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1.
Clin Exp Rheumatol ; 37 Suppl 120(5): 40-47, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31621575

RESUMO

MicroRNAs are small double-stranded RNAs, which negatively regulate gene expression and have been shown to have key roles in both chondrocyte development and cartilage homeostasis with age. Deletion of all microRNAs in chondrocytes leads to skeletal growth defects in mice, whilst deletion of specific microRNAs, e.g. miR-140, leads to premature articular cartilage degradation and increased susceptibility to posttraumatic osteoarthritis. Studies comparing microRNA expression in normal human articular cartilage compared to osteoarthritic cartilage show differential expression, but varying sample groups make interpretation difficult. MicroRNAs have been proposed as circulating biomarkers of osteoarthritis, but again, this differs amongst patient cohorts. Many micro-RNAs have been shown to have roles in chondrocyte phenotype via signalling pathways, apoptosis, autophagy and senescence. Modulating microRNAs in the joint has been shown to reduce osteoarthritis in animal models and translating this to man as a novel therapeutic strategy will be key.


Assuntos
Autofagia , Cartilagem Articular , MicroRNAs , Osteoartrite , Animais , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Condrócitos/metabolismo , Condrócitos/patologia , Humanos , Masculino , Camundongos , MicroRNAs/genética , MicroRNAs/fisiologia , Osteoartrite/genética , Osteoartrite/metabolismo
2.
Toxicol Lett ; 314: 18-26, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31299270

RESUMO

Epidemiological investigations indicate that effects related to prenatal adverse environments on the organs of the offspring could continue to adulthood. This study intends to confirm that prenatal nicotine exposure (PNE) increases the susceptibility of osteoarthritis (OA) in the male offspring, and to explore the potential intrauterine programming mechanism. During pregnancy, rats were divided into a PNE group and a control group. After birth, rats were given a high-fat diet for 6 months and long-distance running for 6 weeks. The rats were euthanized at 18 months after birth (PM18) and on gestational day 20 (GD20), respectively. Knee joints were collected for histochemistry, immunohistochemistry, and quantitative polymerase chain reaction (qPCR) assays. Histological analyses and the Mankin's score showed increased cartilage destruction and accelerated OA progression in adult offspring from the PNE group. Immunohistochemistry results showed decreased expression of transforming growth factor beta (TGFß) signaling pathway. Furthermore, the expression of apoptosis factors (caspase-3 and caspase-8), inflammatory factors [interleukin (IL)-1, IL-6] and matrix degradation enzymes [matrix metalloproteinase (MMP)-3, MMP-13] were also significantly increased. Traced back to the intrauterine period, it was found that the number of chondrocytes and the contents of Col2A1 and aggrecan in the matrix in the PNE group were decreased. And, the expression of the TGFß signaling pathway was inhibited. These results suggested that PNE enhanced the susceptibility of OA in male elderly offspring rats by down-regulating TGFß signaling, which increased articular cartilage local inflammation, matrix degradation, and cell apoptosis. This study confirmed the developmental origin of OA, and clarified the congenital and the living environment impact on the occurrence and development of OA. Our findings provide a theoretical and experimental basis for OA early prevention.


Assuntos
Articulações/efeitos dos fármacos , Nicotina/toxicidade , Agonistas Nicotínicos/toxicidade , Osteoartrite/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Fatores Etários , Agrecanas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 8/metabolismo , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Condrócitos/patologia , Condrogênese/efeitos dos fármacos , Colágeno Tipo II/metabolismo , Feminino , Idade Gestacional , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Articulações/metabolismo , Articulações/patologia , Masculino , Exposição Materna , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Osteoartrite/metabolismo , Osteoartrite/patologia , Gravidez , Ratos Wistar , Fatores de Risco , Fatores Sexuais
3.
Cell Physiol Biochem ; 53(1): 172-185, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31264811

RESUMO

BACKGROUND/AIMS: MicroRNAs (miRs) are transcribed as stem-loop precursors harboring two different miRs on either side of the structure. Both miRs can modulate levels of cellular transcripts based on sequence complementarity between the miR and the mRNA target. The miR of the current study, miR-675, is encoded in the H19 gene with high expression in fetal/placental tissues but low levels in most adult tissues except for skeletal muscle and articular cartilage. miR-675 has a supportive role in expression of the major collagen component of articular cartilage (COL2A1) but it is unknown which arm contributes to this effect. Objectives: To determine the active arm of miR-675 in human articular chondrocytes. To evaluate effects of overexpression of both arms of miR-675 on MMP1 and MMP13, two enzymes involved in breakdown of COL2A1. To investigate whether abundance of both arms of miR-675 is dynamic. METHODS: miR-arm activity was determined by association with the AGO2 complex using immunoprecipitation with an AGO2 specific antibody. miR overexpression and inhibition was used to identify indirect downstream effects on two targets of the Matrix-Metalloprotease family, MMP1 and MMP13. Data was evaluated by qPCR and enzymatic activity assays. Early passage human articular chondrocytes (up to passage 2) obtained from cartilage from both healthy and osteoarthritis affected tissue were used. To evaluate miR-675 levels in a different model, myotube differentiation was employed. RESULTS: We show that both arms of miR-675 have opposing effects on MMP1 and MMP13; however only one arm, miR-675-3' is active in human articular chondrocytes. We demonstrate that during myotube differentiation, high expression of both arms of miR-675 is observed as well as an increase in expression of MMP1. CONCLUSION: We show that both arms of miR-675 result in opposing effects on two downstream molecules MMP1 and MMP13. We propose that miR abundance may arise as response to direct target transcript levels and are thus dynamic to meet the requirements of the cellular environment.


