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1.
FASEB J ; 35(10): e21851, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34547121

RESUMO

It has been known that moderate mechanical loading, like that caused by exercise, promotes bone formation. However, its underlying mechanisms remain elusive. Here we showed that moderate running dramatically improved trabecular bone in mice tibias with an increase in bone volume fraction and trabecular number and a decrease in trabecular pattern factor. Results of immunohistochemical and histochemical staining revealed that moderate running mainly increased the number of osteoblasts but had no effect on osteoclasts. In addition, we observed a dramatic increase in the number of colony forming unit-fibroblast in endosteal bone marrow and the percentage of CD45- Leptin receptor+ (CD45- LepR+ ) endosteal mesenchymal progenitors. Bioinformatics analysis of the transcriptional data from gene expression omnibus (GEO) database identified chemokine c-c-motif ligands (CCL2) as a critical candidate induced by mechanical loading. Interestingly, we found that CCL2 was up-regulated mainly in osteoblastic cells in the tibia of mice after moderate running. Further, we found that mechanical loading up-regulated the expression of CCL2 by activating ERK1/2 pathway, thereby stimulating migration of endosteal progenitors. Finally, neutralizing CCL2 abolished the recruitment of endosteal progenitors and the increased bone formation in mice after 4 weeks running. These results therefore uncover an unknown connection between osteoblasts and endosteal progenitors recruited in the increased bone formation induced by mechanical loading.


Assuntos
Osso Esponjoso/citologia , Quimiocina CCL2/metabolismo , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Osteogênese , Condicionamento Físico Animal , Animais , Osso Esponjoso/metabolismo , Movimento Celular , Quimiocina CCL2/genética , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo
2.
Microb Cell Fact ; 20(1): 102, 2021 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001083

RESUMO

As treatment of Staphylococcus aureus (S. aureus) osteomyelitis is often hindered by the development of antibiotic tolerance, novel antibacterial therapeutics are required. Here we found that the cell-free supernatant of Bacillus subtilis (B. subtilis CFS) killed planktonic and biofilm S. aureus, and increased S. aureus susceptibility to penicillin and gentamicin as well. Further study showed that B. subtilis CFS suppressed the expression of the genes involved in adhesive molecules (Cna and ClfA), virulence factor Hla, quorum sensing (argA, argB and RNAIII) and biofilm formation (Ica and sarA) in S. aureus. Additionally, our data showed that B. subtilis CFS changed the membrane components and increased membrane permeabilization of S. aureus. Finally, we demonstrated that B. subtilis CFS increased considerably the susceptibility of S. aureus to penicillin and effectively reduced S. aureus burdens in a mouse model of implant-associated osteomyelitis. These findings support that B. subtilis CFS may be a potential resistance-modifying agent for ß-lactam antibiotics against S. aureus.


Assuntos
Antibacterianos/farmacologia , Bacillus subtilis/crescimento & desenvolvimento , Meios de Cultura/farmacologia , Osteomielite/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Animais , Antibacterianos/administração & dosagem , Antibacterianos/química , Bacillus subtilis/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Biofilmes/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Meios de Cultura/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Osteomielite/tratamento farmacológico , Percepção de Quorum , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética
3.
Infect Immun ; 89(5)2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33619031

