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1.
Arch Oral Biol ; 109: 104570, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31568992

RESUMO

OBJECTIVE: The aim of this study is to investigate the effects of 17ß-Estradiol (E2) at different concentrations combined with cyclical compressive stress on the proliferation and differentiation of mandibular condylar chondrocytes (MCCs). DESIGN: MCCs, isolated from female Sprague-Dawley rats, were exposed to E2 at different concentration, cyclical compressive stress or the combination, effects of which on MCCs proliferation and differentiation were detected. RESULTS: E2 at physiological concentration (10-9 mol/L) has lower proliferative effects on MCCs, compared with non-physiological concentration (10-12 mol/L or 10-6 mol/L). For MCCs differentiation, effects of E2 at different concentration are totally opposite: E2 at 10-9 mol/L promotes MCCs differentiation, but at 10-12 mol/L or 10-6 mol/L, it inhibits MCCs differentiation. When combined with E2 at 10-9 mol/L, cyclical compressive stress shows synergistic effect on proliferation and differentiation. However, when combined with E2 at 10-12 mol/L or 10-6 mol/L cyclical compressive stress reverses the inhibition in MCCs differentiation provoked by E2 at 10-12 mol/L or 10-6 mol/L. CONCLUSION: Effects of E2 combined with cyclical compressive stress on MCCs proliferation and differentiation are different, which suggests that orthodontist should take fully consideration of the levels of E2 and adopt comprehensive strategies, so as to achieve better orthodontic effect.


Assuntos
Condrócitos/citologia , Estradiol/farmacologia , Côndilo Mandibular/citologia , Pressão , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Feminino , Ratos , Ratos Sprague-Dawley
2.
Cell Physiol Biochem ; 53(4): 623-637, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31550089

RESUMO

BACKGROUND/AIMS: In articular cartilage, chondrocytes are the predominant cell type. A long-term stay in space can lead to bone loss and cartilage breakdown. Due to the poor regenerative capacity of cartilage, this may impair the crewmembers' mobility and influence mission activities. Beside microgravity other factors such as cosmic radiation and vibration might be important for cartilage degeneration. Vibration at different frequencies showed various effects on cartilage in vivo, but knowledge about its impact on chondrocytes in vitro is sparse. METHODS: Human chondrocytes were exposed to a vibration device, simulating the vibration profile occurring during parabolic flights, for 24 h (VIB) and compared to static controls. Phase-contrast microscopy, immunofluorescence, F-actin and TUNEL staining as well as quantitative real-time PCR were performed to examine effects on morphology, cell viability and shape as well as gene expression. The results were compared to earlier studies using semantic analyses. RESULTS: No morphological changes or cytoskeletal alterations were observed in VIB and no apoptotic cells were found. A reorganization and increase in fibronectin were detected in VIB samples by immunofluorescence technique. PXN, VCL, ANXA1, ANXA2, BAX, and BCL2 revealed differential regulations. CONCLUSION: Long-term VIB did not damage human chondrocytes in vitro. The reduction of ANXA2, and up-regulation of ANXA1, PXN and VCL mRNAs suggest that long-term vibration might even positively influence cultured chondrocytes.


Assuntos
Condrócitos/metabolismo , Vibração , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Anexina A2/genética , Anexina A2/metabolismo , Linhagem Celular , Condrócitos/citologia , Condrócitos/patologia , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Redes Reguladoras de Genes , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Vimentina/genética , Vimentina/metabolismo
3.
Int J Mol Sci ; 20(18)2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31514268