Assuntos
Condrócitos/metabolismo , Regulação da Expressão Gênica , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 1 da Matriz/genética , MicroRNAs/genética , Osteoartrite/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Cartilagem Articular/citologia , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Células Cultivadas , Criança , Condrócitos/citologia , Condrócitos/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite/patologia , Regulação para Cima , Adulto Jovem
4.
Curr Pharm Biotechnol ; 20(6): 517-524, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31057106

RESUMO

BACKGROUND: The study aimed to investigate the effects of the active ingredient, nimodipine, on chondrocyte proliferation and extracellular matrix (ECM) structures in cartilage tissue cells. METHODS: Chondrocyte cultures were prepared from tissues resected via surgical operations. Nimodipine was then applied to these cultures and molecular analysis was performed. The data obtained were statistically calculated. RESULTS: Both, the results of the (3-(4,5 dimethylthiazol2-yl)-2,5-diphenyltetrazolium (MTT) assay and the fluorescence microscope analysis [a membrane permeability test carried out with acridine orange/ propidium iodide staining (AO/PI)] confirmed that the active ingredient, nimodipine, negatively affects the cell cultures. CONCLUSION: Nimodipine was reported to suppress cellular proliferation; chondroadherin (CHAD) and hypoxia-inducible factor-1 alpha (HIF-1α) expression thus decreased by 2.4 and 1.7 times, respectively, at 24 hrs when compared to the control group (p < 0.05). Furthermore, type II collagen (COL2A1) expression was not detected (p < 0.05). The risk that a drug prescribed by a clinician in an innocuous manner to treat a patient by relieving the symptoms of a disease may affect the proliferation, differentiation, and viability of other cells and/or tissues at the molecular level, beyond its known side effects or adverse events, should not be forgotten.


Assuntos
Bloqueadores dos Canais de Cálcio/toxicidade , Proliferação de Células/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Nimodipina/toxicidade , Cartilagem/efeitos dos fármacos , Cartilagem/patologia , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Condrócitos/metabolismo , Condrócitos/patologia , Colágeno Tipo II/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/patologia , Proteínas da Matriz Extracelular/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Pessoa de Meia-Idade , Cultura Primária de Células
5.
Artif Cells Nanomed Biotechnol ; 47(1): 1766-1771, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31066587

RESUMO

BACKGROUND: Understanding the mechanism of chondrocytes degeneration could provide a new potential therapeutic idea for rheumatoid arthritis (RA) treatment. MicroRNA-27b-3p (miR-27b-3p) has been shown to regulate a variety of cell behaviors in various cell types. However, the role of miR-27b-3p in RA remains unknown. MATERIALS AND METHODS: Expression of miR-27b-3p and HIPK2 in cartilage tissues and chondrocytes was characterized using qRT-PCR and Western blot. MiR-27b-3p was overexpressed or suppressed in chondrocytes to observe the potential role of miR-27b-3p. RESULTS: We found declined miR-27b-3p and elevated HIPK2 in RA tissues and cells using qRT-PCR. Dual-luciferase reporter assay validated HIPK2 is a direct target of miR-27b-3p, confirmed by Western blot results. Pearson correlation presented that there was a significantly negative correlation between miR-27b-3p and HIPK2 mRNA. Overexpression of miR-27b-3p significantly reduced the expression of pro-apoptotic protein c-caspase3 and increased the expression of anti-apoptotic Bcl-2; however, downregulation of miR-27b-3p has a significant effect of inducing apoptosis. Furthermore, overexpression of miR-27b-3p combined with recombinant HIPK2 protein showed the inhibitory effect of miR-27b-3p was abolished by HIPK2. CONCLUSION: We found declined miR-27b-3p and elevated HIPK2 in RA tissues and cells. Further in vitro studies demonstrated that miR-27b might inhibit chondrocyte apoptosis and thus attenuate RA development by directly inhibiting HIPK2 expression.