RESUMO

To investigate the molecular pathogenesis of bone with osteomyelitis, we developed implant-associated osteomyelitis (IAOM) models in mice. An orthopedic stainless pin was surgically placed in the right femoral midshaft of mice, followed by an inoculation of Staphylococcus aureus into the medullary cavity. Typical characteristics of IAOM, like periosteal reaction and intraosseous abscess, occurred by day 14 postinfection. By day 28 postinfection, necrotic abscess, sequestrum formation, and deformity of the whole femur were observed. Transcriptional analysis identified 101 and 1,702 differentially expressed genes (DEGs) between groups by days 3 and 14 postinfection, respectively. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed the enrichment of pathways in response to the bacterium, receptor-ligand activity, and chemokine signaling by day 3 postinfection. However, by day 14 postinfection, the enrichment switched to angiogenesis, positive regulation of cell motility and migration, skeletal system development, and cytokine-cytokine receptor interaction. Furthermore, protein-protein interaction network analysis identified 4 cytokines (interleukin 6 [IL-6], Cxcl10, gamma interferon [IFN-γ], and Cxcl9) associated with IAOM at an early stage of infection. Overall, as the pathological changes in this mouse model were consistent with those in human IAOM, our model may be used to investigate the mechanism and treatment of IAOM. Furthermore, the data for transcriptome sequencing and bioinformatic analysis will be an important resource for dissecting the molecular pathogenesis of bone with IAOM.


Assuntos
Osteomielite/etiologia , Infecções Relacionadas à Prótese/genética , Infecções Relacionadas à Prótese/microbiologia , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus , Transcriptoma , Animais , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Biologia Computacional/métodos , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ontologia Genética , Camundongos
5.
PeerJ ; 8: e9484, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32742785

RESUMO

Background: Bone marrow adipocyte (BMA), closely associated with bone degeneration, shares common progenitors with osteoblastic lineage. However, the intrinsic mechanism of cells fate commitment between BMA and osteogenic lineage remains unclear. Methods: Gene Expression Omnibus (GEO) dataset GSE107789 publicly available was downloaded and analyzed. Differentially expressed genes (DEGs) were analyzed using GEO2R. Functional and pathway enrichment analyses of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes were conducted by The Database for Annotation, Visualization and Integrated Discovery and Gene set enrichment analysis software. Protein-protein interactions (PPI) network was obtained using STRING database, visualized and clustered by Cytoscape software. Transcriptional levels of key genes were verified by real-time quantitative PCR in vitro in Bone marrow stromal cells (BMSCs) undergoing adipogenic differentiation at day 7 and in vivo in ovariectomized mice model. Results: A total of 2,869 DEGs, including 1,357 up-regulated and 1,512 down-regulated ones, were screened out from transcriptional profile of human BMSCs undergoing adipogenic induction at day 7 vs. day 0. Functional and pathway enrichment analysis, combined with modules analysis of PPI network, highlighted ACSL1, sphingosine 1-phosphate receptors 3 (S1PR3), ZBTB16 and glypican 3 as key genes up-regulated at the early stage of BMSCs adipogenic differentiation. Furthermore, up-regulated mRNA expression levels of ACSL1, S1PR3 and ZBTB16 were confirmed both in vitro and in vivo. Conclusion: ACSL1, S1PR3 and ZBTB16 may play crucial roles in early regulation of BMSCs adipogenic differentiation.

6.
Food Chem Toxicol ; 145: 111689, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32810588

RESUMO

α-hemolysin (Hla) is considered an essential virulent factor for Staphylococcus aureus (S. aureus) toxicity, the mechanism by which Hla affect bone metabolism is poorly understood. In this study, 2-month-old C57BL/6 mice were treated with Hla (40 µg/kg, i.p.) or S. aureus (1 × 106 CFU/ml, 100 µl, i.v.) with the presence or absence of methyl-ß-cyclodextrin (MßCD) (300 mg/kg, i.p.). MicroCT analysis showed progressive bone loss from week 2 to week 4 after Hla treatment, accompanied by a decreased osteoblasts and increased osteoclasts in femoral metaphysis in mice. Further, Hla stimulated the expression of Caveolin-1 in vivo and in vitro, activated lipid rafts accumulation in cell membrane of bone marrow stromal cells (BMSCs), and suppressed osteogenesis of BMSCs. Destruction of lipid rafts with MßCD or inhibition of Caveolin-1 with Daidzein blocked the detrimental effect of Hla on osteogenesis of BMSCs. Importantly, treating mice with MßCD rescued the loss of osteoblasts and increased osteoclastogenesis induced by Hla as well as the bone loss induced by S. aureus infection. Together, we demonstrate that Hla induces bone destruction directly by suppressing osteogenesis and indirectly by stimulating osteoclastogenesis, and that lipid rafts may mediate the detrimental effect of Hla and S. aureus on osteogenesis and bone formation.