RESUMO

In the development of the skeleton, the long bones are arising from the process of endochondral ossification (EO) in which cartilage is replaced by bone. This complex process is regulated by various factors including genetic, epigenetic, and environmental elements. It is recognized that DNA methylation, higher-order chromatin structure, and post-translational modifications of histones regulate the EO. With emerging understanding, non-coding RNAs (ncRNAs) have been identified as another mode of EO regulation, which is consist of microRNAs (miRNAs or miRs) and long non-coding RNAs (lncRNAs). There is expanding experimental evidence to unlock the role of ncRNAs in the differentiation of cartilage cells, as well as the pathogenesis of several skeletal disorders including osteoarthritis. Cutting-edge technologies such as epigenome-wide association studies have been employed to reveal disease-specific patterns regarding ncRNAs. This opens a new avenue of our understanding of skeletal cell biology, and may also identify potential epigenetic-based biomarkers. In this review, we provide an updated overview of recent advances in the role of ncRNAs especially focus on miRNA and lncRNA in the development of bone from cartilage, as well as their roles in skeletal pathophysiology.


Assuntos
Cartilagem/crescimento & desenvolvimento , Cartilagem/metabolismo , RNA não Traduzido/genética , Animais , Condrócitos/citologia , Condrócitos/metabolismo , Epigênese Genética , Lâmina de Crescimento/metabolismo , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , RNA não Traduzido/metabolismo
4.
Cell Prolif ; 52(6): e12653, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31489992

RESUMO

OBJECTIVES: Bioreactor-based production systems have the potential to overcome limitations associated with conventional tissue engineering manufacturing methods, facilitating regulatory compliant and cost-effective production of engineered grafts for widespread clinical use. In this work, we established a bioreactor-based manufacturing system for the production of cartilage grafts. MATERIALS & METHODS: All bioprocesses, from cartilage biopsy digestion through the generation of engineered grafts, were performed in our bioreactor-based manufacturing system. All bioreactor technologies and cartilage tissue engineering bioprocesses were transferred to an independent GMP facility, where engineered grafts were manufactured for two large animal studies. RESULTS: The results of these studies demonstrate the safety and feasibility of the bioreactor-based manufacturing approach. Moreover, grafts produced in the manufacturing system were first shown to accelerate the repair of acute osteochondral defects, compared to cell-free scaffold implants. We then demonstrated that grafts produced in the system also facilitated faster repair in a more clinically relevant chronic defect model. Our data also suggested that bioreactor-manufactured grafts may result in a more robust repair in the longer term. CONCLUSION: By demonstrating the safety and efficacy of bioreactor-generated grafts in two large animal models, this work represents a pivotal step towards implementing the bioreactor-based manufacturing system for the production of human cartilage grafts for clinical applications. Read the Editorial for this article on doi:10.1111/cpr.12625.


Assuntos
Reatores Biológicos , Condrócitos/citologia , Engenharia Tecidual , Tecidos Suporte , Doença Aguda , Animais , Cartilagem Articular/patologia , Doença Crônica , Feminino , Modelos Animais , Ovinos , Engenharia Tecidual/métodos
5.
Int J Mol Sci ; 20(18)2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31492046

RESUMO

Human induced pluripotent stem cells (hiPSCs) play an important role in research regarding regenerative medicine. Particularly, chondrocytes differentiated from hiPSCs seems to be a promising solution for patients suffering from osteoarthritis. We decided to perform chondrogenesis in a three-week monolayer culture. Based on transcriptome analysis, hiPSC-derived chondrocytes (ChiPS) demonstrate the gene expression profile of cells from early chondrogenesis. Chondrogenic progenitors obtained by our group are characterized by significantly high expression of Hox genes, strongly upregulated during limb formation and morphogenesis. There are scanty literature data concerning the role of microRNAs in early chondrogenesis, especially in chondrogenic differentiation of hiPSCs. The main aim of this study was to investigate the microRNA expression profile and to select microRNAs (miRNAs) taking part in early chondrogenesis. Our findings allowed for selection crucial miRNAs engaged in both diminishing pluripotency state and chondrogenic process (inter alia hsa-miR-525-5p, hsa-miR-520c-3p, hsa-miR-628-3p, hsa-miR-196b-star, hsa-miR-629-star, hsa-miR-517b, has-miR-187). These miRNAs regulate early chondrogenic genes such as: HOXD10, HOXA11, RARB, SEMA3C. These results were confirmed by RT-qPCR analysis. This work contributes to a better understanding of the role of miRNAs directly involved in chondrogenic differentiation of hiPSCs. These data may result in the establishment of a more efficient protocol of obtaining chondrocyte-like cells from hiPSCs.


