: 10-17, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30339788

RESUMO

Intervertebral disc (IVD) degeneration has a complex multifactorial origin and it is tightly associated with changes in the secretion of proteoglycans and collagen of the Nucleus Pulposus (NP) extracellular matrix. Chronic infection by Herpes virus has been previously associated with disc degeneration after detection of Herpes Simplex Virus type-1 (HSV-1) and CMV DNA in human excised disc samples. The aim of the present study was to assess the effect of HSV-1 infection on proteoglycan synthesis employing human Nucleus Pulposus (HNPCs) cells as a model of intervertebral disc degeneration. During lytic HSV-1 infection, a significant reduction of Decorin expression was observed 8 h post infection (h.p.i) which furthered deteriorated at 24 h.p.i. Biglycan was also reduced but only 24 h.p.i. Collagen type II, although demonstrated a downward trend, it was not statistically significant, whereas both Versican and Aggrecan showed a substantial decrease at 24 h.p.i. Hyaluronan production was not significantly affected. In a non-productive HSV-1 infection, a substantial reduction of Decorin, Biglycan, Versican and Aggrecan expression was found, similarly to our findings from the lytic infection. Furthermore, collagen type II expression was completely abolished. HAS1 expression was not affected, whereas HAS 2 and 3 were found to be significantly reduced. These results indicate that HSV-1 infection of human NP cells yields a complex effect on host extracellular cell function. The viral-induced changes in proteoglycan and collagen type II concentration may affect cell-matrix interactions and lead to a dysfunctional intervertebral disc which may trigger or promote the degeneration process.


Assuntos
Matriz Extracelular/metabolismo , Herpes Simples/genética , Herpes Simples/virologia , Herpesvirus Humano 1 , Núcleo Pulposo/citologia , Núcleo Pulposo/metabolismo , Animais , Biomarcadores , Linhagem Celular , Expressão Gênica , Herpes Simples/metabolismo , Humanos , Disco Intervertebral/citologia , Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/diagnóstico por imagem , Degeneração do Disco Intervertebral/etiologia , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/patologia , Imagem por Ressonância Magnética/métodos , Microscopia de Fluorescência , Proteoglicanas/metabolismo
8.
Medicine (Baltimore) ; 97(44): e12977, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30383649

RESUMO

This study aims to explore the optimized digestive method of collagenase to nucleus pulposus (NP) cells by observing the digestive effects of type I and II collagenase in different concentrations to NP in degenerated intervetebral discs.NP were collected from 18 human herniated intervertebral disc samples, and digested by type I and II collagenase, which were separated or combined in different concentrations. NP cells were counted using an inverted microscope, and the activities were determined by trypan blue staining at 4, 8, 16, and 24 hours after digestion. The growth of NP cells was also observed.The amount of NP cells with combined collagenases was greater than that separated in an identical concentration. With the combined collagenases at 4 and 8 hours, the higher concentration, the greater the amount of NP cells became. The amount of cells in extremely low concentrations of collagenase increased after 16 and 24 hours, and its activities remained at a higher level.The optimized digestion of extremely low concentrations of type I and II collagenase combined could save enzymes, was less harmful to NP cells, and was more adapted to separated and cultured NP cells.


Assuntos
Separação Celular/métodos , Colagenases/farmacologia , Degeneração do Disco Intervertebral/patologia , Núcleo Pulposo/citologia , Adulto , Contagem de Células/métodos , Técnicas de Cultura de Células/métodos , Sobrevivência Celular , Células Cultivadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
9.
Cell Physiol Biochem ; 50(5): 1687-1697, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30384362

