RESUMO
Osteoarthritis (OA) is a common, age-related, and painful disease characterized by cartilage destruction, osteophyte formation, and synovial hyperplasia. This study revealed that circPDE4D, a circular RNA derived from human linear PDE4D, plays a critical role in maintaining the extracellular cellular matrix (ECM) during OA progression. circPDE4D was significantly downregulated in OA cartilage tissues and during stimulation with inflammatory cytokines. The knockdown of circPDE4D predominantly contributed to Aggrecan loss and the upregulation of matrix catabolic enzymes, including MMP3, MMP13, ADAMTS4, and ADAMTS5, but not proliferation or apoptosis. In a murine model of destabilization of the medial meniscus (DMM), the intraarticular injection of circPDE4D alleviated DMM-induced cartilage impairments. Mechanistically, we found that circPDE4D exerted its effect by acting as a sponge for miR-103a-3p and thereby regulated FGF18 expression, which is a direct target of miR-103a-3p. In conclusion, our findings highlight a novel protective role of circPDE4D in OA pathogenesis and indicate that the targeting of the circPDE4D-miR-103a-3p-FGF18 axis might provide a potential and promising approach for OA therapy.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Fatores de Crescimento de Fibroblastos/genética , MicroRNAs/genética , Osteoartrite/genética , Interferência de RNA , RNA Circular , Biomarcadores , Células Cultivadas , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Osteoartrite/metabolismo , Osteoartrite/patologiaRESUMO
Following the publication of the original paper [1], one corresponding author was inadvertently missed.
RESUMO
OBJECTIVES: FBXO6, a component of the ubiquitin E3 ligases, has been shown to bind high mannose N-linked glycoproteins and act as ubiquitin ligase subunits. Most proteins in the secretory pathway, such as matrix metalloproteinases, are modified with N-glycans and play important roles in the development of osteoarthritis (OA). However, whether FBXO6 exerts regulatory effects on the pathogenesis of OA remains undefined. METHODS: The expression of FBXO6 was examined in the cartilage of human and multiple mouse OA models. The role of FBXO6 in cartilage degeneration was analysed with global FBXO6-/- mice, transgenic Col2a1-CreERT2;FBXO6f/f mice. The FBXO6 interacting partner MMP14 and its regulatory transcriptional factor SMAD2/3 were identified and validated in different pathological models as well as SMAD2-/- mice. RESULTS: The expression of FBXO6 decreased in the cartilage from human OA samples, anterior cruciate ligament transaction (ACLT) -induced OA samples, spontaneous OA STR/ort samples and aged mice samples. Global knockout or conditional knockout of FBXO6 in cartilage promoted experimental OA process. The molecular mechanism study revealed that FBXO6 decreased MMP14 by ubiquitination and degradation, leading to inhibited proteolytic activation of MMP13. Interestingly, FBXO6 expression is regulated by transforming growth factor ß (TGFß)-SMAD2/3 signalling pathway. Therefore, the overexpression of FBXO6 protected mice from post-injury OA development. CONCLUSIONS: TGFß-SMAD2/3 signalling pathway suppressed MMP13 activation by upregulating of FBXO6 transcription and consequently promoting MMP14 proteasomal degradation. Inducement of FBXO6 expression in OA cartilage might provide a promising OA therapeutic strategy.
Assuntos
Matriz Extracelular/patologia , Metaloproteinase 14 da Matriz/metabolismo , Osteoartrite/patologia , Fator de Crescimento Transformador beta/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Matriz Extracelular/metabolismo , Humanos , Camundongos , Osteoartrite/metabolismo , Ubiquitinação/fisiologiaRESUMO
Balance of osteoclast formation is regulated by the receptor activator of NF-κB ligand and extracellular negative regulators such as IFN-γ and IFN-ß. However, very little is known about the intrinsic negative regulatory factors of osteoclast differentiation. Recently, the paired-box homeodomain transcription factor Pax6 was shown to negatively regulate receptor activator of NF-κB ligand-mediated osteoclast differentiation. However, the mechanism underlying this regulation is still unclear. In this study, we show that a p38 inhibitor (VX-745) up-regulates the expression of Pax6 during osteoclast differentiation. Subsequently, we found that ß-catenin could bind to the proximal region of Pax6 promoter to induce its expression, and this action could be impaired by p38-induced ubiquitin-mediated degradation of ß-catenin. Our results suggest that Pax6 is regulated by a novel p38/ß-catenin pathway. Pax6 can further regulate the nuclear translocation of NF of activated T cells, cytoplasmic 1. Our study indicates that this novel p38/ß-catenin/Pax6 axis contributes to negative regulation of osteoclastogenesis. In addition, our study proposes a novel approach to treat osteoclast-related diseases through the use of VX-745 complemented with the ß-catenin activator SKL2001.-Jie, Z., Shen, S., Zhao, X., Xu, W., Zhang, X., Huang, B., Tang, P., Qin, A., Fan, S., Xie, Z. Activating ß-catenin/Pax6 axis negatively regulates osteoclastogenesis by selectively inhibiting phosphorylation of p38/MAPK.