Assuntos
Condrogênese/genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , MicroRNAs/genética , Diferenciação Celular/genética , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Humanos , Transcriptoma
6.
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
7.
Cell Prolif ; 52(5): e12666, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31407423

RESUMO

OBJECTIVES: Cartilaginous tissue degradation occurs because of the lack of survival of chondrocytes. Here, we ascertained whether bakuchiol (BAK) has the capability of activating chondrocyte proliferation. MATERIALS AND METHODS: The effect of BAK on the proliferation of rat chondrocytes at a concentration of 10 and 20 µmol/L was investigated. The molecular mechanisms involving target binding and signalling pathways were elucidated by RNA-sequencing, qPCR, molecular docking and Western blotting. Matrigel mixed with bakuchiol was implanted locally into rat knee articular cartilage defects to verify the activation of chondrocytes due to bakuchiol in vivo. RESULTS: Bakuchiol implantation resulted in the activation of rat chondrocyte proliferation in a dose-dependent manner. RNA-sequencing revealed 107 differentially expressed genes (DEGs) with 75 that were up-regulated and 32 that were down-regulated, indicating increased activation of the PI3K-Akt and cell cycle pathways. Activation of the phosphorylation of Akt, ERK1/2 and their inhibitors blocked the proliferative effect of bakuchiol treatment, confirming its direct involvement in these signal transduction pathways. Molecular docking and siRNA silencing revealed that estrogen receptor-α (ERα) was the target of bakuchiol in terms of its cell proliferative effect via PI3K activation. Two weeks after implantation of bakuchiol, the appearance and physiological structure of the articular cartilage was more integrated with abundant chondrocytes and cartilage matrix compared to that of the control. CONCLUSIONS: Bakuchiol demonstrated significant bioactivity towards chondrocyte proliferation via the PI3K-Akt and ERK1/2 pathways mediated by estrogen receptor activation and exhibited enhanced promotion of the remodelling of injured cartilage.


Assuntos
Cartilagem Articular/fisiologia , Proliferação de Células/efeitos dos fármacos , Fenóis/farmacologia , Receptores Estrogênicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Cartilagem Articular/diagnóstico por imagem , Cartilagem Articular/patologia , Condrócitos/citologia , Condrócitos/metabolismo , Regulação para Baixo/efeitos dos fármacos , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Regeneração/efeitos dos fármacos
9.
Artif Cells Nanomed Biotechnol ; 47(1): 3188-3193, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31366242

RESUMO

Objective: To investigate the effects of miR-15a on proliferation and apoptosis of human knee articular chondrocytes and explore its underlying mechanism. Methods: qRT-PCR was used to detect the expression of miR-15a in normal chondrocytes and knee arthritic chondrocytes; miR-con (transfected miR-con), miR-15a (transfected miR-15a mimics), anti-miR-con group (transfected anti-miR-con), anti-miR-15a group (transfected anti-miR-15a mimics), pcDNA group (transfected pcDNA), pcDNA-SMAD2 group (transfected pcDNA-SMAD2), the miR-15a + pcDNA group (co-transfected miR-15a and pcDNA), miR-15a + pcDNA-SMAD2 group (co-transfected miR-15a mimics and pcDNA-SMAD2), were transfected into knee articular chondrocytes by liposome method, respectively. The cell proliferation and apoptosis of each group were detected by MTT assay and flow cytometry. The protein expression of SMAD2 was detected by Western blot. The fluorescence activity of each group was detected by dual luciferase reporter gene assay. Results: The expression of miR-15a in knee arthritis chondrocytes was significantly increased (p < .05) compared with that in normal chondrocytes. Moreover, overexpression of miR-15a and silencing of SMAD2 inhibited proliferation and promoted apoptosis in knee arthritis chondrocyte. MiR-15a targeted SMAD2. Overexpression of SMAD2 reversed the inhibitory effects on proliferation and promotion effects on apoptosis induced by miR-15a in knee arthritis chondrocytes. Conclusion: miR-15a can inhibit the proliferation and promote apoptosis of knee arthritis chondrocytes. The mechanism may be related to SMAD2, which will provide a new target for the treatment of knee arthritis.