RESUMO

BACKGROUND/AIMS: Previous studies have shown that oxidative damage is a main contributor to disc nucleus pulposus (NP) cell apoptosis. Aquaporin-3 (AQP-3) facilitates reactive oxygen species (ROS) scavenging and thus alleviates oxidative injury in other cells. This study aims to investigate the role and mechanism of AQP-3 in regulating NP cell apoptosis under oxidative damage. METHODS: Rat NP cells were treated with H2O2 for 48 hours, while control NP cells were free of H2O2. Recombinant AQP-3 lentiviral vectors were used to investigate the effect of enhanced AQP-3 expression levels in NP cells. NP cell apoptosis was assessed by flow cytometry, caspase-3 activity, gene expression of apoptosis-related molecules (Bax, Bcl-2 and caspase-3), and protein expression of cellular apoptosis markers (cleaved PARP and cleaved caspase-3). Additionally, intracellular ROS content and activity of the p38 MAPK pathway were evaluated. RESULTS: Compared with the control NP cells, oxidative damage in the treatment cells significantly increased cell apoptosis ratios and caspase-3 activity, upregulated gene expression of Bax and caspase-3, downregulated gene expression of Bcl-2, and increased protein expression of cleaved PARP and cleaved caspase-3, as well as increased intracellular ROS content and activity of the p38 MAPK pathway. However, AQP-3 overexpression partly alleviated cell apoptosis, decreased intracellular ROS content, and inhibited the p38 MAPK pathway in NP cells under oxidative damage. CONCLUSION: Oxidative damage can significantly downregulate AQP-3 expression. Enhancing AQP-3 expression in NP cells partly attenuates cellular apoptosis through regulating the p38 MAPK pathway under oxidative damage.


Assuntos
Apoptose , Aquaporina 3/metabolismo , Estresse Oxidativo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Apoptose/efeitos dos fármacos , Aquaporina 3/genética , Caspase 3/metabolismo , Células Cultivadas , Peróxido de Hidrogênio/farmacologia , Núcleo Pulposo/citologia , Núcleo Pulposo/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Proteína X Associada a bcl-2/metabolismo
10.
Cell Physiol Biochem ; 50(4): 1510-1521, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30359987

RESUMO

BACKGROUND/AIMS: Periodic mechanical stress has been shown to promote extracellular matrix (ECM) synthesis and cell migration of nucleus pulposus (NP) cells, however, the mechanisms need to be fully elucidated. The present study aimed to investigate the signal transduction pathway in the regulation of NP cells under periodic mechanical stress. METHODS: Primary rat NP cells were isolated and seeded on glass slides, and then treated in our self-developed periodic stress field culture system. To further explore the mechanisms, data were analyzed by scratch-healing assay, quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis, western blotting, and co-immunoprecipitation assay. RESULTS: Under periodic mechanical stress, the mRNA expression of ECM collagen 2A1 (Col2A1) and aggrecan, and migration of NP cells were significantly increased (P < 0.05 for each), associating with increases in the phosphorylation of Src, GIT1, and ERK1/2 (P < 0.05 for each). Pretreatment with the Src inhibitor PP2 reduced periodic mechanical stress-induced ECM synthesis and cell migration of NP cells (P < 0.05 for each), while the phosphorylation of GIT1 and ERK1/2 were inhibited. ECM synthesis, cell migration, and phosphorylation of ERK1/2 were inhibited after pretreatment with the small interfering RNA for GIT1 in NP cells under periodic mechanical stress (P < 0.05 for each), whereas the phosphorylation of Src was not affected. Pretreatment with the ERK1/2 inhibitor PD98059 reduced periodic mechanical stress-induced ECM synthesis and cell migration of NP cells (P < 0.05 for each). Co-immunoprecipitation assay showed that there was a direct interaction between Src and GIT1 and between GIT1 and ERK1/2. CONCLUSION: In conclusion, periodic mechanical stress induced ECM expression and migration of NP cells via Src-GIT1-ERK1/2 signaling pathway, playing an important role in regulation of NP cells.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfoproteínas/metabolismo , Estresse Mecânico , Quinases da Família src/metabolismo , Agrecanas/metabolismo , Animais , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Colágeno Tipo II/metabolismo , Matriz Extracelular/metabolismo , Flavonoides/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Núcleo Pulposo/citologia , Núcleo Pulposo/metabolismo , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Fosforilação/efeitos dos fármacos , Pirimidinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Quinases da Família src/antagonistas & inibidores
11.
Cell Physiol Biochem ; 49(6): 2463-2482, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30261504