Assuntos
Osteoclastos/metabolismo , Osteogênese/fisiologia , Fator de Transcrição PAX6/metabolismo , Fosforilação/fisiologia , beta Catenina/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Reabsorção Óssea/metabolismo , Diferenciação Celular/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Ligante RANK/metabolismo , Regulação para Cima/fisiologiaRESUMO
The endogenous metabolite itaconate has emerged as a regulator of macrophage function that limits inflammation. However, its effect on cell differentiation and osteoclast-related diseases is unclear. Here, for the first time, we explored the effect of itaconate and its cell-permeable itaconate derivative, 4-octyl itaconate (OI) on osteoclast differentiation in vitro and in vivo. Firstly, we demonstrated that itaconate concentration was lower in estrogen-deficient mice. OI released itaconate and induced the expression of nuclear factor-erythroid 2-related factor 2 (Nrf2) in bone marrow-derived macrophages during osteoclastogenesis. Furthermore, OI significantly suppressed the early, middle, and late stages of osteoclastogenesis induced by receptor activator of NF-κB ligand in vitro, as confirmed by tartrate-resistant acid phosphatase staining. Moreover, it significantly inhibited fibrous actin ring formation and bone resorption in vitro. Mechanistically, we observed that OI enhanced Nrf2 expression by suppressing its association with ubiquitin via inhibition of the E3 ubiquitin ligase (Hrd1). OI also inhibited LPS-induced the reactive oxygen species and inflammatory responses via Hrd1. An estrogen deficiency (via ovariectomy)-induced osteoporosis model was also established. Here, on micro-computed tomography and histologic analysis showed that OI effectively suppressed ovariectomy-induced bone loss. In summary, OI, an itaconate derivative, can inhibit osteoclastogenesis in vitro and in vivo, indicating that OI could be a potential drug to treat osteoclast-related diseases; our results also link itaconate to the development of osteoporosis.-Sun, X., Zhang, B., Pan, X., Huang, H., Xie, Z., Ma, Y., Hu, B., Wang, J., Chen, Z., Shi, P. Octyl itaconate inhibits osteoclastogenesis by suppressing Hrd1 and activating Nrf2 signaling.
Assuntos
Fator 2 Relacionado a NF-E2/metabolismo , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Succinatos/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Estrogênios/deficiência , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Osteoclastos/citologia , Osteoclastos/metabolismo , Osteoporose/prevenção & controle , Ovariectomia/efeitos adversosRESUMO
Osteosarcoma is the most common bone malignancy, and it seriously affects the quality of life of affected children and adolescents. Glabridin (GLA), a major component of licorice root extract, has been reported to exert antitumor effects against a variety of tumor types; however, its effects on osteosarcoma have not been elucidated. In the current study, we investigate the effects and potential antimetastatic mechanisms of GLA on osteosarcoma in vitro and in vivo. Flow cytometry showed that GLA induced G2/M cell cycle phase arrest and promoted cell apoptosis. Transwell and wound-healing assays showed that GLA significantly decreased the migration and invasion of osteosarcoma cells. Further western blotting and quantitative real-time polymerase chain reaction showed that the expression of matrix metalloproteinase (MMP)-2 and MMP-9 in MG63 and HOS cells were reduced after GLA treatment. Moreover, western blotting demonstrated that GLA downregulated the phosphorylation of p38 mitogen-activated protein kinases and c-Jun N-terminal kinase. A coimmunoprecipitation assay illustrated that formation of cAMP response element-binding protein (CREB)-activating protein 1 (AP1) complexes and the DNA binding activities of CREB and AP1 in MG63 and HOS cells were impaired following treatment with GLA. Finally, GLA inhibited tumor growth and suppressed osteosarcoma cell metastasis in vivo. Overall, our findings highlight the potential of GLA as a therapeutic agent for the prevention and treatment of tumor metastasis.
