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1.
Cell Mol Biol (Noisy-le-grand) ; 69(15): 174-179, 2023 Dec 31.
Artículo en Inglés | MEDLINE | ID: mdl-38279454

RESUMEN

Osteoarthritis (OA) is the most common joint disease in the elderly and is characterized by progressive and irreversible degeneration of articular cartilage, particularly cartilage loss and callus formation. This study would like to investigate the important role and the molecular mechanism of OA progression following interleukin 1ß (IL-1ß)-induced chondrocyte injury regulated by TXNIP. In this study, high-purity mouse chondrocytes were obtained by enzymatic two-step digestion for primary culture. Toluidine blue staining and type II collagen immunofluorescence were used to identify cells through histochemical staining after slide mounting. The relative expression of TXNIP was detected by immunohistochemical staining and qRT-PCR.Aiming at the shRNA sequence of the TXNIP gene, the shRNA expression vector was constructed and packaged with lentivirus to form the lentiviral vector shTXNIP. After inhibiting the expression of TXNIP by transfecting shTXNIP into normal mouse chondrocytes, the CCK-8 kit was used for detecting its effect on cell proliferation after transfection, and the effect on chondrocyte apoptosis was detected by flow cytometry. The staining kit was used to detect the effect of TXNIP knockout on chondrocyte aging, and the differential expression of TNF, IL-6, MMP3, MMP13, ADAMTS-5 and type II collagen genes in chondrocytes was detected by RT-PCR and Western-bolt. Western blot was used to detect the expression of upstream-related protein P-ERK, downstream-related protein NLRP3 and Caspase1 after inflammatory injury of mouse articular chondrocytes. Results showed that the expression level of TXNIP in chondrocytes induced by different concentrations of il-1ß was proportional to the concentration. After silencing TXNIP by shRNA, cell proliferation increased, chondrocyte apoptosis was weakened, and chondrocyte aging was weakened. The differential expression of genes such as TNF, IL-6, MMP3, MMP13, ADAMTS-5 and type II collagen and the differential expression of protein levels were relatively decreased. In addition, the expression of the upstream-related protein P-ERK did not change much when TXNIP was silenced, and the expression levels of the downstream-related proteins NLRP3 and Caspase1 were slightly reduced. In conclusion, silencing TXNIP can inhibit il-1ß-induced chondrocyte apoptosis and aging, and has a positive effect on cell proliferation. However, this study has not clarified the molecular mechanism involved in TXNIP and the process of its signaling expression pathway.


Asunto(s)
Cartílago Articular , Osteoartritis , Humanos , Ratones , Animales , Anciano , Condrocitos/metabolismo , Interleucina-1beta/farmacología , Interleucina-1beta/metabolismo , Metaloproteinasa 13 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/farmacología , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Interleucina-6/metabolismo , Inflamación/genética , Inflamación/metabolismo , Osteoartritis/genética , Osteoartritis/metabolismo , ARN Interferente Pequeño/metabolismo , Cartílago Articular/metabolismo , Apoptosis/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo
2.
Oncol Res ; 20(7): 319-26, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23879172

RESUMEN

The mTOR pathway is a central control of cell growth, proliferation, metabolism, and survival, and is deregulated in most cancers. Cancer cells are addicted to increased activity of mTOR kinase-mediated signaling pathways, leading to numerous inhibitors of mTOR signaling in preclinic and clinical trials for cancer therapy. Phosphorus-containing sirolimus (FIM-A), which targets mTOR signaling, inhibits cancer cell growth in vitro. Here we report that FIM-A reduces the angiogenesis and proliferation of osteosarcoma both in vitro and in vivo. In cultured osteosarcoma cell lines, FIM-A inhibited cell proliferation and arrested cells in the G1 phase of the cell cycle, accompanied with reduction of VEGF and HIF-1alpha. With in vivo mouse osteosarcoma xenografts, FIM-A treatment resulted in the inhibition of mTORC1 signaling as demonstrated by the decreased phosphorylation of p70S6K1 and 4E-BP1. Consistent with this finding, FIM-A significantly decreased the average tumor volume, nuclei staining of PCNA, and the number of intratumoral microvessels. Our data demonstrated that targeting mTORC1 by FIM-A inhibited the growth of osteosarcoma in vitro and in vivo, providing the basis for further development of FIM-A as a therapy for osteosarcoma patients.