RESUMO

BACKGROUND/AIMS: Intervertebral discs consist of an extracellular matrix (ECM) with a central gelatinous nucleus pulposus (NP) enclosed in an outer layer known as the annulus fibrosus. ECM metabolic disorders result in loss of boundary between the annulus fibrosus and NP, which can lead to intervertebral disc degeneration (IDD). Proinflammatory cytokines, such as interleukin (IL)-1ß, mediate the progression of IDD. Nicotinamide phosphoribosyltransferase (Nampt) catalyzes the first step in the biosynthesis of nicotinamide adenine dinucleotide (NAD) and is known to be induced by IL-1ß. APO866 is an inhibitor of NAD biosynthesis and is involved in autophagy. LC3 (microtubule-associated protein 1 light chain 3) is a key regulator of autophagy and is used as an indicator of increased autophagy. Herein, we investigate the role of APO866 in regulating autophagy in NP cells and IL-1ß mediated NP cell degeneration and apoptosis. METHODS: NP cells were extracted from IDD tissues and cultured in DMEM/F12 medium. Nampt was induced by different concentrations of IL-1ß (0, 0.5, 1, 5, 10 ng/mL) for 24 h or NP cells were treated with 10 ng/mL IL-1ß for 0, 6, 12, 48 h. QRT-PCR and western blots were used to detect Nampt and ECM-related protein expression in NP tissue of patients with IDD and in NP cells. Confocal analysis was used to detect membrane-bound LC3, Aggrecan, and Collagen II. RESULTS: Nampt is expressed in NP tissue at higher levels in severe grades of IDD (Grade IV and V) compared with low grades (Grade II and III). In NP cells, 10 ng/mL IL-1ß induced Nampt expression for 48 h, increased expression of the degradative-associated proteins, ADAMTS4/5 and MMP-3/13, and decreased expression of ECM-related proteins, Aggrecan and Collagen II. However, the Nampt inhibitor APO866 blocked IL-1ß induction, and the knockdown of Nampt expression increased the expression of ECM proteins that were inhibited by IL-1ß. Moreover, evidence provided by the autophagic markers LC3 and Beclin-1 indicated that APO866 induced NP cell autophagy. Furthermore, although APO866 inhibited the downregulated expression of ECM-related proteins by IL-1ß, this function was blocked by autophagy inhibitor, 3-methyladenine. CONCLUSION: APO866 protects NP cells and induces autophagy by inhibiting IL-1ß-induced NP cell degeneration and apoptosis, which may have therapeutic potential in IDD.


Assuntos
Acrilamidas/farmacologia , Autofagia/efeitos dos fármacos , Interleucina-1beta/farmacologia , Degeneração do Disco Intervertebral/patologia , Nicotinamida Fosforribosiltransferase/metabolismo , Piperidinas/farmacologia , Proteína ADAMTS4/metabolismo , Agrecanas/metabolismo , Células Cultivadas , Colágeno Tipo II/metabolismo , Citocinas/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Feminino , Humanos , Degeneração do Disco Intervertebral/metabolismo , Masculino , Metaloproteinase 3 da Matriz/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Pessoa de Meia-Idade , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Nicotinamida Fosforribosiltransferase/genética , Núcleo Pulposo/citologia , Núcleo Pulposo/efeitos dos fármacos , Núcleo Pulposo/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo
12.
Oxid Med Cell Longev ; 2018: 3459612, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30050653

RESUMO

Nucleus pulposus-derived mesenchymal stem cells (NPMSCs) have shown a good prospect in the regeneration of intervertebral disc (IVD) tissues. However, fresh NPMSCs are not always readily available for basic research and clinical applications. Therefore, there is a need for an effective long-term cryopreservation method for NPMSCs. The aim of this study was to determine whether adding icariin (ICA) to the conventional cryoprotectant containing dimethyl sulfoxide (DMSO) had a better cryoprotective effect for NPMSCs. The results showed that the freezing solution containing ICA along with DMSO significantly increased the postthawed cell viability, decreased the apoptosis rate, improved cell adherence, and maintained the mitochondrial functions, as compared to the freezing solution containing DMSO alone. And the inhibition of oxidative stress and upregulation of heat shock proteins (HSPs) in the presence of ICA also confirmed the beneficial effect of ICA. Furthermore, ICA had no cytotoxicity and did not alter the characteristics of postthawed NPMSCs. In conclusion, these results suggested that the addition of ICA to the conventional freezing medium could improve the viability and function of the cryopreserved human NPMSCs and provided an optimal formulated freezing solution for human NPMSC cryopreservation.


