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1.
Adv Clin Exp Med ; 28(10): 1293-1300, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31538414

RESUMO

BACKGROUND: MicroRNA (miRNA) is a kind of non-coding small RNA with a negative regulating function. Some miRNAs play a role in regulating the differentiation and function of osteoblasts, chondrocytes and osteoclasts. OBJECTIVES: In this study, we analyzed the role of miR-29a and dickkopf-1 (DKK-1) in osteoblast differentiation. MATERIAL AND METHODS: Specimens were collected from the surgical resection of pathological ankylosing spondylitis (AS) tissue and some normal tissues. The expression of miR-29a, DKK-1 and ß-catenin in normal and AS tissues were detected with real-time polymerase chain reaction (RT-PCR) and western blotting. Cell proliferation was detected with a Cell Counting Kit-8, cell migration and invasion were determined using a Transwell system and cell apoptosis was analyzed with flow cytometry. The luciferase reporter gene plasmid pGL3-DKK-1 and a point-mutation of the luciferase reporter gene plasmid mut-pGL3-DKK-1 were constructed. RESULTS: It was found that miR-29a could promote the proliferation of hFOB1.19 cells, while DKK-1 inhibited their proliferation. Also, miR-29a was able to inhibit the apoptosis of hFOB1.19 cells, while DKK-1 was able to promote the apoptosis of hFOB1.19 cells. When it comes to the invasion and migration of hFOB1.19 cells, miR-29a was found to promote it, while DKK-1 did not. CONCLUSIONS: These findings will lead to a better understanding of the proliferation and differentiation of osteoblasts and will provide new insights for the treatment of this disease.


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , MicroRNAs/metabolismo , Osteoblastos/metabolismo , Via de Sinalização Wnt/fisiologia , Apoptose/fisiologia , Proliferação de Células , Humanos , MicroRNAs/genética , Proteínas Wnt/metabolismo , beta Catenina/metabolismo
2.
Eur J Endocrinol ; 181(5): 525-537, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31536965

RESUMO

Objective: To evaluate the effect of insulin resistance in obesity on the expression in whole blood of mRNA and miRNA affecting bone homeostasis as well as to estimate the influence of oral glucose load (OGTT) on serum osteocalcin concentration in obese individuals with and without insulin resistance. Design: Cross-sectional study. Methods: Carboxylated (cOC), undercarboxylated (ucOC) and total osteocalcin were measured by ELISA in the serum of obese subjects with insulin resistance (n = 41) and obese subjects without insulin resistance (n = 41) (control group) during OGTT. Analysis of gene expression (microarray) and miRNAs (real-time PCR) was performed in venous blood (representating samples) collected before OGTT from obese with insulin resistance and controls. Results: Obese subjects with insulin resistance (higher HOMA-IR and lower oral glucose insulin sensitivity index) presented significantly increased expression of WNT signalling inhibitors (DKK1, DKK2, SOST, SFRP1) and downregulation of the key factor in WNT signalling - ß catenin participating in osteoblasts differentiation. Expression of miRNA involved in osteoblastogenesis was also inhibited (miR-29b, miR-181a, miR-210, miR-324-3p). During OGTT, contrary to the control group, subjects with insulin resistance presented suppression of cOC and total OC decrease after 1 and 2 h of oral glucose load. Conclusions: Obese subjects with insulin resistance may have defects in osteoblastogenesis that was demonstrated via key signalling molecules mRNA downregulation, and increased expression of WNT antagonists as well as inhibition of expression of miRNA participating in the regulation of osteoblast differentiation. Disturbed osteoblastogenesis in insulin-resistant subjects results in the suppression of blood carboxylated and total osteocalcin decrease during OGTT.


Assuntos
Remodelação Óssea/fisiologia , Resistência à Insulina/fisiologia , MicroRNAs/sangue , RNA Mensageiro/sangue , Adulto , Estudos Transversais , Ensaio de Imunoadsorção Enzimática , Feminino , Teste de Tolerância a Glucose , Humanos , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , Obesidade/etiologia , Obesidade/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Via de Sinalização Wnt/fisiologia
3.
J Agric Food Chem ; 67(37): 10285-10295, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31443611