Assuntos
Apoptose/genética , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Proteínas de Transporte/genética , Condrócitos/patologia , MicroRNAs/genética , Proteínas Serina-Treonina Quinases/genética , Animais , Sequência de Bases , Proliferação de Células/genética , Progressão da Doença , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL
6.
Life Sci ; 228: 158-166, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31055086

RESUMO

AIMS: Chondrocyte apoptosis is the most common pathological feature of cartilage in osteoarthritis (OA). Excessive mechanical stress can induce chondrocyte apoptosis and destroy cartilage tissue. Transient receptor potential channel vanilloid 4 (TRPV4) is a mechanosensitive ion channel that mediates chondrocyte response to mechanical stress. Here, we investigated the potential role of TRPV4 in chondrocyte apoptosis induced by excessive mechanical stress. MAIN METHODS: Using a rat OA anterior cruciate-ligament transection (ALCT) model, we detected immunolocalization of calmodulin protein and mRNA and protein levels of TRPV4, calmodulin, and cleaved caspase-8 in articular cartilage. Primary chondrocytes were isolated and cultured in vitro, and Fluo-4AM staining was used to assess intracellular Ca2+ levels in order to evaluate TRPV4-mediated Ca2+ influx. Flow cytometry and western blot were performed to detect apoptosis and apoptosis-related protein levels in chondrocytes, respectively. KEY FINDINGS: TRPV4 was upregulated in ALCT-induced OA articular cartilage, and we found that administration of a TRPV4 inhibitor attenuated cartilage degeneration. Additionally, TRPV4 specifically mediated extracellular Ca2+ influx, leading to chondrocyte apoptosis in vitro, which was inhibited by transfection of TRPV4 small-interfering RNA or administration of a TRPV4 inhibitor. Moreover, increased Ca2+ influx triggered apoptosis by upregulating FAS-associated protein with death domain and cleaved caspase-3, -6, -7, and -8 levels, with these effects abolished by TRPV4 knockdown or TRPV4 inhibition. SIGNIFICANCE: These results indicated that TRPV4 was upregulated in OA articular cartilage, and that excessive mechanical stress might induce chondrocyte apoptosis via TRPV4-mediated Ca2+ influx, suggesting TRPV4 as a potential drug target in OA.


Assuntos
Ligamento Cruzado Anterior/patologia , Apoptose , Condrócitos/patologia , Osteoartrite/patologia , Estresse Mecânico , Canais de Cátion TRPV/metabolismo , Animais , Ligamento Cruzado Anterior/metabolismo , Células Cultivadas , Condrócitos/metabolismo , Modelos Animais de Doenças , Masculino , Osteoartrite/genética , Osteoartrite/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Canais de Cátion TRPV/análise , Canais de Cátion TRPV/genética , Regulação para Cima
7.
Life Sci ; 228: 274-284, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31077718

RESUMO

AIMS: Previous studies have demonstrated that transcriptional silencing of miRNAs due to DNA hypermethylation is associated with different pathologies. It has also been reported that abnormal expression of miR-140-5p and miR-146a is linked to osteoarthritis (OA) progression. In this study, we investigated the role of DNA methylation on miR-140-5p and miR-146a expression in OA. MAIN METHODS: miR-140-5p and miR-146a expression was investigated by qRT-PCR. The methylation status of miR-140 and miR-146a regulatory regions was analyzed using qMSP and bisulfite sequencing analysis. SMAD-3 and NF-kB binding to miR-140 and miR-146a regulatory regions was assessed by ChIP assay and knockdown experiments. OA-related genes' expression was evaluated in 5-AzadC, miRNAs inhibitor and 5-AzadC/miRNAs inhibitor-treated cells. KEY FINDINGS: Hypermethylation of specific CpG sites in miR-140 and miR-146a regulatory regions was associated with downregulation of miR-140-5p and miR-146a in OA chondrocytes and synoviocytes, respectively. 5-AzadC-induced miR-140-5p and miR-146a upregulation was observed in OA chondrocytes and synoviocytes. Moreover, we found decreased binding affinity of SMAD-3 and NF-kB transcription factors on the hypermethylated miR-140-5p and miR-146a regulatory regions, respectively. Downregulation of MMP-13 and ADAMTS-5 in 5-AzadC-treated OA chondrocytes was prevented by miR-140-5p inhibitor transfection. Similarly, 5-AzadC-treated OA synoviocytes showed decreased expression of IRAK-1, IL1Β and IL-6, which was reversed following 5-AzadC-/miR-146a inhibitor treatment. SIGNIFICANCE: Our results strongly suggest the impact of DNA methylation on miR-140-5p and miR-146a suppression in OA chondrocytes and synoviocytes, contributing to OA pathogenesis.