Assuntos
Proteínas de Bactérias/metabolismo , Células da Medula Óssea/citologia , Proteínas Hemolisinas/metabolismo , Microdomínios da Membrana/metabolismo , Osteogênese , Infecções Estafilocócicas/fisiopatologia , Staphylococcus aureus/metabolismo , Animais , Proteínas de Bactérias/genética , Células da Medula Óssea/metabolismo , Células da Medula Óssea/microbiologia , Regulação para Baixo , Proteínas Hemolisinas/genética , Interações Hospedeiro-Patógeno , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos/citologia , Osteoclastos/metabolismo , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética
7.
Artigo em Inglês | MEDLINE | ID: mdl-32793507

RESUMO

Effective management of infectious osteomyelitis relies on timely microorganism identification and appropriate antibiotic therapy. Extracellular vesicles (EVs) carry protein and genetic information accumulated rapidly in the circulation upon infection. Rat osteomyelitis models infected by Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Escherichia coli were established for the present study. Serum EVs were isolated 3 days after infection. The size and number of serum EVs from infected rats were significantly higher than those from controls. In addition, bacterial aggregation assay showed that the S. aureus and E. coli formed large aggregates in response to the stimulation of serum EVs from S. aureus-infected and E. coli-infected rats, respectively. Treatment of EVs-S. epidermidis led to large aggregates of S. epidermidis and E. coli, whereas stimulation of EVs-P. aeruginosa to large aggregates of S. aureus and P. aeruginosa. To evaluate the changes in EVs in osteomyelitis patients, 28 patients including 5 S. aureus ones and 21 controls were enrolled. Results showed that the size and number of serum EVs from S. aureus osteomyelitis patients were higher than those from controls. Further analysis using receiver operating characteristic curves revealed that only the particle size might be a potential diagnostic marker for osteomyelitis. Strikingly, serum EVs from S. aureus osteomyelitis patients induced significantly stronger aggregation of S. aureus and a cross-reaction with P. aeruginosa. Together, these findings indicate that the size and number of serum EVs may help in the diagnosis of potential infection and that EVs-bacteria aggregation assay may be a quick test to identify infectious microorganisms for osteomyelitis patients.


Assuntos
Vesículas Extracelulares , Osteomielite , Infecções Estafilocócicas , Animais , Biomarcadores , Escherichia coli , Humanos , Osteomielite/diagnóstico , Ratos , Infecções Estafilocócicas/diagnóstico , Staphylococcus aureus
8.
Stem Cell Res Ther ; 11(1): 270, 2020 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-32631432

RESUMO

BACKGROUND: Prenatal dexamethasone exposure (PDE) induces low birth weight and retardation of fetal bone development which are associated with lower peak bone mass in adult offspring. Here we evaluated whether and how PDE affects postnatal long bone growth in mouse offspring. METHODS: Pregnant mice were injected subcutaneously with dexamethasone (1.2 mg/kg/day) every morning from gestational days (GD) 12-14. Femurs and tibias of 2-, 4-, 6-, and 12-week-old female offspring were harvested for histological, immunofluorescence, flow cytometric analysis, or microcomputed tomography (µCT) measurement. RESULTS: PDE leads to impaired bone remodeling as well as decreased bone mass in the long bone of female mouse offspring. During postnatal bone growth, significant decrease of CD45-CD29+CD105+Sca-1+ bone marrow mesenchymal stem cells (BMSCs) and CD45-Nestin+ cells, loss of type H vessels, and increment of cellular senescence were found in metaphysis of long bone in mouse offspring after PDE. We further show that eliminating the excessive senescent cells with dasatinib (5 mg/kg/day) and quercetin (50 mg/kg/day) during GD 12-14 rescues the above toxic effect of PDE on the postnatal long bone growth in female mouse offspring. CONCLUSION: Cellular senescence mediates the toxic effect of PDE on postnatal long bone growth in mouse offspring, and inhibition of cellular senescence may be proposed for treating the retardation of bone growth caused by PDE.