RESUMO

Fluoride (F) is capable of promoting abnormal proliferation and differentiation in primary cultured mouse osteoblasts (OB cells), although the underlying mechanism responsible remains rare. This study aimed to explore the roles of wingless and INT-1 (Wnt) signaling pathways and screen appropriate doses of calcium (Ca2+) to alleviate the sodium fluoride (NaF)-induced OB cell toxicity. For this, we evaluated the effect of dickkopf-related protein 1 (DKK1) and Ca2+ on mRNA levels of wingless/integrated 3a (Wnt3a), low-density lipoprotein receptor-related protein 5 (LRP5), dishevelled 1 (Dv1), glycogen synthase kinase 3ß (GSK3ß), ß-catenin, lymphoid enhancer binding factor 1 (LEF1), and cellular myelocytomatosis oncogene (cMYC), as well as Ccnd1 (Cyclin D1) in OB cells challenged with 10-6 mol/L NaF for 24 h. The demonstrated data showed that F significantly increased the OB cell proliferation rate. Ectogenic 0.5 mg/L DKK1 significantly inhibited the proliferation of OB cells induced by F. The mRNA expression levels of Wnt3a, LRP5, Dv1, LEF1, ß-catenin, cMYC, and Ccnd1 were significantly increased in the F group, while significantly decreased in the 10-6 mol/L NaF + 0.5 mg/L DKK1 (FY) group. The mRNA expression levels of Wnt3a, LRP5, ß-catenin, and cMYC were significantly decreased in the 10-6 mol/L NaF + 2 mmol/L CaCl2 (F+CaII) group. The protein expression levels of Wnt3a, Cyclin D1, cMYC, and ß-catenin were significantly increased in the F group, whereas they were decreased in the F+CaII group. However, the mRNA and protein expression levels of GSK3ß were significantly decreased in the F group while significantly increased in the F+CaII group. In summary, F activated the canonical Wnt/ß-catenin pathway and changed the related gene expression and ß-catenin protein location in OB cells, promoting cell proliferation. Ca2+ supplementation (2 mmol/L) reversed the expression levels of genes and proteins related to the canonical Wnt/ß-catenin pathway.


Assuntos
Cálcio/metabolismo , Fluoretos/efeitos adversos , Osteoblastos/efeitos dos fármacos , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Suplementos Nutricionais/análise , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Masculino , Camundongos , Osteoblastos/classificação , Osteoblastos/metabolismo , Proteínas Wnt/genética , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/genética
4.
Shanghai Kou Qiang Yi Xue ; 28(2): 128-132, 2019.
Artigo em Chinês | MEDLINE | ID: mdl-31384895

RESUMO

PURPOSE: To investigate the effect of lipopolysaccharides(LPS) extracted from Porphyromonas endodontalis(P.e) on the expression of macrophage inflammatory protein-2 (MIP-2) mRNA and protein levels in MC3T3-E1 cells and the influence of resveratrol on the expression of MIP-2 protein in P.e-LPS induced cells. METHODS: MC3T3-E1 cells were treated with different concentrations of P.e-LPS(0-50 mg/L) and 20 mg/L P.e-LPS for different time (0-48 h). The expression of MIP-2 mRNA and protein was detected by real-time RT-PCR and enzyme linked immunosorbent assay (ELISA). MC3T3-E1 cells were pretreated with resveratrol for 1 h in the presence of 20 mg/L P.e-LPS for 24 h,which was detected by ELISA. Statistical analysis was performed using one-way ANOVA and Dunnett t test with SPSS 13.0 software package. RESULTS: Treatment of MC3T3-El cells with different concentrations of P.e-LPS(0-50 mg/L) caused a significantly increase in MIP-2 mRNA and protein expression in dose-dependent manners.The expression of MIP-2 protein increased from (41.86±2.49) ng/L to (3126.74±158.30) ng/L, and the difference was significant(P<0.05). In the observation time (0-48 h), the impact of 20 mg/L P.e-LPS on induction of MIP-2 in MC3T3-El cells exhibited a time-dependent manner. At 48 h, the maximal induction of MIP-2 protein expression was (2102.55±123.27) ng/L(P<0.01). Incubation of cells with 10 µmol/L resveratrol for 1h significantly decreased the expression of MIP-2 protein from (1805.33±67.54) ng/L to(813.82±47.21) ng/L, and the difference was significant(P<0.05). CONCLUSIONS: The results suggest that P.e-LPS may mediate MIP-2 expression in MC3T3-E1 cells, and resveratrol has a significant inhibitory effect on this process.


Assuntos
Quimiocina CXCL2 , Lipopolissacarídeos , Osteoblastos , Resveratrol , Animais , Quimiocina CXCL2/efeitos dos fármacos , Quimiocina CXCL2/metabolismo , Camundongos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Porphyromonas endodontalis , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Resveratrol/farmacologia
5.
Bone Joint J ; 101-B(8): 915-921, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31362550

RESUMO

AIMS: Altered alignment and biomechanics are thought to contribute to the progression of osteoarthritis (OA) in the native compartments after medial unicompartmental knee arthroplasty (UKA). The aim of this study was to evaluate the bone activity and remodelling in the lateral tibiofemoral and patellofemoral compartment after medial mobile-bearing UKA. PATIENTS AND METHODS: In total, 24 patients (nine female, 15 male) with 25 medial Oxford UKAs (13 left, 12 right) were prospectively followed with sequential 99mTc-hydroxymethane diphosphonate single photon emission CT (SPECT)/CT preoperatively and at one and two years postoperatively, along with standard radiographs and clinical outcome scores. The mean patient age was 62 years (40 to 78) and the mean body mass index (BMI) was 29.7 kg/m2 (23.6 to 42.2). Mean osteoblastic activity was evaluated using a tracer localization scheme with volumes of interest (VOIs). Normalized mean tracer values were calculated as the ratio between the mean tracer activity in a VOI and background activity in the femoral diaphysis. RESULTS: Significant reduction of normalized tracer activity was observed one year postoperatively in tibial and femoral VOIs adjacent to the joint line in the lateral compartment. Patellar VOIs and remaining femoral VOIs demonstrated a significant, diminished normalized tracer activity at final follow-up. CONCLUSION: The osteoblastic bone activity in the native compartments decreased significantly after treatment of medial end-stage OA with a UKA, implying reduced stress to the subchondral bone in the retained compartments after a UKA. Cite this article: Bone Joint J 2019;101-B:915-921.