Assuntos
Aminas/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Movimento Celular/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Osteossarcoma/tratamento farmacológico , Fator de Transcrição AP-1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Apoptose/efeitos dos fármacos , Neoplasias Ósseas/enzimologia , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Nus , Complexos Multiproteicos , Invasividade Neoplásica , Osteossarcoma/enzimologia , Osteossarcoma/genética , Osteossarcoma/patologia , Fosforilação , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: As a subclass of noncoding RNAs, circular RNAs (circRNAs) have been demonstrated to play a critical role in regulating gene expression in eukaryotes. Recent studies have revealed the pivotal functions of circRNAs in cancer progression. However, little is known about the role of circTADA2A, also named hsa_circ_0043278, in osteosarcoma (OS). METHODS: CircTADA2A was selected from a previously reported circRNA microarray comparing OS cell lines and normal bone cells. QRT-PCR was used to detect the expression of circTADA2A in OS tissue and cell lines. Luciferase reporter, RNA immunoprecipitation (RIP), RNA pull-down and fluorescence in situ hybridization (FISH) assays were performed to confirm the binding of circTADA2A with miR-203a-3p. OS cells were stably transfected with lentiviruses, and Transwell migration, Matrigel invasion, colony formation, proliferation, apoptosis, Western blotting, and in vivo tumorigenesis and metastasis assays were employed to evaluate the roles of circTADA2A, miR-203a-3p and CREB3. RESULTS: Our findings demonstrated that circTADA2A was highly expressed in both OS tissue and cell lines, and circTADA2A inhibition attenuated the migration, invasion and proliferation of OS cells in vitro as well as tumorigenesis and metastasis in vivo. A mechanistic study revealed that circTADA2A could readily sponge miR-203a-3p to upregulate the expression of CREB3, which was identified as a driver gene in OS. Furthermore, miR-203a-3p inhibition or CREB3 overexpression could reverse the circTADA2A silencing-induced impairment of malignant tumor behavior. CONCLUSIONS: CircTADA2A functions as a tumor promoter in OS to increase malignant tumor behavior through the miR-203a-3p/CREB3 axis, which could be a novel target for OS therapy.
Assuntos
Neoplasias Ósseas/patologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , MicroRNAs/genética , Osteossarcoma/patologia , RNA/genética , Animais , Neoplasias Ósseas/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Citoplasma/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Masculino , Camundongos , Metástase Neoplásica , Transplante de Neoplasias , Osteossarcoma/genética , RNA Circular , Regulação para CimaRESUMO
BACKGROUND: CircMYO10 is a circular RNA generated by back-splicing of gene MYO10 and is upregulated in osteosarcoma cell lines, but its functional role in osteosarcoma is still unknown. This study aimed to clarify the mechanism of circMYO10 in osteosarcoma. METHODS: CircMYO10 expression in 10 paired osteosarcoma and chondroma tissues was assessed by quantitative reverse transcription polymerase chain reaction (PCR). The function of circMYO10/miR-370-3p/RUVBL1 axis was assessed regarding two key characteristics: proliferation and endothelial-mesenchymal transition (EMT). Bioinformatics analysis, western blotting, real-time PCR, fluorescence in situ hybridization, immunoprecipitation, RNA pull-down assays, luciferase reporter assays, chromatin immunoprecipitation, and rescue experiments were used to evaluate the mechanism. Stably transfected MG63 cells were injected via tail vein or subcutaneously into nude mice to assess the role of circMYO10 in vivo. RESULTS: CircMYO10 was significantly upregulated, while miR-370-3p was downregulated, in osteosarcoma cell lines and human osteosarcoma samples. Silencing circMYO10 inhibited cell proliferation and EMT in vivo and in vitro. Mechanistic investigations revealed that miR-370-3p targets RUVBL1 directly, and inhibits the interaction between RUVBL1 and ß-catenin/LEF1 complex while circMYO10 showed a contrary effect via the inhibition of miR-370-3p. RUVBL1 was found to be complexed with chromatin remodeling and histone-modifying factor TIP60, and lymphoid enhancer factor-1 (LEF1) to promote histone H4K16 acetylation (H4K16Ac) in the vicinity of the promoter region of gene C-myc. Chromatin immunoprecipitation methods showed that miR-370-3p sponge promotes H4K16Ac in the indicated region, which is partially abrogated by RUVBL1 small hairpin RNA (shRNA) while circMYO10 showed a contrary result via the inhibition of miR-370-3p. Either miR-370-3p sponge or ShRUVBL1 attenuated circMYO10-induced phenotypes in osteosarcoma cell lines. MiR-370-3p inhibition abrogated the inhibition of proliferation, EMT of osteosarcoma cells in vitro and in vivo seen upon circMYO10 suppression via Wnt/ß-catenin signaling. CONCLUSIONS: CircMYO10 promotes osteosarcoma progression by regulating miR-370-3p/RUVBL1 axis to promote chromatin remodeling and thus enhances the transcriptional activity of ß-catenin/LEF1 complex, which indicates that circMYO10 may be a potential therapeutic target for osteosarcoma treatment.