Assuntos
Metilação de DNA , Regulação da Expressão Gênica , MicroRNAs/genética , Osteoartrite/genética , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Condrócitos/metabolismo , Condrócitos/patologia , Ilhas de CpG , Humanos , Pessoa de Meia-Idade , Osteoartrite/patologia , Sinoviócitos/metabolismo , Sinoviócitos/patologia
8.
Artif Cells Nanomed Biotechnol ; 47(1): 1043-1049, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30942091

RESUMO

Osteoarthritis (OA) is a major age-related disease, which may be caused by the accumulation of advanced glycation end-products (AGEs). Excessive degradation of type II collagen and aggrecan by matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin type 1 motif (ADAMTS) induced by AGEs is a pivotal event in the pathogenesis of osteoarthritis. In addition, activation of the nuclear factor-κB (NF-κB) pathway induces the expression of a cascade of proinflammatory cytokines, such as interleukin (IL)-1ß and tumor necrosis factor-α (TNF-α). In the present study, we investigated the effects of salicin, one of the main constituents of aspirin and a derivative of Alangium chinense, on AGE-induced degradation of the articular extracellular matrix in SW1353 human chondrocytes. Our findings reveal a novel beneficial role of salicin in rescuing degradation of type II collagen and aggrecan, reducing oxidative stress, attenuating expression of proinflammatory cytokines, and inhibiting activation of the NF-κB proinflammatory signaling pathway in chondrocytes stimulated with AGEs. Salicin may thus have potential as a safe and effective therapy against the development and progression of OA.


Assuntos
Agrecanas/metabolismo , Álcoois Benzílicos/farmacologia , Condrócitos/efeitos dos fármacos , Colágeno Tipo II/metabolismo , Glucosídeos/farmacologia , Produtos Finais de Glicação Avançada/farmacologia , Osteoartrite/tratamento farmacológico , Proteólise/efeitos dos fármacos , Álcoois Benzílicos/uso terapêutico , Quimiocinas/metabolismo , Condrócitos/metabolismo , Condrócitos/patologia , Glucosídeos/uso terapêutico , Humanos , NF-kappa B/metabolismo , Osteoartrite/metabolismo , Osteoartrite/patologia , Transdução de Sinais/efeitos dos fármacos
9.
Artif Cells Nanomed Biotechnol ; 47(1): 1241-1247, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30945573

RESUMO

Osteoarthritis (OA) is a common degenerative joint disease worldwide. Long non-coding RNAs (lncRNAs) have been widely confirmed to involve in the modulation of OA progression. However, the underlying mechanisms of lncRNA FOXD2-AS1 in OA remain unclear. In the present study, we showed FOXD2-AS1 expression was upregulated and positively associated with the severity of OA patients. IL-1ß and/or TNF-α treatment could increase FOXD2-AS1 expression in chondrocytes. FOXD2-AS1 overexpression induced cell proliferation, inflammation and extracellular matrix (ECM) degradation in chondrocytes. Mechanistically, we found that FOXD2-AS1 upregulated the expression level of TLR4 by sponging miR-27a-3p. In addition, we revealed that miR-27a-3p mimics could abolish the effects of FOXD2-AS1 overexpression on cell proliferation, inflammation, and ECM degradation in chondrocytes. Therefore, we demonstrated that FOXD2-AS1 could play a crucial role in the progression of OA, at least partially, by regulating miR-27a-3p/TLR4 axis.


Assuntos
Condrócitos/patologia , MicroRNAs/genética , Osteoartrite/genética , Osteoartrite/patologia , RNA Longo não Codificante/genética , Linhagem Celular , Proliferação de Células/genética , Progressão da Doença , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Receptor 4 Toll-Like/genética
10.
Biomed Res Int ; 2019: 3904923, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30949498

RESUMO

Background and Aims: A growing body of research has demonstrated that the degeneration of chondrocytes is the primary cause of osteoarthritis (OA). Parathyroid hormone-related protein (PTHrP) can alleviate the degeneration of chondrocytes via promotion of chondrocyte proliferation and inhibition of terminal differentiation, but the underlying mechanism remains unknown. This study aimed to identify the microRNAs (miRNAs) that may target PTHrP and regulate the proliferation and terminal differentiation of chondrocytes. Methods: Bioinformatic analysis was used to predict which miRNAs target PTHrP. We collected human knee cartilage specimens to acquire the primary chondrocytes, which we then used to test the expression and function of the targeted miRNAs. To explore the effects of miR-15a-5p on the putative binding sites, specific mimics or inhibitors were transfected into the chondrocytes. Furthermore, a dual-luciferase reporter gene assay and chondrocyte degeneration-related factors were used to verify the possible mechanism. Results: The expression of PTHrP was upregulated in the OA chondrocytes, whilst miR-15a-5p was downregulated in the OA chondrocytes. A negative correlation was observed between PTHrP and miR-15a-5p. The knockdown of miR-15a-5p promoted the growth of chondrocytes and inhibited calcium deposition, whilst overexpression of miR-15a-5p reversed this trend. The effect of miR-15a-5p overexpression was neutralised by PTHrP. Dual-luciferase reporter assays revealed that PTHrP can be used as a novel targeting molecule for miR-15a-5p. Conclusions: miR-15a-5p promotes the degeneration of chondrocytes by targeting PTHrP and, in addition to helping us understand the development of OA, may be a potential biomarker of OA.