Assuntos
Artroplastia do Joelho/efeitos adversos , Fêmur/metabolismo , Hemiartroplastia/efeitos adversos , Articulação do Joelho/metabolismo , Osteoartrite do Joelho/cirurgia , Osteoblastos/metabolismo , Tíbia/metabolismo , Adulto , Idoso , Artroplastia do Joelho/instrumentação , Artroplastia do Joelho/métodos , Biomarcadores/metabolismo , Remodelação Óssea , Feminino , Fêmur/diagnóstico por imagem , Seguimentos , Hemiartroplastia/instrumentação , Hemiartroplastia/métodos , Humanos , Articulação do Joelho/diagnóstico por imagem , Articulação do Joelho/cirurgia , Prótese do Joelho , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/fisiopatologia , Estudos Prospectivos , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único , Tíbia/diagnóstico por imagem , Resultado do Tratamento
6.
Pan Afr Med J ; 33: 37, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31384352

RESUMO

Introduction: Osteoporosis is characterized by low bone mass and density, as well as change in microarchitecture of bone tissue leading to decreased bone strength. In vitro research shows nicotine can increase osteoblast activity and proliferation, also suppress osteoclast activity. Therefore we explore nicotine anti-resorptive property by in vivo true experimental and randomized posttest only controlled group research that was conducted in 18-20 weeks old Rattus norvegicus. Methods: Twenty-five female rats were divided into five groups, with 5 rats per group. The first group represented normal rats (Sham), while the second to fifth group underwent bilateral ovariectomy. The second group serves as positive control group (ovariectomy-only/OVX). The third to fifth group serve as dose 1 (P1-0.25mg/kg), dose 2 (P2-0.5 mg/kg), and Dose 3 (P3-0.75 mg/kg) treatment group receiving daily per-oral nicotine for 28 days, started 3 weeks post- ovariectomy. After 28 days treatment, the serum was checked. Results: Nicotine has dose-dependent manner on serum osteocalcin and serum DPD level. Level of osteocalcin in P2 group was significantly lower (Mann-Whitney, p = 0.008) compared to OVX group (59.4% lower). Level of DPD in all group was not significantly different (ANOVA, p < 0.05) but shows lowest level in P2 group. For serum calcitonin level, there's no significant different between groups. Conclusion: Nicotine at right low-dose might be able to inhibit osteoclast activity, thus open a possibility of anti-resorptive property of nicotine.


Assuntos
Densidade Óssea/efeitos dos fármacos , Nicotina/farmacologia , Osteoclastos/efeitos dos fármacos , Osteoporose/prevenção & controle , Aminoácidos/sangue , Animais , Biomarcadores/metabolismo , Remodelação Óssea/efeitos dos fármacos , Osso e Ossos/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Nicotina/administração & dosagem , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteocalcina/sangue , Osteoclastos/metabolismo , Osteoporose/patologia , Ovariectomia , Ratos , Ratos Wistar
7.
Yonsei Med J ; 60(8): 751-759, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31347330

RESUMO

PURPOSE: This study aimed to explore the effects and mechanisms of long non-coding RNA (lncRNA) anti-differentiation non-coding RNA (ANCR) on the osteogenesis of osteoblast cells in postmenopausal osteoporosis (PMOP). MATERIALS AND METHODS: Mice models of PMOP were established. ANCR expression and intracellular calcium ions were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and laser confocal microscopy, respectively. ANCR was silenced in osteoblast cells from PMOP mice by the transfection of siRNA-ANCR (si-ANCR). The proliferation and apoptosis of osteoblast cells was analyzed by MTT and flow cytometry, respectively. Alkaline phosphatase (ALP) activity and calcium nodules were examined by ALP and alizarin red staining assay, respectively. The expression of enhancer of zeste homolog 2 (EZH2), runt related transcription factor 2 (RUNX2), and OSTERIX was detected by qRT-PCR and Western blot. Furthermore, an osteogenesis model was constructed in mice, and osteoid formation was observed by hematoxylin-eosin (HE) staining. The interaction between lncRNA-ANCR and EZH2 was further identified by RNA pull-down assay. RESULTS: ANCR expression and intracellular calcium ions were increased in PMOP mice. Si-ANCR significantly increased the proliferation, ALP activity, calcium deposition of osteoblast cells and decreased apoptosis. ANCR and EZH2 were down-regulated by si-ANCR, while RUNX2 and OSTERIX were upregulated. Si-ANCR also promoted osteoid formation in mice treated with hydroxyapatite-tricalcium phosphate. In addition, ANCR specifically bound to EZH2. CONCLUSION: Silencing ANCR promotes the osteogenesis of PMOP osteoblast cells. The specific binding of ANCR with EZH2 suppressed RUNX2, thereby inhibiting osteogenesis.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Inativação Gênica , Osteoblastos/metabolismo , Osteogênese , Osteoporose Pós-Menopausa/genética , Osteoporose Pós-Menopausa/patologia , RNA Longo não Codificante/genética , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Osteoblastos/patologia , Osteogênese/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos , Fator de Transcrição Sp7/metabolismo
8.
Chem Biol Interact ; 310: 108748, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31306638