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Proteínas de Transporte/genética , Montagem e Desmontagem da Cromatina , DNA Helicases/genética , MicroRNAs/genética , Miosinas/genética , Osteossarcoma/genética , Osteossarcoma/metabolismo , RNA Circular , Regiões 3' não Traduzidas , Animais , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Progressão da Doença , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Histonas/metabolismo , Humanos , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Metilação , Camundongos , Metástase Neoplásica , Osteossarcoma/patologia , Regiões Promotoras Genéticas , Ligação Proteica , Interferência de RNA , Via de Sinalização Wnt , beta Catenina/metabolismoRESUMO
OBJECTIVES: Circular RNAs (circRNA) expression aberration has been identified in various human diseases. In this study, we investigated whether circRNAs could act as competing endogenous RNAs to regulate the pathological process of osteoarthritis (OA). METHODS: CircRNA deep sequencing was performed to the expression of circRNAs between OA and control cartilage tissues. The regulatory and functional role of CircSERPINE2 upregulation was examined in OA and was validated in vitro and in vivo, downstream target of CircSERPINE2 was explored. RNA pull down, a luciferase reporter assay, biotin-coupled microRNA capture and fluorescence in situ hybridisation were used to evaluate the interaction between CircSERPINE2 and miR-1271-5 p, as well as the target mRNA, E26 transformation-specific-related gene (ERG). The role and mechanism of CircSERPINE2 in OA was also explored in rabbit models. RESULTS: The decreased expression of CircSERPINE2 in the OA cartilage tissues was directly associated with excessive apoptosis and imbalance between anabolic and catabolic factors of extracellular matrix (ECM). Mechanistically, CircSERPINE2 acted as a sponge of miR-1271-5 p and functioned in human chondrocytes (HCs) through targeting miR-1271-5 p and ERG. Intra-articular injection of adeno-associated virus-CircSERPINE2-wt alleviated OA in the rabbit model. CONCLUSIONS: Our results reveal an important role for a novel circRNA-CircSERPINE2 in OA progression. CircSERPINE2 overexpression could alleviate HCs apoptosis and promote anabolism of ECM through miR-1271-ERG pathway. It provides a potentially effective therapeutic strategy for OA progression.
Assuntos
MicroRNAs/metabolismo , Osteoartrite/genética , Serpina E2/fisiologia , Animais , Apoptose/genética , Artrite Experimental/terapia , Cartilagem Articular/metabolismo , Estudos de Casos e Controles , Linhagem Celular , Células Cultivadas , Condrócitos/metabolismo , Matriz Extracelular/metabolismo , Feminino , Marcação de Genes , Terapia Genética/métodos , Humanos , Masculino , MicroRNAs/genética , Terapia de Alvo Molecular/métodos , Osteoartrite/metabolismo , Osteoartrite/patologia , Osteoartrite/terapia , RNA Circular/metabolismo , Coelhos , Serpina E2/genéticaRESUMO
BACKGROUND: Osteoclasts are key determinant cellular components implicated in the development and progression of disorders driven by bone damage. Herein, we studied the upshot of T007, an antagonist of peroxisome proliferator-activated receptor-gamma (PPARγ), on osteoclastogenesis using cell and animal models. RESULTS: The in vitro assays revealed that T007 hindered the osteoclastogenesis caused by the treatment with the receptor activator of nuclear factor-κB ligand (RANKL) through inhibiting the levels of PPARγ in cells. The PPARγ siRNA partially reproduced the inhibitory action of T007. The opposite findings were produced after PPARγ overexpression. Furthermore, T007 prevented from bone loss in a mouse model of osteoporosis induced by ovariectomy (OVX). These findings implied that T007 is a potential efficient drug for the prophylaxis and cure of osteoclast-related disorders. CONCLUSIONS: Taken together, our findings demonstrated that T007 impedes osteoclastogenesis and will be useful for the therapy of bone related diseases, essentially osteoporosis.