Assuntos
Condrócitos/metabolismo , Regulação da Expressão Gênica , MicroRNAs/metabolismo , Osteoartrite/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo/biossíntese , Condrócitos/patologia , Humanos , MicroRNAs/genética , Osteoartrite/genética , Osteoartrite/patologia , Proteína Relacionada ao Hormônio Paratireóideo/genética
11.
Biomed Res Int ; 2019: 5689613, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30931327

RESUMO

Sinapic acid (SA) modulates the nuclear factor-erythroid 2-related factor 2 (Nrf2) signaling pathway in chondrocytes. In order to test the hypothesis that SA is protective against the development of osteoarthritis (OA), primary mouse chondrocytes were treated in vitro with SA and the promoter transactivation activity of heme oxygenase 1 (HO-1), nuclear translocation of Nrf2, and protein expression of HO-1 were assayed. To test the hypothesis in vivo, a destabilization of the medial meniscus (DMM) model was used to induce OA in the knees of mice and SA was delivered orally to the experimental group. The chondrocytes were harvested for further analysis. The expression of HO-1 was similarly upregulated in cartilage from both the experimental mice and human chondrocytes from osteoarthritic knees. SA was found to enhance the promoter transactivation activity of heme oxygenase 1 (HO-1) and increase the expression of Nrf2 and HO-1 in primary chondrocytes. Histopathologic scores showed that the damage induced by the DMM model was significantly lower in the SA treatment group. The addition of a HO-1 inhibitor with SA did not show additional benefit over SA alone in terms of cartilage degradation or histopathologic scores. The expression of TNF-α, IL-1ß, IL-6, MMP-1, MMP-3, MMP-13, ADAMTS4, and ADAMTS5 was significantly reduced both in vitro and in vivo by the presence of SA. Protein expressions of HO-1 and Nrf2 were substantially increased in knee cartilage of mice that received oral SA. Our results suggest that SA should be further explored as a preventative treatment for OA.


Assuntos
Ácidos Cumáricos/administração & dosagem , Heme Oxigenase-1/genética , Fator 2 Relacionado a NF-E2/genética , Osteoartrite do Joelho/tratamento farmacológico , Administração Oral , Idoso , Animais , Cartilagem/efeitos dos fármacos , Cartilagem/patologia , Condrócitos/efeitos dos fármacos , Condrócitos/patologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Meniscos Tibiais/efeitos dos fármacos , Meniscos Tibiais/fisiopatologia , Camundongos , Osteoartrite do Joelho/genética , Osteoartrite do Joelho/patologia , Regiões Promotoras Genéticas/efeitos dos fármacos
12.
Biomed Res Int ; 2019: 1434538, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30993110

RESUMO

Osteoarthritis (OA) is a chronic degenerative joint disease, where chondrocyte apoptosis is responsible for cartilage degeneration. Bax is a well-known proapoptotic protein of the Bcl-2 family, involved in a large number of physiological and pathological processes. However, the regulation mechanisms of Bax underlying chondrocyte apoptosis in OA remain unknown. In the present study, we determined the role of Bax in human OA and chondrocyte apoptosis. The results showed that Bax was upregulated in chondrocytes from the articular cartilage of OA patients and in cultured chondrocyte-like ATDC5 cells treated by IL-1ß. Bax was identified to be the direct target of miR-29a by luciferase reporter assay and by western blotting. Inhibition of miR-29a by the mimics protested and overexpression by miR-29a inhibitors aggravated ATDC5 apoptosis induced by IL-1ß. These data reveal that miR-29a/Bax axis plays an important role in regulating chondrocyte apoptosis and suggest that targeting the proapoptotic protein Bax and increasing expression levels of miR-29a emerge as potential approach for protection against the development of OA.


Assuntos
Apoptose , Condrócitos/metabolismo , Regulação da Expressão Gênica , MicroRNAs/biossíntese , Osteoartrite/metabolismo , Proteína X Associada a bcl-2/biossíntese , Idoso , Idoso de 80 Anos ou mais , Condrócitos/patologia , Humanos , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Osteoartrite/genética , Osteoartrite/patologia , Proteína X Associada a bcl-2/genética
13.
Nat Commun ; 10(1): 1914, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015473

RESUMO

Degradation of extracellular matrix (ECM) underlies loss of cartilage tissue in osteoarthritis, a common disease for which no effective disease-modifying therapy currently exists. Here we describe BNTA, a small molecule with ECM modulatory properties. BNTA promotes generation of ECM components in cultured chondrocytes isolated from individuals with osteoarthritis. In human osteoarthritic cartilage explants, BNTA treatment stimulates expression of ECM components while suppressing inflammatory mediators. Intra-articular injection of BNTA delays the disease progression in a trauma-induced rat model of osteoarthritis. Furthermore, we identify superoxide dismutase 3 (SOD3) as a mediator of BNTA activity. BNTA induces SOD3 expression and superoxide anion elimination in osteoarthritic chondrocyte culture, and ectopic SOD3 expression recapitulates the effect of BNTA on ECM biosynthesis. These observations identify SOD3 as a relevant drug target, and BNTA as a potential therapeutic agent in osteoarthritis.