RESUMO

BACKGROUND: Fracture healing is a very important process after fracture. Tanshinone IIA (Tan IIA) has been reported to possess beneficial impact on osteoblasts growth. Our study investigated the effects of Tan IIA on fracture healing. METHODS: In vitro, mouse pre-osteoblast MC3T3-E1 cells were treated with Tan IIA. Then, the protein levels of Runx2, Osx, Collagen I, JNK and c-Jun, alkaline phosphatase (ALP) activity and calcium deposition were detected, respectively. Furthermore, the roles of microRNA-424 (miR-424) and Bone morphogenetic protein 2 (BMP-2) in Tan IIA-caused MC3T3-E1 cell differentiation were probed. In vivo, mice open osteotomy at femur diaphysis model was established. The callus area, callus intensity, low-density bone volume/callus total volume (BV1/TV), tissue mineral density (TMD) and bone mineral density (BMD) were tested. RESULTS: In vitro, Tan IIA promoted MC3T3-E1 cell differentiation via increasing the Runx2, Osx and collagen I expression, along with enhancing ALP activity and calcium deposition. In addition, Tan IIA activated JNK pathway in MC3T3-E1 cells, while inhibition of JNK pathway mitigated the Tan IIA-caused MC3T3-E1 cell differentiation. Moreover, Tan IIA declined the miR-424 expression in MC3T3-E1 cells. Overexpression of miR-424 also weakened the Tan IIA-caused MC3T3-E1 cell differentiation. BMP-2 was a target gene of miR-424. BMP-2 silence reversed the Tan IIA-caused activation of JNK pathway. In vivo, Tan IIA increased the callus area, callus intensity, BV1/TV, TMD and BMD. CONCLUSION: Tan IIA could promote fracture healing. In vitro, Tan IIA promoted MC3T3-E1 cell differentiation might be via down-regulating miR-424, up-regulating BMP-2 and then activating JNK pathway.


Assuntos
Diterpenos de Abietano/farmacologia , Consolidação da Fratura/efeitos dos fármacos , Animais , Proteína Morfogenética Óssea 2/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Diterpenos de Abietano/uso terapêutico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , MicroRNAs/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/patologia
9.
Cell Biochem Funct ; 37(6): 432-442, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31318458

RESUMO

Advanced glycation end products (AGEs) are naturally occurring molecules that start to accumulate from embryonic developmental stages and form as part of normal ageing. When reducing sugars interact with and modify proteins or lipids, AGE production occurs. AGE formation accelerates in chronic hyperglycemic conditions, and high AGE levels have been associated with the pathogenesis of various diseases. In addition, enhanced levels of AGEs have been linked to delayed wound healing as seen in patients with diabetes mellitus. Research has provided numerous ways in which a high AGE concentration results in impaired wound healing, including oxidative stress, structural and functional changes to proteins important in wound repair, an enhanced inflammatory response by activation of transcription factors, and possible exaggerated apoptosis of cells necessary to the wound repair process. Apoptosis is a naturally occurring cell death process that is significant for normal tissue functioning and plays an important role in wound repair by preventing a prolonged inflammatory response and excessive scar formation. Abnormal apoptosis affects wound healing, resulting in slow healing wounds. This review will summarize the role of AGEs in wound healing, focusing on the mechanisms by which AGEs lead to apoptosis in various cell types. The review provides the way forward for medical research and molecular studies as it focuses on the mechanisms by which AGEs induce apoptosis in various cell types, including fibroblasts, osteoblasts, neuronal cells, and endothelial cells. Reviewing the mechanisms of AGE-linked apoptosis is important in understanding the impact of high AGE levels in delayed wound healing in diabetic patients due to abnormal apoptosis of cells necessary to the wound healing process.


Assuntos
Apoptose , Produtos Finais de Glicação Avançada/metabolismo , Cicatrização , Animais , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Neurônios/metabolismo , Neurônios/patologia , Osteoblastos/metabolismo , Osteoblastos/patologia
10.
Cell Biochem Funct ; 37(6): 400-407, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31328801