Assuntos
Dexametasona , Efeitos Tardios da Exposição Pré-Natal , Animais , Desenvolvimento Ósseo , Senescência Celular , Dexametasona/toxicidade , Feminino , Camundongos , Gravidez , Ratos , Ratos Wistar , Microtomografia por Raio-X
9.
J Orthop Translat ; 24: 12-22, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32518750

RESUMO

Background: Abnormal bone formation in subchondral bone resulting from uncoupled bone remodeling is considered a central feature in osteoarthritis (OA) pathogenesis. H-type vessels can couple angiogenesis and osteogenesis. We previously revealed that elevated H-type vessels in subchondral bone were correlated with OA and focal adhesion kinase (FAK) in MSCs is critical for H-type vessel formation in osteoporosis. The aim of this study was to explore the correlation between H-type vessels and MSCs in OA pathogenesis through regulation of H-type vessel formation using defactinib (an FAK inhibitor). Methods: In vivo: 3-month-old male C57BL/6J (WT) mice were randomly divided into three groups: sham controls, vehicle-treated ACLT mice, and defactinib-treated ACLT mice (25 mg/kg, intraperitoneally weekly). In vitro: we explored the role of conditioned medium (CM) of MSCs from subchondral bone of different groups on the angiogenesis of endothelial cells (ECs). Flow cytometry, Western blotting, ELISA, real time (RT)-PCR, immunostaining, CT-based microangiography, and bone micro-CT (µCT) were used to detect changes in relative cells and tissues. Results: This study demonstrated that inhibition of H-type vessels with defactinib alleviated OA by inhibiting H-type vessel-linked MSCs in subchondral bone. During OA pathogenesis, H-type vessels and MSCs formed a positive feedback loop contributing to abnormal bone formation in subchondral bone. Elevated H-type vessels provided indispensable MSCs for abnormal bone formation in subchondral bone. Flow cytometry and immunostaining results confirmed that the amount of MSCs in subchondral bone was obviously higher in vehicle-treated ACLT mice than that in sham controls and defactinib-treated ACLT mice. In vitro, p-FAK in MSCs from subchondral bone of vehicle-treated ALCT mice increased significantly relative to other groups. Further, the CM from MSCs of vehicle-treated ACLT mice enhanced angiogenesis of ECs through FAK-Grb2-MAPK-linked VEGF expression. Conclusions: Our results demonstrate that defactinib inhibits OA by suppressing the positive feedback loop between H-type vessels and MSCs in subchondral bone. The translational potential of this article: Our results provide a mechanistic rationale for the use of defactinib as an effective candidate for OA treatment.

10.
Front Microbiol ; 11: 1301, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32595631

RESUMO

Staphylococcus aureus (S. aureus) infection-induced osteomyelitis is a great challenge in clinic treatment. Identification of the essential genes and biological processes that are specifically changed in mononuclear cells at an early stage of S. aureus osteomyelitis is of great clinical significance. Based on transcriptional dataset GSE16129 available publicly, a bioinformatic analysis was performed to identify the differentially expressed genes of osteomyelitis caused by S. aureus infection. ERBB2, TWIST1, and NANOG were screened out as the most valuable osteomyelitis-related genes (OMRGs). A mice model of implant-associated S. aureus osteomyelitis was used to verify the above genes. We found significantly up-regulated expression of TWIST1 in macrophages and accumulation of macrophages around the infected implant. Meanwhile, S. aureus infection increased the expression of TWIST1, MMP9, and MMP13, and stimulated the migration and phagocytosis function of Raw 264.7 cells. Additionally, knock-down of the expression of TWIST1 by siRNA could significantly down-regulate MMP9 and MMP13 and suppress the migration and phagocytosis ability of macrophages in response to S. aureus infection. Furthermore, we found that NF-κB signaling was activated in Raw 264.7 cells by S. aureus and that inhibition of NF-κB signaling by Bay11-7082 blocked the expression of TWIST1, MMP9, and MMP13 as well as cell migration and phagocytosis evoked by S. aureus. Our findings demonstrate that NF-κB/TWIST1 is necessary for migration and phagocytosis of macrophages in response to S. aureus infection. Our study highlights the essential role of NF-κB/TWIST1 in early innate immune response to S. aureus infection in bone.