Asunto(s)
Antineoplásicos/farmacología , Neoplasias Óseas/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Neovascularización Patológica/tratamiento farmacológico , Osteosarcoma/tratamiento farmacológico , Sirolimus/análogos & derivados , Inhibidores de la Angiogénesis/farmacología , Animales , Línea Celular Tumoral , Femenino , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Endogámicos BALB C , Complejos Multiproteicos/antagonistas & inhibidores , Fósforo , Sirolimus/química , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Ensayos Antitumor por Modelo de Xenoinjerto
3.
Medicine (Baltimore) ; 101(31): e29972, 2022 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-35945747

RESUMEN

The study aimed to evaluate the clinical significance of thyroid hormone-responsive (THRSP) and explore its relevant pathways in thyroid carcinoma (THCA). The gene expression data of THRSP were obtained and the prognostic significance of THRSP in THCA was analyzed through various bioinformatics databases. Then, the factors influencing THRSP mRNA expression were explored, and the function of THRSP in predicting the lymph node metastasis (LNM) stage was determined. We further performed the enrichment analysis and constructed a protein-protein interaction (PPI) network to examine potential regulatory pathways associated with THRSP. THRSP gene expression was significantly increased in THCA compared with the normal tissues. High THRSP mRNA expression had a favorable overall survival (OS) in THCA patients (P < .05). Additionally, the mRNA expression of THRSP was related to stage, histological subtype, and methylation among THCA patients (all P < .05). Besides, THRSP served as a potent predictor in discriminating the LNM stage of thyroid cancer patients. According to Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene set enrichment analysis (GSEA) on THRSP-associated genes, THRSP was positively related to metabolic pathways. The upregulation of THRSP predicted a good OS in THCA patients. Furthermore, THRSP might inhibit THCA progression through positive regulation of metabolism-associated pathways.


Asunto(s)
Neoplasias de la Tiroides , Biología Computacional , Humanos , Metástasis Linfática , Mapas de Interacción de Proteínas , ARN Mensajero/metabolismo , Hormonas Tiroideas/metabolismo , Neoplasias de la Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/genética
4.
Front Pharmacol ; 12: 726361, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34566650

RESUMEN

Osteolytic bone disorders are characterized by an overall reduction in bone mineral density which enhances bone ductility and vulnerability to fractures. This disorder is primarily associated with superabundant osteoclast formation and bone resorption activity. Nicorandil (NIC) is a vasodilatory anti-anginal drug with ATP-dependent potassium (KATP) channel openings. However, NIC is adopted to manage adverse cardiovascular and coronary events. Recent research has demonstrated that NIC also possesses anti-inflammatory peculiarity through the regulation of p38 MAPK and NF-κB signaling pathways. Both MAPK and NF-κB signaling pathways play pivotal roles in RANKL-induced osteoclast formation and bone resorption function. Herein, we hypothesized that NIC may exert potential biological effects against osteoclasts, and revealed that NIC dose-dependently suppressed bone marrow macrophage (BMM) precursors to differentiate into TRAP + multinucleated osteoclasts in vitro. Furthermore, osteoclast resorption assays demonstrated anti-resorptive effects exhibited by NIC. NIC had no impact on osteoblast differentiation or mineralization function. Based on Biochemical analyses, NIC relieved RANKL-induced ERK, NF-κB and p38 MAPK signaling without noticeable effects on JNK MAPK activation. However, the attenuation of NF-κB and p38 MAPK activation was sufficient to hamper the downstream induction of c-Fos and NFATc1 expression. Meanwhile, NIC administration markedly protected mice from ovariectomy (OVX)-induced bone loss through in vivo inhibition of osteoclast formation and bone resorption activity. Collectively, this work demonstrated the potential of NIC in the management of osteolytic bone disorders mediated by osteoclasts.