RESUMO

There are two types of bisphosphonates (BPs), nitrogen-containing (N-BPs) and those free from nitrogen (non-N-BPs). Although N-BPs show greater inhibition of bone resorption than non-N-BPs, their effects are likely accompanied with inflammation, which non-N-BPs mitigate. We examined the competitive effects of zoledronate (ZOL), an N-BP, and etidronate (ETI), a non-N-BP, in osteoblasts. ZOL, but not ETI, markedly reduced alkaline phosphatase activity and cell viability in osteoblastic MC3T3-E1 and Saos2 cells, while that inhibition was relieved by simultaneous administration of ETI, possibly because of competition with ZOL for cellular uptake. However, phosphonoformate, an inhibitor of the phosphonate transporters SLC20A and SLC34A, did not mitigate the reducing effects of ZOL, suggesting that those transporters are not involved in BP uptake in osteoblastic cells. Additionally, ZOL reduced fibroblastic NIH3T3 and C3H10T1/2 cell viability, which was relieved by administration of both ETI and phosphonoformate. Transporter gene expression levels were significantly lower in osteoblasts as compared with fibroblasts, which may account for the distinct effects of phosphonoformate with different cell types. Together, our results suggest existence of a common uptake route of N-BPs and non-N-BPs into osteoblastic cells that is unrelated to the SLC20A and SLC34A families. SIGNIFICANCE OF THE STUDY: N-BP ZOL was shown to suppress differentiation and viability of osteoblasts. ZOL-induced cell viability suppression was also observed in fibroblasts, which was markedly relieved by addition of the non-N-BP ETI. Additionally, mitigation of the effects of ZOL was achieved with phosphonoformate, a sodium-phosphate cotransporter inhibitor, in fibroblastic cells but not osteoblasts. Expression levels of SLC20A and SLC34A family genes were significantly lower in osteoblasts as compared with fibroblasts. These observations suggest that incorporation of N-BPs and non-N-BPs in osteoblasts is mediated via common transporters that appear to be distinct from SLC20A and 34A, which operate in fibroblasts.


Assuntos
Difosfonatos/farmacologia , Osteoblastos/efeitos dos fármacos , Proteínas Cotransportadoras de Sódio-Fosfato/antagonistas & inibidores , Células 3T3 , Animais , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Camundongos , Osteoblastos/citologia , Osteoblastos/metabolismo , Relação Estrutura-Atividade
11.
Nat Commun ; 10(1): 2958, 2019 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-31273195

RESUMO

RNAi-based bone anabolic gene therapy has demonstrated initial success, but many practical challenges are still unmet. Here, we demonstrate that a recombinant adeno-associated virus 9 (rAAV9) is highly effective for transducing osteoblast lineage cells in the bone. The adaptor protein Schnurri-3 (SHN3) is a promising therapeutic target for osteoporosis, as deletion of shn3 prevents bone loss in osteoporotic mice and short-term inhibition of shn3 in adult mice increases bone mass. Accordingly, systemic and direct joint administration of an rAAV9 vector carrying an artificial-microRNA that targets shn3 (rAAV9-amiR-shn3) in mice markedly enhanced bone formation via augmented osteoblast activity. Additionally, systemic delivery of rAAV9-amiR-shn3 in osteoporotic mice counteracted bone loss and enhanced bone mechanical properties. Finally, we rationally designed a capsid that exhibits improved specificity to bone by grafting the bone-targeting peptide motif (AspSerSer)6 onto the AAV9-VP2 capsid protein. Collectively, our results identify a bone-targeting rAAV-mediated gene therapy for osteoporosis.


Assuntos
Reabsorção Óssea/complicações , Reabsorção Óssea/prevenção & controle , Osso e Ossos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dependovirus/metabolismo , Inativação Gênica , Osteoporose/complicações , Animais , Reabsorção Óssea/patologia , Osso e Ossos/virologia , Capsídeo/metabolismo , Cartilagem/virologia , Modelos Animais de Doenças , Deleção de Genes , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Sorogrupo
12.
Biochemistry (Mosc) ; 84(6): 686-692, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31238868

RESUMO

Resveratrol has been shown to stimulate differentiation of osteoblastic MC3T3-E1 cells in vitro; however, the mechanisms underlying the anabolic effect of resveratrol on osteoblasts remain largely unknown. Our study was aimed to investigate the molecular mechanism of resveratrol-induced differentiation of MC3T3-E1 cells. MC3T3-E1 cells were treated for 8 days with different concentrations of resveratrol (10-8-10-6 M) and 10-6 M cyclosporine A (CsA), a specific inhibitor of the calcineurin/NFAT pathway. According to the results of pilot studies of cell proliferation and alkaline phosphatase activity, 10-7 M concentration of resveratrol was used in subsequent experiments. The levels of mRNA expression of the osteosis-related genes CaN, NFATc1, and Runx2 were analyzed by real-time RT-PCR; the levels of the corresponding proteins were estimated by Western blot analysis. Resveratrol upregulated expression of the CaN, NFATc1, and Runx2 genes at both mRNA and protein levels compared to the control group (p < 0.05), while CsA reduced the effects of resveratrol (p < 0.05). Using immunohistochemical staining, we showed that resveratrol induced NFATc1 accumulation in the cell nuclei, and treatment with CsA inhibited resveratrol-mediated induction of NFATc1, suggesting that the calcineurin/NFATc1 signaling pathway plays an important role in the regulatory effect of resveratrol on osteoblasts.