Assuntos
Anti-Inflamatórios/farmacologia , Benzamidas/farmacologia , Cartilagem Articular/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Depuradores de Radicais Livres/farmacologia , Fatores Imunológicos/farmacologia , Osteoartrite/tratamento farmacológico , Sulfonamidas/farmacologia , Animais , Cartilagem Articular/imunologia , Cartilagem Articular/patologia , Condrócitos/efeitos dos fármacos , Condrócitos/imunologia , Condrócitos/patologia , Citocinas/genética , Citocinas/imunologia , Modelos Animais de Doenças , Progressão da Doença , Matriz Extracelular/imunologia , Matriz Extracelular/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Injeções Intra-Articulares , Masculino , Osteoartrite/genética , Osteoartrite/imunologia , Osteoartrite/patologia , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Superóxido Dismutase/genética , Superóxido Dismutase/imunologia , Superóxidos/antagonistas & inibidores , Superóxidos/metabolismo , Transcriptoma/imunologia
14.
Life Sci ; 226: 164-172, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30970265

RESUMO

AIMS: Osteoarthritis (OA) is a chronic and degenerative joint disease prevalent in the elderly, which is characterized by hypertrophy and reactive hyperplasia of articular cartilage. Autophagy has been reported to inhibit inflammation and reduce chondrocyte apoptosis in OA. As the microRNA (miRNA)-335-5p has been linked to both inflammation and autophagy, this study aimed to investigate its potential role in regulating autophagy during the pathogenesis of OA. MAIN METHODS: Quantitative real-time PCR (qRT-PCR) was used to detect miRNA-335-5p expression in normal and OA human chondrocytes. Following transfection of human OA chondrocytes with double-stranded miRNA-335-5p mimic/inhibitor, qRT-PCR, western blotting, and immunofluorescence were used to determine expression levels of the inflammatory mediators IL-1ß, IL-6, and TNF-α, and the autophagic markers Beclin-1, autophagy-related protein 5 (ATG5), and ATG7. The autophagy inhibitor 3-methyladenine (3-MA) was used to link the anti-inflammatory effects of miRNA-335-5p to autophagy. KEY FINDINGS: The expression of miRNA-335-5p was significantly lower in OA chondrocytes than in normal chondrocytes. Transfection of human OA chondrocytes with the miRNA-335-5p mimic led to a remarkable increase in viability, a significant increase in autophagy-related factors, and a reduction in inflammatory mediators. Importantly, treatment of miRNA-335-5p-overexpressing OA chondrocytes with the autophagy inhibitor 3-MA restored the expression of inflammatory mediators. SIGNIFICANCE: We conclude that miRNA-335-5p can significantly alleviate inflammation in human OA chondrocytes by activating autophagy. Therefore, miRNA-335-5p has potential for future use in the clinical diagnosis and treatment of OA.


Assuntos
Condrócitos/metabolismo , MicroRNAs/biossíntese , Osteoartrite/metabolismo , Apoptose/genética , Artroplastia do Joelho , Autofagia/fisiologia , Proteína 5 Relacionada à Autofagia/biossíntese , Proteína 7 Relacionada à Autofagia/biossíntese , Proteína Beclina-1/biossíntese , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Condrócitos/patologia , Humanos , Mediadores da Inflamação/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Osteoartrite/genética , Osteoartrite/patologia , Cultura Primária de Células , Transdução de Sinais
15.
Gene ; 704: 134-141, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30981839

RESUMO

To maintain normal function of cartilage tissue normally, the presence of a sufficient amount of type II collagen and aggrecan is essential, and their synthesis is tightly regulated. Therefore, understanding the mechanisms that control the expression of type II collagen and aggrecan would be useful for understanding gene expression changes in diseases such as osteoarthritis. Recently, we have identified two pairs of enhancer elements, termed E1 and E2 in the type II collagen gene and Ea and Eb in the aggrecan gene. However, their different mechanisms of action remained unclear. Thus, the central aim of this study was to clarify the different transcriptional regulation mediated through each enhancer element. To this end, we established different stable reporter cell lines that express a reporter gene under the control of different enhancer elements using a silent reporter system we previously constructed. Using these cell lines, we found that dexamethasone, forskolin, and trichostatin A affect the gene expression of type II collagen and aggrecan via different enhancer elements. Moreover, we clarified that E1 and E2 enhancer activities are regulated through distinct epigenetic modifications by histone deacetylase 10 and sirtuin 6.