Assuntos
Apoptose/genética , Condrócitos/citologia , Articulação do Joelho/citologia , MicroRNAs/genética , Proteína Smad2/genética , Artrite/genética , Artrite/patologia , Sequência de Bases , Proliferação de Células/genética , Condrócitos/patologia , Regulação da Expressão Gênica/genética , Humanos , Articulação do Joelho/patologia
10.
Artif Cells Nanomed Biotechnol ; 47(1): 3259-3264, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31368822

RESUMO

Impairment of type II collagen caused by MMPs in response to overproduction of IL-1ß is an important step in the pathological progression of osteoarthritis (OA). Lunasin, a well-known peptide present in the soybean, has displayed a positive impact on numerous physiological functions. Little information in the effects of lunasin on cartilage degradation has been sought in clinical research before. Here, we report that lunasin suppressed the increase in MMP-3 and MMP-13 caused by IL-1ß. In addition, we found that lunasin could prevent the decrease in TIMP-1 and TIMP-2 expressions caused by IL-1ß. Notably, lunasin suppressed reduction of type II collagen, the basis for articular cartilage. Lunasin also attenuated activation of the JAK2/STAT1/IRF-1 pathway. These effects of lunasin suggest that it might become a promising therapeutic agent for chondro-protective therapy.


Assuntos
Colágeno Tipo II/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , Proteínas de Plantas/farmacologia , Cartilagem Articular/citologia , Proliferação de Células/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Relação Dose-Resposta a Droga , Humanos , Interleucina-1beta/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
11.
Biofabrication ; 11(4): 045016, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31342915

RESUMO

An advantage of bioprinting is the ability to incorporate cells into the hydrogel bioink allowing for precise control over cell placement within a construct. Previous work found that the printability of collagen bioinks is highly dependent on their rheological properties. The effect of cell density on collagen rheological properties and, therefore, printability has not been assessed. Therefore, the objective of this study was to determine the effects of incorporating cells on the rheology and printability of collagen bioinks. Primary chondrocytes, at densities relevant to cartilage tissue engineering (up to 100 × 106 cells ml-1), were incorporated into 8 mg ml-1 collagen bioinks. Bioink rheological properties before, during, and after gelation as well as printability were assessed. Cell-laden printed constructs were also cultured for up to 14 d to assess longer-term cell behavior. The addition of cells resulted in an increase in the storage modulus and viscosity of the collagen before gelation. However, the storage modulus after gelation and the rate of gelation decreased with increasing cell density. Theoretical models were compared to the rheological data to suggest frameworks that could be used to predict the rheological properties of cell-laden bioinks. Printability testing showed that improved printability could be achieved with higher cell densities. Fourteen-day culture studies showed that the printing process had no adverse effects on the viability or function of printed cells. Overall, this study shows that collagen bioinks are conducive to bioprinting with a wide range of cell densities while maintaining high printability and chondrocyte viability and function.