Assuntos
Antioxidantes/farmacologia , Calcineurina/metabolismo , Diferenciação Celular/efeitos dos fármacos , Fatores de Transcrição NFATC/metabolismo , Osteoblastos/efeitos dos fármacos , Resveratrol/farmacologia , Células 3T3 , Animais , Calcineurina/genética , Proliferação de Células/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Camundongos , Fatores de Transcrição NFATC/genética , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real
13.
Biotechnol Appl Biochem ; 66(4): 690-697, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31173404

RESUMO

Osteoporosis is one of the clinical complications of long-term treatment with glucocorticoids (GCs), characterized by systemic damage of bone mass and osteoblast dysfunction. Hydrogen sulfide was found to be involved in GCs-induced osteoblast dysfunction. Osteoblastic MC3T3-E1 cell and mitochondrial function were determined by cell viability, M-CSF level, and ALP activity and superoxide production, membrane potential, and ATP level, respectively. The purpose of this research was to explore the impact of NaHS on osteoblastic MC3T3-E1 cell function as well as on Sirt1 and PGC1α expression in dexamethasone (DEX)-treated osteoblast cells. DEX-treated MC3T3-E1 cells exhibited decreased cell viability and ALP activity, as well as increased M-CSF level; all these changes were dramatically attenuated by NaHS. DEX-treated cells also displayed mitochondrial dysfunction, namely decreased mitochondrial membrane potential and ATP generation and increased superoxide generation, which were partly reversed by NaHS. We confirmed decreased Sirt1 and PGC1α protein expression in DEX-treated MC3T3-E1 cells by Western blot, which was also partly reversed by NaHS. Silencing of Sirt1 abrogated the protective effect of NaHS against DEX-induced cell damage and mitochondrial dysfunction. NaHS alleviates DEX-induced osteoblastic MC3T3-E1 cell injury by improving mitochondrial function.


Assuntos
Dexametasona/farmacologia , Mitocôndrias/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteoblastos/patologia , Sulfetos/farmacologia , Células 3T3 , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Camundongos , Mitocôndrias/metabolismo , Osteoblastos/metabolismo
14.
Chem Biol Interact ; 309: 108706, 2019 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-31194955

RESUMO

Oxidative-stress-induced osteoblast dysfunction plays an important role in the development and progression of osteoporosis. BTB and CNC homology 1 (Bach1) has been suggested as a critical regulator of oxidative stress; however, whether Bach1 plays a role in regulating oxidative-stress-induced osteoblast dysfunction remains unknown. Thus, we investigated the potential role and mechanism of Bach1 in regulating oxidative-stress-induced osteoblast dysfunction. Osteoblasts were treated with hydrogen peroxide (H2O2) to mimic a pathological environment for osteoporosis in vitro. H2O2 exposure induced Bach1 expression in osteoblasts. Functional experiments demonstrated that Bach1 silencing improved cell viability and reduced cell apoptosis and reactive oxygen species (ROS) production in H2O2-treated cells, while Bach1 overexpression produced the opposite effects. Notably, Bach1 inhibition upregulated alkaline phosphatase activity and osteoblast mineralization. Mechanism research revealed that Bach1 inhibition increased the activation of nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling and upregulated heme oxygenase 1 and NAD(P)H:quinone oxidoreductase 1 mRNA expression. The Bach1 inhibition-mediated protective effect was partially reversed by silencing Nrf2 in H2O2-exposed osteoblasts. Taken together, these results demonstrate that Bach1 inhibition alleviates oxidative-stress-induced osteoblast apoptosis and dysfunction by enhancing Nrf2/ARE signaling activation, findings that suggest a critical role for the Bach1/Nrf2/ARE regulation axis in osteoporosis progression. Our study suggests that Bach1 may serve as a potential therapeutic target for treating osteoporosis.


Assuntos
Elementos de Resposta Antioxidante/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Peróxido de Hidrogênio/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina Básica/antagonistas & inibidores , Fatores de Transcrição de Zíper de Leucina Básica/genética , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo , Camundongos , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/genética , Osteoblastos/citologia , Osteoblastos/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo
15.
Nat Commun ; 10(1): 2829, 2019 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-31249296

RESUMO

Extracellular vesicles (EVs) are involved in the regulation of cell physiological activity and the reconstruction of extracellular environment. Matrix vesicles (MVs) are a type of EVs released by bone-related functional cells, and they participate in the regulation of cell mineralization. Here, we report bioinspired MVs embedded with black phosphorus (BP) and functionalized with cell-specific aptamer (denoted as Apt-bioinspired MVs) for stimulating biomineralization. The aptamer can direct bioinspired MVs to targeted cells, and the increasing concentration of inorganic phosphate originating from BP can facilitate cell biomineralization. The photothermal effect of the Apt-bioinspired MVs can also promote the biomineralization process by stimulating the upregulated expression of heat shock proteins and alkaline phosphatase. In addition, the Apt-bioinspired MVs display outstanding bone regeneration performance. Our strategy provides a method for designing bionic tools to study the mechanisms of biological processes and advance the development of medical engineering.