Assuntos
Criopreservação/métodos , Flavonoides/farmacologia , Núcleo Pulposo/citologia , Actinas/metabolismo , Apoptose/efeitos dos fármacos , Western Blotting , Adesão Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Crioprotetores , Humanos , Técnicas In Vitro , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
13.
Int J Mol Sci ; 19(6)2018 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-29899321

RESUMO

A discrimination of the highly specialised annulus fibrosus (AF) and nucleus pulposus (NP) cells in the mature human intervertebral disc (IVD) is thus far still not possible in a reliable way. The aim of this study was to identify molecular markers that distinguish AF and NP cells in human disc tissue using microarray analysis as a screening tool. AF and NP samples were obtained from 28 cervical discs. First, all samples underwent quality sorting using two novel scoring systems for small-sized disc tissue samples including macroscopic, haptic and histological evaluation. Subsequently, samples with clear disc characteristics of either AF or NP that were free from impurities of foreign tissue (IVD score) and with low signs of disc degeneration on cellular level (DD score) were selected for GeneChip analysis (HGU1332P). The 11 AF and 9 NP samples showed distinctly different genome-wide transcriptomes. The majority of differentially expressed genes (DEGs) could be specifically assigned to the AF, whereas no DEG was exclusively expressed in the NP. Nevertheless, we identified 11 novel marker genes that clearly distinguished AF and NP, as confirmed by quantitative gene expression analysis. The novel established scoring systems and molecular markers showed the identity of AF and NP in disc starting material and are thus of great importance in the quality assurance of cell-based therapeutics in regenerative treatment of disc degeneration.


Assuntos
Anel Fibroso/metabolismo , Núcleo Pulposo/metabolismo , Transcriptoma , Adulto , Idoso , Anel Fibroso/citologia , Anel Fibroso/patologia , Biomarcadores/metabolismo , Biópsia/normas , Feminino , Perfilação da Expressão Gênica/normas , Humanos , Masculino , Pessoa de Meia-Idade , Núcleo Pulposo/citologia , Núcleo Pulposo/patologia
14.
Mol Med Rep ; 18(3): 2681-2688, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29956812

RESUMO

Osteoporosis is closely associated with intervertebral disc degeneration. While parathyroid hormone (PTH) 1­34, which is an established drug used to treatosteoporosis, is thought to inhibit the disc degeneration associated with osteoporosis, the precise mechanism involved remains unclear. In the present study, primary Sprague­Dawley rat nucleus pulposus cells (NPCs) were cultured, phenotyped and then treated with dexamethasone (DXM) for 48 h. Cell area analysis and ß­galactosidase staining were used to investigate the effect of DXM on the senescence of NPCs. In addition, the protein levels of LC3­II, Beclin­1, P62, p­mTOR and p­p70S6k were determined by western blotting and analyzing the regulatory effect of PTH upon autophagy and the mTOR signaling pathway in cells treated with DXM. Following autophagic inhibition induced by ATG5 siRNA transfection, the regulatory effect of PTH on senescence in NPCs were investigated in addition to the potential role of autophagy. As the concentration of DXM increased, the size of the NPCs was significantly enlarged and the proportion of cells with positive ß­galactosidase staining increased significantly (P<0.05). In terms of protein expression, PTH treatment led to an increase in LC3­II and Beclin­1 proteins, a reduction in P62 protein, and inhibited p­mTOR and p­p70S6k protein expression in DXM­treated NPCs (P<0.05). PTH attenuated the effect of DXM according to the cell size and percentage of ß­galactosidase­positive cells. However, the inhibition of autophagy via ATG5 siRNA transfection reversed the protective effect of PTH on cell senescence (P<0.05). Collectively, the present findings suggest that PTH may inhibit the senescence of NPCs induced by DXM by activating autophagy via the mTOR pathway.


Assuntos
Autofagia/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Hormônio Paratireóideo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Autofagossomos/metabolismo , Proteína 5 Relacionada à Autofagia/antagonistas & inibidores , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/metabolismo , Células Cultivadas , Dexametasona/farmacologia , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Núcleo Pulposo/citologia , Núcleo Pulposo/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais/efeitos dos fármacos
15.
Cell Physiol Biochem ; 47(1): 257-265, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29768261