Assuntos
Agrecanas/genética , Colágeno Tipo II/genética , Elementos Facilitadores Genéticos/genética , Epigênese Genética/fisiologia , Agrecanas/metabolismo , Animais , Condrócitos/metabolismo , Condrócitos/patologia , Colágeno Tipo II/metabolismo , Regulação da Expressão Gênica , Histona Desacetilases do Grupo III/metabolismo , Regiões Promotoras Genéticas , Ratos , Sirtuínas/metabolismo , Células Tumorais Cultivadas
16.
Life Sci ; 228: 258-265, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30953645

RESUMO

Diabetes is an independent risk factor for knee osteoarthritis (OA), and hyperglycaemia-induced inflammation is considered to play an important role in their connection. The Toll-like receptor 4 (TLR4) regulates inflammatory responses in several pathological conditions including diabetes and OA. However, its role in diabetes-associated OA is poorly understood. In this study, we found that TLR4 expression was higher in OA cartilage from patients with type 2 diabetes mellitus (T2DM) than that from non-T2DM patients. Similarly, its expression was induced in primary mouse chondrocytes treated with high glucose, which suggests that TLR4 upregulation in T2DM-associated OA cartilage may originate from hyperglycaemia stimulation. We further discovered that TLR4 promoted high glucose-induced catabolic and inflammatory responses in chondrocytes, and mechanistically, these effects could be explained by the exacerbated activation of the transcription factor nuclear factor kappa B (NF-κB) pathway, since its inhibition by Bay 11-7082 abrogated TLR4 effects on high glucose-treated chondrocytes. Taken together, these findings may reveal a promotive role of TLR4 in regulating hyperglycaemia-induced catabolism and inflammation in T2DM-associated OA, and also implicate that TLR4 inhibition might be of therapeutic significance in treating T2DM-associated OA.


Assuntos
Condrócitos/imunologia , Diabetes Mellitus Tipo 2/imunologia , Glucose/imunologia , NF-kappa B/imunologia , Osteoartrite/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Células Cultivadas , Condrócitos/patologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/patologia , Humanos , Inflamação/complicações , Inflamação/imunologia , Inflamação/patologia , Masculino , Camundongos Endogâmicos C57BL , Osteoartrite/complicações , Osteoartrite/patologia
17.
Eur Cell Mater ; 37: 214-232, 2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30900738

RESUMO

Nasal chondrocytes (NCs) have gained increased recognition for cartilage tissue regeneration. To assess NCs as a source for cell therapy treatment of intervertebral disc (IVD) degeneration, tissue-forming properties of NCs under physiological conditions mimicking the degenerated IVD were compared to those of mesenchymal stromal cells (MSCs) and articular chondrocytes (ACs), two cell sources presently used in clinical trials. Cells were cultured in a combination of low glucose, hypoxia, acidity and inflammation for 28 d. Depending on the conditions, cells were either cultured in the absence of instructive growth factors or underwent chondrogenic instructional priming by addition of transforming growth factor ß1 (TGFß1) for the first 7 d. Histology, immunohistochemistry, biochemistry, enzyme-linked immunosorbent assay (ELISA) and quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) analyses demonstrated limited cell maintenance and accumulation of cartilaginous extracellular matrix for MSCs in IVD conditions. ACs maintained a steady accumulation of glycosaminoglycans (GAGs) throughout all non-acidic conditions, with and without priming, but could not synthesise type II collagen (Col2). NCs accumulated both GAGs and Col2 in all non-acidic conditions, independent of priming, whereas MSCs strongly diminished their GAG and Col2 accumulation in an inflamed environment. Supplementation with inflammatory cytokines or an acidic environment affected NCs to a lower extent than ACs or MSCs. The data, overall indicating that in an inflamed IVD environment NCs were superior to ACs and MSCs, encourage further assessment of NCs for treatment of degenerative disc disease.


Assuntos
Condrócitos/patologia , Degeneração do Disco Intervertebral/patologia , Nariz/patologia , Adolescente , Adulto , Biomarcadores/metabolismo , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/patologia , Condrócitos/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , DNA/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Feminino , Glucose/farmacologia , Glicosaminoglicanos/metabolismo , Humanos , Inflamação/patologia , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Núcleo Pulposo/patologia , Oxigênio/farmacologia , Receptores de Citocinas/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Adulto Jovem
18.
Int J Mol Med ; 43(5): 2241-2251, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30896805

RESUMO

Osteoarthritis (OA) is the most common type of degenerative joint disease and secreted inflammatory molecules serve a pivotal role in it. Peimine has been reported to have anti­inflammatory activity. In order to investigate the potential therapeutic role of Peimine in OA, mouse articular chondrocytes were treated with IL­1ß and different doses of Peimine in vitro. The data revealed that Peimine not only suppressed IL­1ß­induced production of nitric oxide (NO) and prostaglandin E2, but also reduced the protein levels of inducible NO synthase (iNOS) and cyclooxygenase­2 (COX­2). In addition, Peimine inhibited the IL­1ß­induced mRNA expression of matrix metalloproteinase (MMP)­1, MMP­3, MMP­9, MMP­13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)­4 and ADAMTS­5. Furthermore, Peimine inhibited IL­1ß­induced activation of the mitogen­activated protein kinase (MAPK) pathway. The protective effect of Peimine on IL­1ß­treated chondrocytes was attenuated following activation of the MAPK pathway, as demonstrated by the increased expression levels of MMP­3, MMP­13, ADAMTS­5, iNOS and COX­2 compared with the Peimine group. The in vivo data suggested that Peimine limited the development of OA in the mouse model. In general, the data indicate that Peimine suppresses IL­1ß­induced inflammation in mouse chondrocytes by inhibiting the MAPK pathway, suggesting a promising therapeutic role for Peimine in the treatment of OA.