Assuntos
Benzamidas/farmacologia , Reabsorção Óssea/patologia , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Ovariectomia/efeitos adversos , PPAR gama/antagonistas & inibidores , Piridinas/farmacologia , Ligante RANK/farmacologia , Células 3T3 , Animais , Reabsorção Óssea/complicações , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/etiologia , Diferenciação Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Osteoblastos/efeitos dos fármacos , Osteoblastos/patologia , Osteoclastos/patologia , Osteoporose/complicaçõesRESUMO
High incidence of osteoporotic fractures emphasizes the necessity of developing effective measures to promote osteogenesis. In our study, we investigated a possible role of MAPK-ERK signaling in the TGF-ß-mediated osteoblastic differentiation. Our results indicated that TGF-ß activated the MAPK-ERK pathway and inhibited osteogenesis in mesenchymal pluripotent cell line, C3H10T1/2, and preosteoblastic cell line, MC3T3 cells. And the downregulation of MAPK-ERK signaling using pharmacological inhibitor U0126 and RNA interference rescued osteoblast differentiation suppressed by TGF-ß, which was confirmed by Alkaline phosphatase (ALP) staining and alizarrn red staining, and the enhanced expression of osteogenesic markers. Western blotting analysis indicated that TGF-ß induced protein expression of E3 ubiquitin-protein ligase SMURF1, which contributed to the degradation of RUNX2 and SMAD1 as evidenced by SMURF1 inhibition using RNA interference and proteasome inhibitor MG132. Moreover, we observed that the expression of SMURF1 was decreased, while that of SMAD1 and RUNX2 increased by MAPK-ERK inhibitor U0126 in TGF-ß-treated differentiating preosteoblasts, suggesting that MAPK-ERK regulated the transcription of osteogenesis-related genes. Furthermore, a synergistic effect between U0126 and bone morphogenic protein (BMP)-2 on osteoblast differentiation and bone formation was observed both in cell cultures and experimental animals. In conclusion, our results revealed that TGF-ß inhibited osteoblastic differentiation by inducing the MAPK-ERK pathway which upregulated the expression of ubiquitin ligase SMURF1 and resulted in reduced presence of osteogenic proteins. In addition, the potentiation of BMP-2 on osteogenic activity by ERK1/2 inhibitor U0126 suggests that it may have potential clinical utility for promoting osteogenesis in bone fracture repair.
Assuntos
Diferenciação Celular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Células 3T3 , Animais , Proteína Morfogenética Óssea 2/farmacologia , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/enzimologia , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Proteína Smad1/metabolismo , Transfecção , Ubiquitina-Proteína Ligases/genética , Regulação para CimaRESUMO
BACKGROUND/AIMS: Cartilaginous endplate (CEP) degeneration is an important cause for intervertebral disc (IVD) degeneration that leads to low-back pain. The identification of compounds that may prevent CEP degeneration is of interest for the prevention of IVD degeneration. METHODS: Catabolic protease expression in the CEP of disc degeneration patients was first assessed. The toxicity, function and underlying mechanism of lycorine (LY) on CEP-derived chondrocytes degeneration were assessed in vitro by flow cytometry analysis and western blotting. The concentration and function of LY in rat-tail disc-degeneration models were also assessed by HPLC (High Performance Liquid Chromatography) quantification and histological analysis. RESULTS: In CEP cells, Interleukin (IL)-1ß upregulated the expression of matrix metalloproteinase (MMP)-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 that is critical for the degradation of cartilage extracellular matrix. Interestingly, LY suppressed the expression of these enzymes via the inhibition of nuclear factor-κB (NFκB) signalling and thus prevented IL-1ß-induced endplate cell degeneration in vitro. More importantly, LY also reduced the expression of MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5 in CEP and exerted a protective effect on both CEP and nucleus pulposus (NP) degeneration. In addition to its inhibitory effect on matrix-degrading protease expression, LY treatment also reduced positive regulators of proinflammatory cytokines, such as MIF, which can be secreted by CEP cells and subsequently target NP cells. CONCLUSION: LY could serve as a potential drug for treating IVD disease.