Assuntos
Vesículas Extracelulares/metabolismo , Fósforo/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Biomineralização , Osso e Ossos/química , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Vesículas Extracelulares/química , Feminino , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/química , Osteoblastos/metabolismo , Fosfatos/metabolismo , Fósforo/química , Ratos
16.
Int J Mol Sci ; 20(12)2019 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-31200471

RESUMO

Lactoferrin (LF) is known to modulate the bone anabolic effect. Previously, we and others reported that the effects of LF on the bone may be conferred by the stimulation of transforming growth factor ß (TGF-ß) signaling in the preosteoblast. However, the underlying molecular mechanisms of LF-induced osteogenic differentiation of mesenchymal stem cells (MSCs) has not been identified. In this study, we tested the hypothesis that the effects of LF on osteogenesis of MSCs required mediation by TGF-ß Receptors and activating TGF-ß signaling pathway. Using siRNA silencing technology, the knockdown of TGF-ß Receptor II (TßRII) could significantly attenuate LF's effect on the proliferation rate and alkaline phosphatase (ALP) activity of MSCs. It indicated that LF induced osteogenic activity that is dependent on TßRII in C3H10T1/2. Subsequently, it was shown that LF activated Smad2. Downregulating TGF-ß Receptor I (TßRI) with SB431542 attenuated the expression of p-Smad2 and p-P38, also the LF-induced the osteogenic activity. Besides, the stimulation by LF on the expression of Osteocalcin (OCN), Osteopontin (OPN), Collagen-2a1 (Col2a1), and Fibroblast Growth Factor 2 (FGF2) were abolished by SB431542. These results confirmed that LF induced osteogenic activity though the TGF-ß canonical and noncanonical signaling pathway. This study provided the first evidence of the signaling mechanisms of LF's effect on osteogenesis in MSCs.


Assuntos
Diferenciação Celular , Lactoferrina/farmacologia , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Animais , Linhagem Celular , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteocalcina/genética , Osteocalcina/metabolismo , Osteopontina/genética , Osteopontina/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Proteína Smad2/genética , Proteína Smad2/metabolismo
17.
Int J Mol Sci ; 20(11)2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-31181661

RESUMO

Several lines of evidence suggest that oxidative stress is one of the key pathogenic mechanisms of osteoporosis. We aimed to elucidate the bone protective effects of petunidin, one of the most common anthocyanidins, considering its potent antioxidative activity. Petunidin (>5 µg/mL) significantly inhibited osteoclastogenesis and downregulated c-fos, Nfatc1, Mmp9, Ctsk, and Dc-stamp mRNA expression in RAW264.7 cells. Conversely, petunidin (>16 µg/mL) stimulated mineralized matrix formation and gene expression of Bmp2 and Ocn, whereas it suppressed Mmp13, Mmp2, and Mmp9 mRNA expression and proteolytic activities of MMP13 and MMP9 in MC3T3-E1 cells. Micro-CT and bone histomorphometry analyses of sRANKL-induced osteopenic C57BL/6J mice showed that daily oral administration of petunidin (7.5 mg/kg/day) increased bone volume to tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), the ratio of osteoid volume to tissue volume (OV/TV), osteoid thickness (O.Th), the ratio of osteoid surface to bone surface (OS/BS), the ratio of osteoblast surface to bone surface (Ob.S/BS), and the number of osteoblast per unit of bone surface (N.Ob/BS), and decreased trabecular separation (Tb.Sp), the ratio of eroded surface to bone surface (ES/BS), the ratio of osteoclast surface to bone surface (Oc.S/BS), and number of osteoclast per unit of bone surface (N.Oc/BS), compared to untreated mice. Furthermore, histological sections of the femurs showed that oral administration of petunidin to sRANKL-induced osteopenic mice increased the size of osteoblasts located along the bone surface and the volume of osteoid was consistent with the in vitro osteoblast differentiation and MMP inhibition. These results suggest that petunidin is a promising natural agent to improve sRANKL-induced osteopenia in mice through increased osteoid formation, reflecting accelerated osteoblastogenesis, concomitant with suppressed bone resorption.


Assuntos
Antocianinas/uso terapêutico , Conservadores da Densidade Óssea/uso terapêutico , Osteoblastos/efeitos dos fármacos , Osteogênese , Osteoporose/tratamento farmacológico , Animais , Antocianinas/farmacologia , Conservadores da Densidade Óssea/farmacologia , Proteína Morfogenética Óssea 2/metabolismo , Osso Esponjoso/efeitos dos fármacos , Osso Esponjoso/metabolismo , Feminino , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Osteoporose/metabolismo , Ligante RANK/metabolismo , Células RAW 264.7
18.
Nat Commun ; 10(1): 2226, 2019 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-31110221

RESUMO

Lineage commitment and tumorigenesis, traits distinguishing stem cells, have not been well characterized and compared in mesenchymal stem cells derived from human dental pulp (DP-MSCs) and bone marrow (BM-MSCs). Here, we report DP-MSCs exhibit increased osteogenic potential, possess decreased adipogenic potential, form dentin pulp-like complexes, and are resistant to oncogenic transformation when compared to BM-MSCs. Genome-wide RNA-seq and differential expression analysis reveal differences in adipocyte and osteoblast differentiation pathways, bone marrow neoplasm pathway, and PTEN/PI3K/AKT pathway. Higher PTEN expression in DP-MSCs than in BM-MSCs is responsible for the lineage commitment and tumorigenesis differences in both cells. Additionally, the PTEN promoter in BM-MSCs exhibits higher DNA methylation levels and repressive mark H3K9Me2 enrichment when compared to DP-MSCs, which is mediated by increased DNMT3B and G9a expression, respectively. The study demonstrates how several epigenetic factors broadly affect lineage commitment and tumorigenesis, which should be considered when developing therapeutic uses of stem cells.