11.
Cell Microbiol ; 22(10): e13240, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32584493

RESUMO

Internalisation of Staphylococcus aureus in osteoblasts plays a critical role in the persistence and recurrence of osteomyelitis, the mechanisms involved in this process remain largely unknown. In the present study, evidence of internalised S. aureus in osteoblasts was found in long bone of haematogenous osteomyelitis in mice after 2 weeks of infection. Meanwhile, eliminating extracellular S. aureus by gentamicin can partially rescue bone loss, whereas the remaining intracellular S. aureus in osteoblasts may be associated with continuous bone destruction. In osteoblastic MC3T3 cells, intracellular S. aureus was detectable as early as 15 min after infection, and the internalisation rates increased with the extension of infection time. Additionally, S. aureus invasion stimulated the expression of phosphor-focal adhesion kinase (FAK), phosphor-epidermal growth factor receptor (EGFR) and phosphor-c-Src in a time-dependent way, and blocking EGFR/FAK or c-Src signalling significantly reduced the internalisation rate of S. aureus in osteoblasts. Our findings provide new insights into the mechanism of S. aureus internalisation in osteoblast and raise the potential of targeting EGFR/FAK and c-Src as adjunctive therapeutics for treating chronic S. aureus osteomyelitis.


Assuntos
Receptores ErbB/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Osteoblastos/microbiologia , Osteomielite/microbiologia , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/patogenicidade , Animais , Linhagem Celular , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Staphylococcus aureus/metabolismo
12.
J Cell Physiol ; 235(11): 8653-8666, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32324278

RESUMO

Osteoarthritis (OA), a disease of the entire joint, is characterized by abnormal bone remodeling and coalescent degradation of articular cartilage. We have previously found that elevated levels of H-type vessels in subchondral bone correlate with OA and that focal adhesion kinase (FAK) is critical for H-type vessel formation in osteoporosis. However, the potential role of FAK in OA remains unexplored. Here, we demonstrate that the p-FAK level was dramatically elevated in subchondral bone following anterior cruciate ligament transection (ACLT) in rats. Specific inhibition of FAK signaling with Y15 in subchondral bone resulted in the suppression of subchondral bone deterioration and this effect was mediated by H-type vessel-induced ectopic bone formation. Further, articular cartilage degeneration was also alleviated after Y15 treatment. In vitro, the p-FAK level was significantly elevated in mesenchymal stem cells (MSCs) from vehicle-treated ACLT rats as compared to that in MSCs from sham controls and Y15-treated ACLT rats. Elevated p-FAK level in MSCs promoted vascular endothelial growth factor (VEGF) expression, as demonstrated from the high VEGF level in the blood, subchondral bone, and conditioned medium (CM) of MSCs from vehicle-treated ACLT rats. The CM of MSCs from vehicle-treated ACLT rats might promote the angiogenesis of endothelial cells and the catabolic response of chondrocytes through the FAK-growth factor receptor-bound protein 2-mitogen-activated protein kinase-mediated expression of VEGF. The effect of the CM from MSCs of Y15-treated ACLT rats or that treated with a VEGF-neutralizing antibody on vessel formation and the catabolic response was lowered. Thus, the specific inhibition of FAK signaling may be a promising avenue for the prevention or early treatment of OA.