Assuntos
Alcaloides de Amaryllidaceae/farmacologia , Degeneração do Disco Intervertebral/prevenção & controle , Fenantridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteína ADAMTS4/genética , Proteína ADAMTS4/metabolismo , Proteína ADAMTS5/genética , Proteína ADAMTS5/metabolismo , Alcaloides de Amaryllidaceae/sangue , Alcaloides de Amaryllidaceae/uso terapêutico , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Humanos , Interleucina-1beta/metabolismo , Interleucina-1beta/farmacologia , Disco Intervertebral/metabolismo , Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/patologia , Masculino , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , NF-kappa B/metabolismo , Fenantridinas/sangue , Fenantridinas/uso terapêutico , Ratos , Ratos Sprague-DawleyRESUMO
Intervertebral disc degeneration causes low back pain.Interleukin-1ß (IL-1ß) is a well-known inflammatory mediator that is involved in disc degeneration but its molecular mechanisms on catabolic and anabolic events in nucleus pulposus (NP) cells remain unclear. Krüppel-like factor 5 (KLF5) is associated with inflammation and was previously shown to cause cartilage degradation. In this study, we revealed that KLF5 is involved in IL-1ß activated NF-kB cascade by enhancing both p65 phosphorylation and p65 acetylation. Moreover, the catabolic effect of KLF5 can be abolished by transforming growth factor-ß (TGF-ß) via promoting the proteasomal degradation of KLF5. Therefore, a KLF5 inhibitor ML264 was further proved to synergize with TGF-ß to attenuate IL-1ß-induced intervertebral disc degeneration. These results indicate the critical role of KLF5 in regulating intervertebral disc metabolism and suggest KLF5 inhibitor such as ML264 as potential compound for treatment of degenerative disc disease.
Assuntos
Acrilamidas/farmacologia , Óxidos S-Cíclicos/farmacologia , Interleucina-1beta/farmacologia , Fatores de Transcrição Kruppel-Like/metabolismo , Núcleo Pulposo/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Animais , Cartilagem/patologia , Células Cultivadas , Sinergismo Farmacológico , Humanos , Inflamação , Degeneração do Disco Intervertebral/metabolismo , Masculino , Núcleo Pulposo/citologia , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Ratos , Ratos Sprague-Dawley , Ubiquitina-Proteína Ligases/metabolismoRESUMO
PURPOSE: To evaluate the effects of leptin/leptin receptor (LepR) combined with mechanical stress on the development of ossification of the posterior longitudinal ligament (OPLL), which is a disease characterized by ectopic bone formation of the posterior longitudinal ligament (PLL) and can lead to radiculopathy and myelopathy. METHODS: Six human samples of the PLL were analyzed for the expression of leptin and LepR by RT-PCR and western blotting. PLL cells were stimulated with leptin and mechanical stress delivered via a Flexcell tension system, and osteogenic differentiation was evaluated by RT-PCR and western blotting analysis of osteogenic marker expression as well as by alkaline phosphatase (ALP) staining and alizarin red S staining. Activation of mitogen-activated protein kinase (MAPK), Janus kinase (JAK) 2-signal transducer, activator of transcription (STAT) 3 and phosphatidylinositol 3-kinase (PI3K)-Akt was evaluated by western blotting. RESULTS: Samples from the OPLL group had higher LepR mRNA and protein levels and lower leptin levels than those from healthy controls. Exposure to leptin and Flexcell increased the number of ALP-positive cells and calcium nodules in a dose-dependent manner; this effect was accompanied by upregulation of the osteogenic markers osteocalcin, runt-related transcription factor 2 (RUNX2) and osteopontin. Extracellular signal-regulated kinase, P38 MAPK, JAK2, STAT3, PI3K and Akt signaling, was also activated by the combined effects of leptin and mechanical stress. CONCLUSIONS: Leptin and LepR are differentially expressed in OPLL tissues, and the combined use of leptin/LepR and mechanical stress promotes osteogenic differentiation of PLL cells via MAPK, JAK2-STAT3 and PI3K/Akt signaling. These slides can be retrieved under Electronic Supplementary Material.