Assuntos
Carcinogênese/genética , Polpa Dentária/citologia , Células-Tronco Mesenquimais/patologia , Osteogênese/genética , PTEN Fosfo-Hidrolase/metabolismo , Adipócitos/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Células da Medula Óssea/patologia , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Carcinogênese/patologia , Diferenciação Celular/genética , Células Cultivadas , Criança , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/genética , Polpa Dentária/patologia , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Antígenos de Histocompatibilidade/metabolismo , Código das Histonas/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Osteoblastos/metabolismo , Osteossarcoma/genética , Osteossarcoma/patologia , PTEN Fosfo-Hidrolase/genética , Regiões Promotoras Genéticas/genética , Análise de Sequência de RNA
19.
Life Sci ; 229: 261-276, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31082400

RESUMO

AIM: Myokines are associated with regulation of bone and muscle mass. However, limited information is available regarding the impact of myokines on glucocorticoid (GC) mediated adverse effects on the musculoskeletal system. This study investigates the role of myokine fibroblast growth factor-2 (FGF-2) in regulating GC-induced deleterious effects on bone and skeletal muscle. METHODS: Primary osteoblast cells and C2C12 myoblast cell line were treated with FGF-2 and then exposed to dexamethasone (GC). FGF-2 mediated attenuation of the inhibitory effect of GC on osteoblast and myoblast differentiation and muscle atrophy was assessed through quantitative PCR and western blot analysis. Further, FGF-2 was administered subcutaneously to dexamethasone treated mice to collect bone and skeletal muscle tissue for in vivo analysis of bone microarchitecture, mechanical strength, histomorphometry and for histological alterations in treated tissue samples. KEY FINDINGS: FGF-2 abrogated the dexamethasone induced inhibitory effect on osteoblast differentiation by modulating BMP-2 pathway and inhibiting Wnt antagonist sclerostin. Further, dexamethasone induced atrophy in C2C12 cells was mitigated by FGF-2 as evident from down regulation of atrogenes expression. FGF-2 prevented GC-induced impairment of mineral density, biomechanical strength, trabecular bone volume, cortical thickness and bone formation rate in mice. Additionally, skeletal muscle tissue from GC treated mice displayed weak myostatin immunostaining and reduced expression of atrogenes following FGF-2 treatment. SIGNIFICANCE: FGF-2 mitigated GC induced effects through inhibition of sclerostin and myostatin expression in bone and muscle respectively. Taken together, this study exhibited the role of exogenous FGF-2 in sustaining osteoblastogenesis and inhibiting muscle atrophy in presence of glucocorticoid.


Assuntos
Osso e Ossos/metabolismo , Dexametasona/toxicidade , Fator 2 de Crescimento de Fibroblastos/farmacologia , Glicoproteínas/antagonistas & inibidores , Músculo Esquelético/metabolismo , Doenças Musculoesqueléticas/tratamento farmacológico , Miostatina/antagonistas & inibidores , Animais , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/patologia , Diferenciação Celular , Células Cultivadas , Glucocorticoides/toxicidade , Camundongos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Doenças Musculoesqueléticas/induzido quimicamente , Doenças Musculoesqueléticas/metabolismo , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos
20.
Cell Biochem Funct ; 37(5): 385-394, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31140646

RESUMO

To explore the molecular mechanism of insulin on proliferation and differentiation of MC3T3-E1 cell under high glucose conditions. We first investigated the effect of different concentrations of insulin on the osteoblast cell proliferation and cell differentiation at various time points by MTT analysis, cell cycle analysis, and expression detection of differentiation genes. Then, we used 200 ng/mL of insulin to treat the osteoblast cell at different time points for identifying the common differentially expressed mRNAs among various time points by RNA sequencing. Thirdly, we performed the gene ontology (GO) and the Kyoto Encyclopaedia of Genes and Genomes (KEGG) analysis to explore the biological function of these common differentially expressed mRNAs. The results showed that insulin promoted the cell proliferation and differentiation of osteoblast cell. In RNA sequencing, a total of 31 common differentially expressed mRNAs were identified between different time points. Mt1, Tmem135, Avp, and Dlg2 were found to be associated with the new bone formation. In addition, three important signalling pathways, namely, lysosome, glutamatergic synapse, and chemokine signalling pathways, were found in the KEGG enrichment analysis. Our work demonstrated that insulin could promote the osteoblast cell proliferation and cell differentiation, which may play a key role in bone formation. SIGNIFICANCE OF THE STUDY: Our result showed that insulin could promote the proliferation and differentiation of osteoblast at both cellular and molecular levels, which may promote the new bone formation in the osteoblasts.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Glucose/metabolismo , Insulina/farmacologia , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Células 3T3 , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Camundongos , Osteoblastos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
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