5.
Stem Cell Res Ther ; 12(1): 605, 2021 12 20.
Artículo en Inglés | MEDLINE | ID: mdl-34930462

RESUMEN

BACKGROUND: Enhancer of zeste homolog 2 (EZH2) is a novel oncogene that can specifically trimethylate the histone H3 lysine 27 (H3K27me3) to transcriptionally inhibit the expression of downstream tumor-suppressing genes. As a small molecular inhibitor of EZH2, 3-Deazaneplanocin (DZNep) has been widely studied due to the role of tumor suppression. With the roles of epigenetic regulation of bone cells emerged in past decades, the property and molecular mechanism of DZNep on enhancing osteogenesis had been reported and attracted a great deal of attention recently. This study aims to elucidate the role of DZNep on EZH2-H3K27me3 axis and downstream factors during both osteoclasts and osteoblasts formation and the therapeutic possibility of DZNep on bone defect healing. METHODS: Bone marrow-derived macrophages (BMMs) cells were cultured, and their responsiveness to DZNep was evaluated by cell counting kit-8, TRAP staining assay, bone resorption assay, podosome actin belt. Bone marrow-derived mesenchymal stem cells (BMSC) were cultured and their responsiveness to DZNep was evaluated by cell counting kit-8, ALP and AR staining assay. The expression of nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), Wnt signaling pathway was determined by qPCR and western blotting. Mouse bone defect models were created, rescued by DZNep injection, and the effectiveness was evaluated by X-ray and micro-CT and histological staining. RESULTS: Consistent with the previous study that DZNep enhances osteogenesis via Wnt family member 1(Wnt1), Wnt6, and Wnt10a, our results showed that DZNep also promotes osteoblasts differentiation and mineralization through the EZH2-H3K27me3-Wnt4 axis. Furthermore, we identified that DZNep promoted the receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclast formation via facilitating the phosphorylation of IKKα/ß, IκB, and subsequently NF-κB nuclear translocation, which credit to the EZH2-H3K27me3-Foxc1 axis. More importantly, the enhanced osteogenesis and osteoclastogenesis result in accelerated mice bone defect healing in vivo. CONCLUSION: DZNep targeting EZH2-H3K27me3 axis facilitated the healing of mice bone defect via simultaneously enhancing osteoclastic bone resorption and promoting osteoblastic bone formation.


Asunto(s)
Resorción Ósea , Osteogénesis , Adenosina/análogos & derivados , Animales , Resorción Ósea/patología , Diferenciación Celular , Epigénesis Genética , Ratones , FN-kappa B/metabolismo , Osteoclastos , Osteogénesis/genética , Ligando RANK/genética , Ligando RANK/farmacología
7.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi ; 27(11): 1380-5, 2013 Nov.
Artículo en Zh | MEDLINE | ID: mdl-24501901

RESUMEN

OBJECTIVE: To construct recombinant lentiviral vectors of porcine bone morphogenetic protein 2 (BMP-2) gene and to detect BMP-2 gene activity and bone marrow mesenchymal stem cells (BMSCs) osteogenetic differentiation so as to lay a foundation of the further study of osteochondral tissue engineering. METHODS: BMSCs were isolated from bone marrow of 2-month-old Bama miniature porcines (weighing, 15 kg), and the 2nd generation of BMSCs were harvested for experiments. The porcine BMP-2 gene lentiviral vector was constructed by recombinant DNA technology and was used to transfect BMSCs at multiplicity of infection (MOI) of 10, 25, 50, 100, and 200, then the optimal value of MOI was determined by fluorescent microscope and inverted phase contrast microscope. BMSCs transfected by BMP-2 recombinant lentiviral vectors served as experimental group (BMP-2 vector group); BMSCs transfected by empty vector (empty vector group), and non-transfected BMSCs (non-transfection group) were used as control groups. RT-PCR, immunohistochemistry staining, and Western blot were performed to detect the expressions of BMP-2 mRNA and protein. Then the BMSCs osteogenesis was detected by alkaline phosphatase (ALP) staining, ALP activities, and Alizarin red staining. RESULTS: The recombinant lentiviral vectors of porcine BMP-2 gene was successfully constructed and identified by RT-PCR and gene sequencing, and BMSCs were successfully transfected by BMP-2 recombinant lentiviral vectors. Green fluorescent protein could be seen in the transfected BMSCs, especially at MOI of 100 with best expression. The immunohistochemistry staining and Western blot showed that BMSCs transfected by BMP-2 recombinant lentiviral vectors could express BMP-2 protein continuously and stably at a high level. After cultivation of 2 weeks, the expression of ALP and the form of calcium nodules were observed. CONCLUSION: The porcine BMP-2 gene lentiviral vector is successfully constructed and transfected into the BMSCs, which can express BMP-2 gene and protein continuously and stably at a high level and induce BMSCs differentiation into osteoblasts.