Assuntos
Técnicas de Cultura de Células/métodos , Colágeno/química , Tinta , Reologia , Animais , Bovinos , Contagem de Células , Forma Celular , Sobrevivência Celular , Condrócitos/citologia , Ratos
12.
Int J Mol Sci ; 20(14)2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31331074

RESUMO

In normal articular cartilage, chondrocytes do not readily proliferate or terminally differentiate, and exhibit a low level of metabolism. Hypertrophy-like changes of chondrocytes have been proposed to play a role in the pathogenesis of osteoarthritis by inducing protease-mediated cartilage degradation and calcification; however, the molecular mechanisms underlying these changes are unclear. Glycans are located on the outermost cell surface. Dynamic cellular differentiation can be monitored and quantitatively characterized by profiling the glycan structures of total cellular glycoproteins. This study aimed to clarify the alterations in glycans upon late differentiation of chondrocytes, during which hypertrophy-like changes occur. Primary mouse chondrocytes were differentiated using an insulin-induced chondro-osteogenic differentiation model. Comprehensive glycomics, including N-glycans, O-glycans, free oligosaccharides, glycosaminoglycan, and glycosphingolipid, were analyzed for the chondrocytes after 0-, 10- and 20-days cultivation. The comparison and clustering of the alteration of glycans upon hypertrophy-like changes of primary chondrocytes were performed. Comprehensive glycomic analyses provided complementary alterations in the levels of various glycans derived from glycoconjugates during hypertrophic differentiation. In addition, expression of genes related to glycan biosynthesis and metabolic processes was significantly correlated with glycan alterations. Our results indicate that total cellular glycan alterations are closely associated with chondrocyte hypertrophy and help to describe the glycophenotype by chondrocytes and their hypertrophic differentiation. our results will assist the identification of diagnostic and differentiation biomarkers in the future.


Assuntos
Diferenciação Celular , Condrócitos/citologia , Condrócitos/metabolismo , Glicoproteínas/metabolismo , Polissacarídeos/metabolismo , Animais , Biomarcadores , Diferenciação Celular/genética , Células Cultivadas , Glicosilação , Hipertrofia , Metabolômica/métodos , Camundongos , Osteogênese/genética , Proteômica/métodos
13.
Nat Commun ; 10(1): 3168, 2019 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-31320650

RESUMO

Multipotent mesenchymal stromal cells (MSCs) are required for skeletal formation, maintenance, and repair throughout life; however, current models posit that postnatally arising long-lived adult MSCs replace transient embryonic progenitor populations. We previously reported exclusive expression and function of the embryonic patterning transcription factor, Hoxa11, in adult skeletal progenitor-enriched MSCs. Here, using a newly generated Hoxa11-CreERT2 lineage-tracing system, we show Hoxa11-lineage marked cells give rise to all skeletal lineages throughout the life of the animal and persist as MSCs. Hoxa11 lineage-positive cells give rise to previously described progenitor-enriched MSC populations marked by LepR-Cre and Osx-CreER, placing them upstream of these populations. Our studies establish that Hox-expressing cells are skeletal stem cells that arise from the earliest stages of skeletal development and self-renew throughout the life of the animal.


Assuntos
Adipócitos/citologia , Condrócitos/citologia , Proteínas de Homeodomínio/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Animais , Regeneração Óssea/genética , Osso e Ossos/citologia , Osso e Ossos/embriologia , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Animais , Receptores para Leptina/genética , Fator de Transcrição Sp7/genética
14.
Mol Med Rep ; 20(3): 2843-2850, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31322228

RESUMO

Osteoarthritis (OA) is a degenerative joint disease characterized by articular cartilage degradation and joint inflammation. A previous study showed that microRNA (miR)­671­3p is involved in the development of OA, however, its function and molecular target in chondrocytes during the pathogenesis of OA remain to be fully elucidated. In the present study, miR­671­3p was significantly downregulated in knee OA cartilage tissues compared with normal cartilage tissues. The expression levels of pro­inflammatory cytokines, including interleukin (IL)­1ß, IL­6, IL­8 and tumor necrosis factor (TNF)­α, in the knee OA cartilage tissues were significantly higher than those in the normal cartilage tissues. Through gain­of­function and loss­of­function experiments, miR­671­3p was shown to significantly affect matrix synthesis gene expression, cell proliferation, apoptosis and inflammation in chondrocytes from patients with OA. Subsequent bioinformatics analysis identified potential target sites of the miR­671­3p located in the 3'untranslated region of TNF receptor­associated factor (TRAF3). The results of a dual­luciferase reporter assay showed that TRAF3 is a target gene of miR­671­3p. Western blot analysis demonstrated that miR­671­3p inhibited the gene expression of TRAF3. Furthermore, the restoration of TRAF3 markedly abrogated the effect of miR­671­3p. Taken together, the present study suggests that miR­671­3p may be important in the pathogenesis of OA through targeting TRAF3 and regulating chondrocyte apoptosis and inflammation, which may be a potential molecular target for OA treatment.