Assuntos
Leptina/metabolismo , Ossificação do Ligamento Longitudinal Posterior/metabolismo , Ossificação Heterotópica/metabolismo , Receptores para Leptina/metabolismo , Estresse Mecânico , Fosfatase Alcalina/metabolismo , Western Blotting , Técnicas de Cultura de Células , Diferenciação Celular , Humanos , Ligamentos Longitudinais/citologia , Ligamentos Longitudinais/metabolismo , Ligamentos Longitudinais/patologia , Ossificação do Ligamento Longitudinal Posterior/etiologia , Ossificação Heterotópica/etiologia , Reação em Cadeia da Polimerase em Tempo Real , Transdução de SinaisRESUMO
We studied the separation behaviour of binary granular particles in a vertically vibrated container. The final separation of the binary particle system exhibited the Brazil-Nut (BN) effect, though it was not complete. Particle convection occurred, and four different typical convection modes were observed when the frequency f changed from 20 Hz to 80 Hz at constant dimensionless acceleration Γ = 4πAf(2)/g. However, when Γ changed from 2 to 4 at constant f, the system's convection mode stayed almost the same. In our experiments, one type of particle generally moved much faster than the other, so the former was termed the 'convecting' particle, and the latter was termed the 'non-convecting' particle. To study the separation results qualitatively, we divided the system into vertical layers and calculated the mass distribution of the binary particles along the z axis. The results showed that when f increased at constant Γ or Γ decreased at constant f, the convecting particles, usually the smaller and lighter ones, distributed less to the top side and more to the bottom side of the container. Finally, to explain the experimental results, we derived a mass conservation equation for the convecting particles considering simultaneous convection and diffusion. The equation described the experimental results well. We also analysed the effects of f, Γ, diameter ratio, density ratio, etc., on the final separation results.
RESUMO
Toward the realization of efficient, durable, and sustainable fiber-based perovskite solar cells (fb-PSCs), a comprehensive optimization strategy focused on enhancing electron transport layer (ETL), perovskite (PVK) photovoltaic layer, and hole transport layer (HTL) is presented. A champion PCE of 10.66 % with 37.9 % relative enhancement over control has been achieved in the optimized fb-PSC. A significantly improved mechanical resilience and storage durability are also recorded. Decorating the SnO2 ETL with methylammonium lead triiodide (MAPbI3) strengthened the ETL/PVK interfacial integrity, and doping the MAPbI3 layer with the multi-functional polymer of PJ71 remarkably enhanced the PVK layer's crystallization quality, and effectively passivated the grain boundary defects. A CO2 pre-treatment of the spiro-OMeTAD HTL enhanced its hole conductivity. It is the synergetic combination of these methodologies that mutually contributed to the performance boost of the fb-PSC. The phenomenological model based on layer conductance shows that the PVK layer chiefly influences the device's anti-bending ability, followed by the ETL, and HTL the least impact. To further enhance the PCE of fb-PSCs, optimizing the interface and minimizing the stress-induced defects are essential. These measures, coupled with increasing carrier diffusion length and reducing surface recombination, are key to advancing the fb-PSC performance. An encapsulation with polyolefin elastomer substantially reduced the potential lead leakage of the device, and facilitated its eco-friendly application.
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Background: Osteoporosis is one of the most common bone diseases in middle-aged and elderly populations worldwide. The development of new drugs to treat the disease is a key focus of research. Current treatments for osteoporosis are mainly directed at promoting osteoblasts and inhibiting osteoclasts. However, there is currently no ideal approach for osteoporosis treatment. l-arginine is a semi-essential amino acid involved in a number of cellular processes, including nitric production, protein biosynthesis, and immune responses. We previously reported that l-arginine-derived compounds can play a regulatory role in bone homeostasis. Purpose: To investigate the specific effect of l-arginine on bone homeostasis. Methods: Mildly aged and ovariectomized mouse models were used to study the effects of l-arginine on osteogenesis and angiogenesis, assessed by micro-computed tomography and immunostaining of bone tissue. The effect of l-arginine on osteogenesis, angiogenesis, and adipogenesis was further studied in vitro using osteoblasts obtained from cranial cap bone, endothelial cells, and an adipogenic cell line. Specific methods to assess these processes included lipid staining, cell migration, tube-forming, and wound-healing assays. Protein and mRNA expression was determined for select biomarkers. Results: We found that l-arginine attenuated bone loss and promoted osteogenesis and angiogenesis. l-arginine increased the activity of vascular endothelial cells, whereas it inhibited adipogenesis in vitro. In addition, we found that l-arginine altered the expression of PINK1/Parkin and Bnip3 in the mitochondria of osteoblast-lineage and endothelial cells, thereby promoting mitophagy and protecting cells from ROS. Similarly, l-arginine treatment effectively ameliorated osteoporosis in an ovariectomized mouse model. Conclusion: l-arginine promotes angio-osteogenesis, and inhibits adipogenesis, effects mediated by the PINK1/Parkin- and Bnip3-mediated mitophagy. The Translational Potential of this Article: L-arginine supplementation may be an effective adjunct therapy in the treatment of osteoporosis.