Assuntos
Cevanas/uso terapêutico , Condrócitos/enzimologia , Condrócitos/patologia , Regulação para Baixo , Inflamação/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas ADAMTS/metabolismo , Animais , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/patologia , Sobrevivência Celular/efeitos dos fármacos , Cevanas/farmacologia , Condrócitos/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/biossíntese , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Inflamação/patologia , Interleucina-1beta , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metaloproteinases da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/metabolismo , Osteoartrite/patologia
19.
Int J Nanomedicine ; 14: 1283-1298, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30863061

RESUMO

Purpose: We previously created a self-assembled cartilage-like complex in vitro from only three cartilage components, hyaluronic acid (HA), aggrecan (AG) and type II collagen, without other materials such as cross-linking agents. Based on this self-organized AG/HA/collagen complex, we have created three novel types of biphasic cartilage and bone-like scaffolds combined with hydroxyapatite (HAP) for osteochondral tissue engineering. These scaffolds have been developed from self-assembled cartilage component molecules and HAP at the nanometer scale by manipulating the intermolecular relations. Patients and methods: The surface structure of each self-organized biphasic cartilage and bone-like scaffold was evaluated by scanning electron microscopy, whereas the viscoelasticity was also analyzed in vitro. Three types of artificial cartilage-HAP conjugates were implanted into an osteochondral defect in rat knee joints, and bone and cartilage tissues of the implanted site were examined 4 and 8 weeks after implantation. The tissues were examined histopathologically to evaluate the effects of the implantation on the articular cartilage and subchondral bone tissues. Results: Our in vitro and in vivo data reveal that the self-organized biphasic cartilage and bone-like scaffold conjugated with HAP are superior to the scaffold with no HAP in both cartilage regeneration and subchondral bone regeneration. Conclusion: Our present study indicates that the self-organized biphasic cartilage and bone-like scaffold, which is conjugated with an HAP layer, may have potential not only to repair articular cartilage defects but also to ameliorate the degeneration of subchondral bone in the diseases with osteochondral defect.


Assuntos
Regeneração Óssea/efeitos dos fármacos , Cartilagem Articular/efeitos dos fármacos , Durapatita/farmacologia , Articulação do Joelho/patologia , Engenharia Tecidual/métodos , Idoso , Animais , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrócitos/patologia , Colágeno Tipo II/metabolismo , Elasticidade , Humanos , Articulação do Joelho/efeitos dos fármacos , Masculino , Osteoartrite/patologia , Ratos Sprague-Dawley , Tecidos Suporte/química , Viscosidade
20.
Cell Mol Life Sci ; 76(11): 2077-2091, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30887097

RESUMO

Medial vascular calcification has emerged as a putative key factor contributing to the excessive cardiovascular mortality of patients with chronic kidney disease (CKD). Hyperphosphatemia is considered a decisive determinant of vascular calcification in CKD. A critical role in initiation and progression of vascular calcification during elevated phosphate conditions is attributed to vascular smooth muscle cells (VSMCs), which are able to change their phenotype into osteo-/chondroblasts-like cells. These transdifferentiated VSMCs actively promote calcification in the medial layer of the arteries by producing a local pro-calcifying environment as well as nidus sites for precipitation of calcium and phosphate and growth of calcium phosphate crystals. Elevated extracellular phosphate induces osteo-/chondrogenic transdifferentiation of VSMCs through complex intracellular signaling pathways, which are still incompletely understood. The present review addresses critical intracellular pathways controlling osteo-/chondrogenic transdifferentiation of VSMCs and, thus, vascular calcification during hyperphosphatemia. Elucidating these pathways holds a significant promise to open novel therapeutic opportunities counteracting the progression of vascular calcification in CKD.


Assuntos
Hiperfosfatemia/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Insuficiência Renal Crônica/metabolismo , Transdução de Sinais , Calcificação Vascular/metabolismo , Animais , Fosfatos de Cálcio/química , Fosfatos de Cálcio/metabolismo , Transdiferenciação Celular , Condrócitos/metabolismo , Condrócitos/patologia , Regulação da Expressão Gênica , Humanos , Hiperfosfatemia/complicações , Hiperfosfatemia/genética , Hiperfosfatemia/patologia , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , NF-kappa B/genética , NF-kappa B/metabolismo , Osteoblastos/metabolismo , Osteoblastos/patologia , Ligante RANK/genética , Ligante RANK/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/genética , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/patologia , Calcificação Vascular/complicações , Calcificação Vascular/genética , Calcificação Vascular/patologia
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