RESUMO

BACKGROUND/AIMS: Diabetes mellitus (DM) is a potential etiology of disc degeneration. N-cadherin (N-CDH) helps maintain the cell viability, cell phenotype and matrix biosynthesis of nucleus pulposus (NP) cells. Here, we mainly aimed to investigate whether N-CDH can attenuate high glucose-induced NP cell senescence and its potential mechanism. METHODS: Rat NP cells were cultured in a base culture medium and base culture medium with a 0.2 M glucose concentration. Recombinant lentiviral vectors were used to enhance N-CDH expression in NP cells. Senescence-associated ß-galactosidase (SA-ß-Gal) activity was measured by SA-ß-Gal staining. NP cell proliferation was evaluated by CCK-8 assay. Telomerase activity and intracellular reactive oxygen species (ROS) content were tested by specific chemical kits according to the manufacturer's instructions. G0/G1 cell cycle arrest was evaluated by flow cytometry. Real-time PCR and Western blotting were used to analyze mRNA and protein expressions of senescence markers (p16 and p53) and matrix macromolecules (aggrecan and collagen II). Additionally, p-NF-κB expression was also analyzed by Western blotting to evaluate NF-κB pathway activity. RESULTS: High glucose significantly decreased N-CDH expression, increased ROS generation and NF-κB pathway activity, and promoted NP cell senescence, which was reflected in the increase in SA-ß-Gal activity and senescence marker (p16 and p53) expression, compared to the control group. High glucose decreased telomerase activity and cell proliferation potency. However, N-CDH overexpression partially attenuated NP cell senescence, decreased ROS content and inhibited the activation of the NF-κB pathway under the high glucose condition. CONCLUSION: High glucose decreases N-CDH expression and promotes NP cell senescence. N-CDH overexpression can attenuate high glucose-induced NP cell senescence through the regulation of the ROS/ NF-κB pathway. This study suggests that N-CDH is a potential therapeutic target to slow DM-mediated disc NP degeneration.


Assuntos
Caderinas/metabolismo , Senescência Celular , NF-kappa B/metabolismo , Núcleo Pulposo/citologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Animais , Células Cultivadas , Diabetes Mellitus/metabolismo , Núcleo Pulposo/metabolismo , Ratos
16.
Life Sci ; 205: 26-37, 2018 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-29746847

RESUMO

AIMS: This study aims to investigate the protective effects and potential mechanisms of cyclosporine A (CsA), which efficiently inhibits mitochondrial permeability transition pore (MPTP) opening, on compression-induced apoptosis of human nucleus pulposus mesenchymal stem cells (NP-MSCs). MATERIALS AND METHODS: Human NP-MSCs were subjected to various periods of 1.0 MPa compression. Cell viability was evaluated using cell counting kit-8 (CCK-8) assay. The cellular ultrastructure and ATP level were analyzed via transmission electron microscopy (TEM) and ATP detection kit respectively. The apoptosis ratio was determined using Annexin V/PI dual staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays. The levels of apoptosis-associated molecules (cleaved caspase-3, Bax and Bcl-2) were analyzed by western blot and qRT-PCR. Additionally, MPTP opening, mitochondrial membrane potential (MMP) and the levels of oxidative stress-related indicators (ROS), superoxide dismutase (SOD) and malondialdehyde (MDA) were monitored. KEY FINDINGS: Annexin V/PI dual staining and detection of apoptosis-associated molecules demonstrated that compression significantly up-regulated apoptosis level of NP-MSCs in a time-dependent manner. CsA greatly down-regulated compression-mediated NP-MSC apoptosis and the cell death ratio. Compression also notably exacerbated mitochondrial dysfunction, ATP depletion and oxidative stress in NP-MSCs, all of which were rescued by CsA. SIGNIFICANCE: Our results demonstrated that CsA efficiently inhibited compression-induced NP-MSCs apoptosis by alleviating mitochondrial dysfunction and oxidative stress. These findings provide new insights into intervertebral disc (IVD) degeneration (IVDD), and suggest CsA treatment as a potential strategy for delaying or even preventing IVDD.