Assuntos
Condrócitos/patologia , MicroRNAs/genética , Osteoartrite do Joelho/genética , Fator 3 Associado a Receptor de TNF/genética , Idoso , Apoptose , Proliferação de Células , Condrócitos/citologia , Condrócitos/metabolismo , Regulação para Baixo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/patologia , Regulação para Cima
15.
Gene ; 712: 143959, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31278964

RESUMO

Blockade of Hedgehog signaling can prevent osteoarthritis (OA) syndromes. However, the amelioration of related inflammation condition is limited. The purpose of this study was to observe the effect of combined use of Hedgehog signaling inhibitor GANT-61 and common clinical anti-inflammatory drug indomethacin on cartilage injury and inflammation in experimental OA mice. We found that GANT-61 and indomethacin synergistically attenuate cartilage damage and serum levels of inflammatory cytokines TNF-α, IL-2 and IL-6 in OA mice. Moreover, in vitro treatment of GANT-61 and indomethacin synergistically reduced the mRNA expression of TNF-α, IL-2 and IL-6 in lipopolysaccharide (LPS)-stimulated C28/I2 chondrocytes. Mechasnistic studies showed that GANT-61 and indomethacin synergistically attenuate the expressions of cell pyroptosis-related genes caspase-1, IL-1ß and IL-18 at mRNA and protein level. To conclude, our study showed that GANT-61 and indomethacin had a synergistically ameliorating effect on osteoarthritis by mediating chondrocytes pyroptosis.


Assuntos
Cartilagem/efeitos dos fármacos , Condrócitos/citologia , Proteínas Hedgehog/antagonistas & inibidores , Indometacina/farmacologia , Osteoartrite/tratamento farmacológico , Piridinas/administração & dosagem , Pirimidinas/administração & dosagem , Animais , Anti-Inflamatórios/farmacologia , Cartilagem/patologia , Caspase 1/metabolismo , Linhagem Celular , Citocinas/metabolismo , Modelos Animais de Doenças , Sinergismo Farmacológico , Humanos , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Interleucina-2/metabolismo , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Piroptose , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo
16.
PLoS Genet ; 15(7): e1008215, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31260448

RESUMO

The unfolded protein response (UPR) is a conserved cellular response to the accumulation of proteinaceous material in endoplasmic reticulum (ER), active both in health and disease to alleviate cellular stress and improve protein folding. Multiple epiphyseal dysplasia (EDM5) is a genetic skeletal condition and a classic example of an intracellular protein aggregation disease, whereby mutant matrilin-3 forms large insoluble aggregates in the ER lumen, resulting in a specific 'disease signature' of increased expression of chaperones and foldases, and alternative splicing of the UPR effector XBP1. Matrilin-3 is expressed exclusively by chondrocytes thereby making EDM5 a perfect model system to study the role of protein aggregation in disease. In order to dissect the role of XBP1 signalling in aggregation-related conditions we crossed a p.V194D Matn3 knock-in mouse model of EDM5 with a mouse line carrying a cartilage specific deletion of XBP1 and analysed the resulting phenotype. Interestingly, the growth of mice carrying the Matn3 p.V194D mutation compounded with the cartilage specific deletion of XBP1 was severely retarded. Further phenotyping revealed increased intracellular retention of amyloid-like aggregates of mutant matrilin-3 coupled with dramatically decreased cell proliferation and increased apoptosis, suggesting a role of XBP1 signalling in protein accumulation and/or degradation. Transcriptomic analysis of chondrocytes extracted from wild type, EDM5, Xbp1-null and compound mutant lines revealed that the alternative splicing of Xbp1 is crucial in modulating levels of protein aggregation. Moreover, through detailed transcriptomic comparison with a model of metaphyseal chondrodysplasia type Schmid (MCDS), an UPR-related skeletal condition in which XBP1 was removed without overt consequences, we show for the first time that the differentiation-state of cells within the cartilage growth plate influences the UPR resulting from retention of a misfolded mutant protein and postulate that modulation of XBP1 signalling pathway presents a therapeutic target for aggregation related conditions in cells undergoing proliferation.