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Ubiquitination is a reversible post-translational modification implicated in cell differentiation, homeostasis, and organ development. Several deubiquitinases (DUBs) decrease protein ubiquitination through the hydrolysis of ubiquitin linkages. However, the role of DUBs in bone resorption and formation is still unclear. In this study, we identified DUB ubiquitin-specific protease 7 (USP7) as a negative regulator of osteoclast formation. USP7 combines with tumor necrosis factor receptor-associated factor 6 (TRAF6) and inhibits its ubiquitination by impairing the Lys63-linked polyubiquitin chain. Such impairment leads to the suppression of receptor activator of NF-κB ligand (RANKL)-mediated nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) activation without affecting TRAF6 stability. USP7 also protects the stimulator of interferon genes (STING) against degradation, inducing interferon-ß (IFN-ß) expression in osteoclast formation, thereby inhibiting osteoclastogenesis cooperatively with the classical TRAF6 pathway. Furthermore, USP7 inhibition accelerates osteoclast differentiation and bone resorption both in vitro and in vivo. Contrarily, USP7 overexpression impairs osteoclast differentiation and bone resorption in vitro and in vivo. Additionally, in ovariectomy (OVX) mice, USP7 levels are lower than those in sham-operated mice, suggesting that USP7 plays a role in osteoporosis. Altogether, our data reveal the dual effect of USP7-mediated TRAF6 signal transduction and USP7-mediated protein degradation of STING in osteoclast formation.
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Osteoarthritis (OA) is a degenerative disease with a series of metabolic changes accompanied by many altered enzymes. Here, we report that the down-regulated dimethylarginine dimethylaminohydrolase-1 (DDAH1) is accompanied by increased asymmetric dimethylarginine (ADMA) in degenerated chondrocytes and in OA samples. Global or chondrocyte-conditional knockout of ADMA hydrolase DDAH1 accelerated OA development in mice. ADMA induces the degeneration and senescence of chondrocytes and reduces the extracellular matrix deposition, thereby accelerating OA progression. ADMA simultaneously binds to SOX9 and its deubiquitinating enzyme USP7, blocking the deubiquitination effects of USP7 on SOX9 and therefore leads to SOX9 degradation. The ADMA level in synovial fluids of patients with OA is increased and has predictive value for OA diagnosis with good sensitivity and specificity. Therefore, activating DDAH1 to reduce ADMA level might be a potential therapeutic strategy for OA treatment.
Assuntos
Arginina , Camundongos , Animais , Peptidase 7 Específica de Ubiquitina , Arginina/metabolismoRESUMO
Recently, global natural disasters have occurred frequently and caused serious damage. As an important urban space resource and public service facility, the reasonable planning and layout optimization of shelters is very important to reduce the disaster loss and improve the sustainable development of cities. Based on the review of location theory and models for shelter site selection, this study constructs a bi-level multi-objective location-allocation model, an accessibility, economy, and efficiency (AEE) model, based on sequential decision logic to maximize the economic sustainability and social utility. The model comprehensively considers factors such as the level of decision-making, the utilization efficiency, and capacity constraints of shelters. The gravity model is introduced to simulate the decision-making behavior of evacuees. A calculation example and its solution prove the high practicability and operability of the AEE model in an actual shelter site selection and construction investment, which can achieve the global optimization of evacuation time and the maximization of the use efficiency of the shelters under the financial constraints. It provides a scientific and effective decision-making method for the multi-objective location optimization problem of shelters.