Assuntos
Apoptose/efeitos dos fármacos , Ciclosporina/farmacologia , Imunossupressores/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Doenças Mitocondriais/tratamento farmacológico , Núcleo Pulposo/citologia , Estresse Oxidativo/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Humanos , Degeneração do Disco Intervertebral/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Células-Tronco Mesenquimais/ultraestrutura , Núcleo Pulposo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
17.
Biomed Pharmacother ; 99: 725-734, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29710470

RESUMO

Intervertebral disc degeneration (IDD) is the most common cause leading to low back pain, a highly prevalent, costly and crippling condition worldwide. Overexpression of miR-21 has been shown to promote proliferation of nucleus pulposus (NP) cells. However, it remains unclear whether miR-21 can promote the degradation of type II collagen (Col II) and aggrecan, two main extracellular matrix components within the disc. Here, the miRNA microassay assay identified 29 differentially expressed miRNAs in NP tissues from IDD patients compared with healthy controls. Following qRT-PCR validation, miR-21 expression was significantly upregulated in degenerated NP tissues, and showed a positive correlation with disc degeneration grade. Through gain-of-function and loss-of-function studies in human NP cells, miR-21 was shown to inhibit autophagy and then upregulate the expression of matrix metalloproteinase (MMP)-3 and MMP-9, leading to increased degradation of Col II and aggrecan. Mechanistically, phosphatase and tensin homolog (PTEN) was identified as a direct target of miR-21, and activated PTEN/ Akt/mammalian target of rapamycin (mTOR) signaling pathway was involved in miR-21-induced autophagy inhibition and Col II and aggrecan breakdown. Taken together, these results suggest that miR-21 contributes to Col II and aggrecan catabolism by inhibiting autophagy via the PTEN/Akt/mTOR signaling pathway in human NP cells.


Assuntos
Matriz Extracelular/genética , Degeneração do Disco Intervertebral/genética , MicroRNAs/genética , Núcleo Pulposo/patologia , Adolescente , Idoso , Agrecanas/metabolismo , Autofagia/genética , Estudos de Casos e Controles , Colágeno Tipo II/metabolismo , Feminino , Humanos , Degeneração do Disco Intervertebral/patologia , Masculino , Pessoa de Meia-Idade , Núcleo Pulposo/citologia , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/metabolismo , Adulto Jovem
18.
Cell Physiol Biochem ; 46(2): 644-653, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29617687

RESUMO

BACKGROUND/AIMS: Nucleus pulposus cell (NPC) apoptosis is the main factor in intervertebral disc degeneration (IDD); thus, inhibiting the excessive apoptosis of nucleus pulposus cells may be a potential way to alleviate IDD. The effect of Hemeoxygenase-1 (HO-1) on human NPC apoptosis has never been reported. Our study aimed to investigate the effect and mechanism of HO-1 on apoptosis in human degenerative NPCs. METHODS: Nucleus pulposus tissues were collected from patients with lumbar vertebral fracture (LVF) and IDD. The expression of HO-1 and P65 in intervertebral discs was determined using immunohistochemistry and western blot analysis. Apoptosis of human nucleus pulposus cells was quantified by flow cytometric analysis. A recombinant lentiviral vector overexpressing HO-1 and HO-1-siRNA was used to promote or silence the expression of HO-1 in nucleus pulposus cells. The NF-κB inhibitor PDTC was used to inhibit the NF-κB pathway. RESULTS: Our study demonstrated that compared with normal samples, IDD samples showed down-regulation of HO-1 expression and up-regulation of P65 expression. Overexpression of HO-1 inhibited the increase in nucleus pulposus cell apoptosis after IL-1ß treatment and simultaneously inhibited the expression of p-P65. Furthermore, after treatment with PDTC, the number of apoptotic cells was significantly decreased with or without overexpression of HO-1. CONCLUSION: HO-1 might play a significant role in IDD, and HO-1 protected degenerative human NPCs against apoptosis induced by IL-1ß through the NF-κB pathway. These findings would aid in the development of novel therapeutic approaches for IDD treatment.


Assuntos
Apoptose/efeitos dos fármacos , Heme Oxigenase-1/metabolismo , Interleucina-1beta/farmacologia , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adulto , Idoso , Feminino , Heme Oxigenase-1/antagonistas & inibidores , Heme Oxigenase-1/genética , Humanos , Degeneração do Disco Intervertebral/diagnóstico por imagem , Degeneração do Disco Intervertebral/patologia , Imagem por Ressonância Magnética , Masculino , Núcleo Pulposo/citologia , Núcleo Pulposo/efeitos dos fármacos , Núcleo Pulposo/metabolismo , Fosforilação/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fraturas da Coluna Vertebral/diagnóstico por imagem , Fraturas da Coluna Vertebral/patologia
19.
Cell Physiol Biochem ; 46(2): 482-491, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29614512