Assuntos
Mutação , Osteocondrodisplasias/genética , Osteocondrodisplasias/patologia , Proteína 1 de Ligação a X-Box/genética , Processamento Alternativo , Animais , Apoptose , Proliferação de Células , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático , Perfilação da Expressão Gênica , Humanos , Proteínas Matrilinas/química , Proteínas Matrilinas/genética , Camundongos , Osteocondrodisplasias/metabolismo , Agregados Proteicos , Transdução de Sinais , Resposta a Proteínas não Dobradas , Proteína 1 de Ligação a X-Box/metabolismo
17.
Chem Biol Interact ; 310: 108730, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31260663

RESUMO

The present study shows the basis for the anti-inflammatory effects of statins in interleukin 1ß (IL-1ß) induced SW1353 chondrosarcoma cell-line. The cells were pre-treated with simvastatin (5 µM, 10 µM, and 50 µM), followed by IL-1ß (5 ng/mL) stimulation. Effects of simvastatin on cell viability and cytotoxicity of chondrocytes were measured with WST-1 and lactate dehydrogenase (LDH) assays, respectively. Under inflammatory conditions, in the absence/presence of simvastatin, the changes in matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase-1 (TIMP-1) expression levels were examined. Expression levels of MMP-1, -2, -3, -9, -13, and TIMP-1 and -2 were examined by qPCR. MMP-1, -9, -13, TIMP-1, and -2 levels were also determined by Western blotting. Gelatin zymography was performed to analyze the released and intracellular MMP-2 and MMP-9 activity levels. The results showed that simvastatin downregulated the degradation related genes MMP-3, MMP-13, MMP-2, MMP-9 and TIMP-2 in a dose-dependent manner.


Assuntos
Condrócitos/citologia , Condrossarcoma/patologia , Metaloproteinases da Matriz/metabolismo , Sinvastatina/farmacologia , Anti-Inflamatórios/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Citotoxinas/farmacologia , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-1beta/farmacologia , Metaloproteinases da Matriz/efeitos dos fármacos , Inibidores Teciduais de Metaloproteinases/metabolismo
18.
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
19.
Mol Med Rep ; 20(2): 1523-1530, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31257459

RESUMO

Intervertebral disc degeneration (IVDD) is the main pathological basis of spinal degenerative diseases, and aberrant apoptosis of nucleus pulposus cells (NPCs) is the main cellular process that causes IVDD. In our previous studies, 17ß­estradiol (E2) was demonstrated to protect rat NPCs from interleukin­1ß (IL­1ß)­induced apoptosis via the PI3K/Akt signaling pathway. However, the downstream signaling pathway of PI3K/Akt is currently unclear. The present study aimed to explore the signaling pathways that are downstream of the PI3K/Akt pathway, including mTOR, NF­κB and glycogen synthase kinase­3ß (GSK­3ß). Annexin V/propidium iodide double staining was used to determine the incidence of apoptosis. Cell Counting kit­8 and MTS assays were used to determine the proliferation and viability of NPCs, respectively. Cellular binding was evaluated using a cell­collagen binding assay. Western blotting was used to determine the protein expression levels of mTOR, NF­κB and GSK­3ß, and their phosphorylation levels, as well as the expression levels of active caspase­3. The results revealed that IL­1ß induced NPC apoptosis and increased the early apoptotic rate of NPCs. However, E2 reduced the early apoptosis of NPCs induced by IL­1ß. In addition, E2 suppressed the decrease in cell viability and binding ability caused by IL­1ß cytotoxicity. Western blotting revealed that E2 also reduced the expression of activated caspase­3, and increased the expression of activated mTOR. As a specific inhibitor of mTOR, rapamycin effectively attenuated the effects of E2. These findings indicated that E2 protected NPCs against apoptosis via activation of the mTOR/caspase­3 pathway.