Asunto(s)
Adenoviridae/genética , Proteína Morfogenética Ósea 2/genética , Diferenciación Celular , Vectores Genéticos/genética , Células Madre Mesenquimatosas/citología , Osteogénesis , Factor de Crecimiento Transformador beta/genética , Fosfatasa Alcalina/metabolismo , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Proteína Morfogenética Ósea 2/metabolismo , Proliferación Celular , Células Cultivadas , Femenino , Expresión Génica , Terapia Genética , Masculino , Células Madre Mesenquimatosas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Porcinos , Ingeniería de Tejidos/métodos , Transfección , Factor de Crecimiento Transformador beta/metabolismo
8.
Artículo en Zh | MEDLINE | ID: mdl-23012939

RESUMEN

OBJECTIVE: To prepare collagen-chitosan/nano-hydroxyapatite-collagen-polylactic acid (Col-CS/nHAC-PLA) biomimetic scaffold and to examine its biocompatibility so as to lay the foundation for its application on the treatment of osteochondral defect. METHODS: PLA was dissolved in dioxane for getting final concentration of 8%, and the nHAC power was added at a weight ratio of nHAC to PLA, 1 : 1. The solution was poured into a mold and frozen. CS and Col were dissolved in 2% acetum for getting the final concentrations of 2% and 1% respectively, then compounded at a weight ratio of CS to Col, 20 : 1. The solution was poured into the frozen mold containing nHAC-PLA, and then biomimetic osteochondral scaffold of Col-CS/nHAC-PLA was prepared by freeze-drying. Acute systemic toxicity test, intracutaneous stimulation test, pyrogen test, hemolysis test, cytotoxicity test, and bone implant test were performed to evaluate its biocompatibility. RESULTS: Col-CS/nHAC-PLA had no acute systemic toxicity. Primary irritation index was 0, indicating that Col-CS/nHAC-PLA had very slight skin irritation. In pyrogen test, the increasing temperature of each rabbit was less than 0.6 degrees C, and the increasing temperature sum of 3 rabbits was less than 1.3 degrees C, which was consistent with the evaluation criteria. Hemolytic rate of Col-CS/nHAC-PLA was 1.38% (far less than 5%). The toxicity grade of Col-CS/nHAC-PLA was classified as grade I. Bone implant test showed that Col-CS/nHAC-PLA had good biocompatibility with the surrounding tissue. CONCLUSION: Col-CS/nHAC-PLA scaffold has good biocompatibility, which can be used as an alternative osteochondral scaffold.


Asunto(s)
Quitosano/química , Colágeno/química , Durapatita/química , Ensayo de Materiales/métodos , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Animales , Materiales Biocompatibles , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Quitosano/farmacología , Colágeno/farmacología , Durapatita/farmacología , Femenino , Ácido Láctico/química , Ácido Láctico/farmacología , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones , Microscopía Electrónica de Rastreo , Poliésteres , Polímeros/química , Polímeros/farmacología , Conejos , Pruebas de Toxicidad Aguda/métodos
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