RESUMO

BACKGROUND/AIMS: An adequate matrix production of nucleus pulposus (NP) cells is an important tissue engineering-based strategy to regenerate degenerative discs. Here, we mainly aimed to investigate the effects and mechanism of mechanical compression (i.e., static compression vs. dynamic compression) on the matrix synthesis of three-dimensional (3D) cultured NP cells in vitro. METHODS: Rat NP cells seeded on small intestinal submucosa (SIS) cryogel scaffolds were cultured in the chambers of a self-developed, mechanically active bioreactor for 10 days. Meanwhile, the NP cells were subjected to compression (static compression or dynamic compression at a 10% scaffold deformation) for 6 hours once per day. Unloaded NP cells were used as controls. The cellular phenotype and matrix biosynthesis of NP cells were investigated by real-time PCR and Western blotting assays. Lentivirus-mediated N-cadherin (N-CDH) knockdown and an inhibitor, LY294002, were used to further investigate the role of N-CDH and the PI3K/Akt pathway in this process. RESULTS: Dynamic compression better maintained the expression of cell-specific markers (keratin-19, FOXF1 and PAX1) and matrix macromolecules (aggrecan and collagen II), as well as N-CDH expression and the activity of the PI3K/Akt pathway, in the 3D-cultured NP cells compared with those expression levels and activity in the cells grown under static compression. Further analysis showed that the N-CDH knockdown significantly down-regulated the expression of NP cell-specific markers and matrix macromolecules and inhibited the activation of the PI3K/Akt pathway under dynamic compression. However, inhibition of the PI3K/Akt pathway had no effects on N-CDH expression but down-regulated the expression of NP cell-specific markers and matrix macromolecules under dynamic compression. CONCLUSION: Dynamic compression increases the matrix synthesis of 3D-cultured NP cells compared with that of the cells under static compression, and the N-CDH-PI3K/Akt pathway is involved in this regulatory process. This study provides a promising strategy to promote the matrix deposition of tissue-engineered NP tissue in vitro prior to clinical transplantation.


Assuntos
Caderinas/metabolismo , Força Compressiva/fisiologia , Matriz Extracelular/metabolismo , Animais , Caderinas/antagonistas & inibidores , Caderinas/genética , Células Cultivadas , Cromonas/farmacologia , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Géis/química , Regulação da Expressão Gênica , Queratina-19/genética , Masculino , Morfolinas/farmacologia , Núcleo Pulposo/citologia , Núcleo Pulposo/metabolismo , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Tecidos Suporte/química
20.
Int J Mol Sci ; 19(4)2018 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-29652862

RESUMO

Spinal fusion is a common surgical procedure to address a range of spinal pathologies, like damaged or degenerated discs. After the removal of the intervertebral disc (IVD), a structural spacer is positioned followed by internal fixation, and fusion of the degenerated segment by natural bone growth. Due to their osteoinductive properties, bone morphogenetic proteins (BMP) are applied to promote spinal fusion. Although spinal fusion is successful in most patients, the rates of non-unions after lumbar spine fusion range from 5% to 35%. Clinical observations and recent studies indicate, that the incomplete removal of disc tissue might lead to failure of spinal fusion. Yet, it is still unknown if a secretion of BMP antagonists in intervertebral disc (IVD) cells could be the reason of inhibition in bone formation. In this study, we co-cultured human primary osteoblasts (OB) and IVD cells i.e., nucleus pulposus (NPC), annulus fibrosus (AFC) and cartilaginous endplate cells (CEPC), to test the possible inhibitory effect from IVD cells on OB. Although we could see a trend in lower matrix mineralization in OB co-cultured with IVD cells, results of alkaline phosphatase (ALP) activity and gene expression of major bone genes were inconclusive. However, in NPC, AFC and CEPC beads, an up-regulation of several BMP antagonist genes could be detected. Despite being able to show several indicators for an inhibition of osteoinductive effects due to IVD cells, the reasons for pseudarthrosis after spinal fusion remain unclear.


Assuntos
Fosfatase Alcalina/metabolismo , Técnicas de Cocultura/métodos , Disco Intervertebral/citologia , Osteoblastos/citologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anel Fibroso/citologia , Calcificação Fisiológica , Células Cultivadas , Feminino , Expressão Gênica , Humanos , Masculino , Metaloproteinases da Matriz Secretadas/metabolismo , Núcleo Pulposo/citologia , Osteoblastos/metabolismo
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