Assuntos
Apoptose/efeitos dos fármacos , Caspase 3/genética , Condrócitos/efeitos dos fármacos , Estradiol/farmacologia , Interleucina-1beta/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Animais , Apoptose/genética , Caspase 3/metabolismo , Condrócitos/citologia , Condrócitos/metabolismo , Regulação da Expressão Gênica , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Interleucina-1beta/farmacologia , Masculino , Núcleo Pulposo/citologia , Núcleo Pulposo/efeitos dos fármacos , Núcleo Pulposo/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo
20.
Mol Med Rep ; 20(3): 2633-2640, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31322277

RESUMO

Osteoarthritis (OA) is a type of degenerative joint disease that affects the health of the elderly. OA is characterized by articular cartilage degradation and joint inflammation. The present study aimed to investigate the role and mechanism of microRNA­let­7a (Let­7a) in OA by examining its role in lipopolysaccharide (LPS)­induced cartilage inflammatory injury in ATDC5 cells. ATDC5 cells were treated with various concentrations of LPS. The present results suggested that 5 and 10 µg/ml LPS significantly inhibited ATDC5 cell viability, and 5 µg/ml LPS was selected for further experiments. Reverse transcription­quantitative PCR (RT­qPCR) results suggested that treatment with LPS significantly induced the expression levels of multiple inflammatory factors, including tumor necrosis factor­α (TNF­α), interleukin (IL)­1ß, IL­6 and IL­8, and increased the expression level of Let­7a in ATDC5 cells. IL­6 receptor (IL­6R) was identified to be a direct target of Let­7a using TargetScan and a dual­luciferase reporter assay. Subsequently, Cell Counting Kit­8 and flow cytometry analyses identified that Let­7a inhibitor could significantly promote cell viability and reduce cell apoptosis in ATDC5 cells treated with LPS, and these effects could be reversed by transfection with small interfering (si)RNA­IL­6R. ELISA was used to examine the expression of inflammatory factors in ATDC5 cells following treatment with LPS. Additionally, RT­qPCR and western blotting were performed to detect the mRNA and protein expression level of IL­6R and STAT3. The present results suggested that Let­7a inhibitor significantly reduced the expression level of TNF­α, IL­1ß, IL­6 and IL­8 in ATDC5 cells, and this effect was reversed by transfecting siRNA­IL­6R. Moreover, RT­qPCR and western blot assay results suggested that Let­7a inhibitor significantly increased the expression level of IL­6R and phosphorylated STAT3, and these effects could be reversed by siRNA­IL­6R. Collectively, Let­7a inhibitor increased cell proliferation, reduced apoptosis and inhibited inflammatory response in ATDC5 cells treated with LPS. The present study provided a new potential therapeutic target for OA treatment.


Assuntos
Condrócitos/imunologia , Inflamação/genética , Lipopolissacarídeos/imunologia , MicroRNAs/genética , Receptores de Interleucina-6/genética , Linhagem Celular , Sobrevivência Celular , Condrócitos/citologia , Condrócitos/metabolismo , Regulação para Baixo , Humanos , Inflamação/imunologia , MicroRNAs/imunologia , Receptores de Interleucina-6/imunologia , Regulação para Cima
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