Assuntos
Cartilagem Articular/metabolismo , Quinase 1 de Adesão Focal/antagonistas & inibidores , Proteína-Tirosina Quinases de Adesão Focal/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Osteoartrite/tratamento farmacológico , Alendronato/farmacologia , Animais , Ligamento Cruzado Anterior/patologia , Remodelação Óssea/efeitos dos fármacos , Remodelação Óssea/fisiologia , Osso e Ossos/patologia , Condrócitos/metabolismo , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Masculino , Osteoartrite/patologia , Ratos Sprague-Dawley
13.
J Biomater Appl ; 35(1): 97-107, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32233720

RESUMO

Fabrication of osteoconductive scaffold with osteoinductive capability and appropriate resorption rate is of great significance for treating bone defects. To achieve this aim, strontium-substituted calcium sulfate hemihydrate (Sr-CSH) and hydroxyapatite (HA) were mixed to develop a novel composite. Sr-CSH containing 5% and 10% strontium was mixed with HA at the weight ratio of 6:4, respectively. Female Sprague-Dawley rats underwent bone defect surgery in left tibia were randomly assigned to three different treatment groups filled with CSH/HA, 5% and 10% Sr-CSH/HA. Micro-CT analysis showed increased new bone formation in 10% Sr-CSH/HA group compared to CSH/HA group. In addition, histological analysis showed large amounts of chondrocytes and osteoblasts within the pores of Sr-CSH/HA composites as a result of the CSH resorption. Further, CFU-F assay demonstrated the increased amount of bone marrow mesenchymal stromal cells (BMSCs) colonies in 10% Sr-CSH/HA group. In primary BMSCs, extraction from Sr-CSH/HA composite significantly increased the migration of cells, up-regulated the expression of osteoblastic marker genes, and increased the area of mineralized nodules. Together, Sr-CSH/HA may promote bone formation by recruiting and stimulating osteogenic differentiation of BMSCs. Therefore, this composite may be proposed as an ideal substitute to repair bone defects.


Assuntos
Regeneração Óssea , Sulfato de Cálcio/química , Hidroxiapatitas/química , Células-Tronco Mesenquimais/citologia , Estrôncio/química , Tecidos Suporte/química , Animais , Regeneração Óssea/efeitos dos fármacos , Sulfato de Cálcio/farmacologia , Proliferação de Células/efeitos dos fármacos , Feminino , Hidroxiapatitas/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Ratos Sprague-Dawley , Estrôncio/farmacologia
14.
Stem Cell Res Ther ; 11(1): 131, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32197645

RESUMO

BACKGROUND: Hypercholesterolemia increases the risk of tendon pain and tendon rupture. Tendon-derived stem cells (TDSCs) play a vital role in the development of tendinopathy. Our previous research found that high cholesterol inhibits tendon-related gene expression in TDSCs. Whether high cholesterol has other biological effects on TDSCs remains unknown. METHODS: TDSCs isolated from female SD rats were exposed to 10 mg/dL cholesterol for 24 h. Then, cell apoptosis was assessed using flow cytometry and fluorescence microscope. RFP-GFP-LC3 adenovirus transfection was used for measuring autophagy. Signaling transduction was measured by immunofluorescence and immunoblotting. In addition, Achilles tendons from ApoE -/- mice fed with a high-fat diet were histologically assessed using HE staining and immunohistochemistry. RESULTS: In this work, we verified that 10 mg/dL cholesterol suppressed cell proliferation and migration and induced G0/G1 phase arrest. Additionally, cholesterol induced apoptosis and autophagy simultaneously in TDSCs. Apoptosis induction was related to increased expression of cleaved caspase-3 and BAX and decreased expression of Bcl-xL. The occurrence of autophagic flux and accumulation of LC3-II demonstrated the induction of autophagy by cholesterol. Compared with the effects of cholesterol treatment alone, the autophagy inhibitor 3-methyladenine (3-MA) enhanced apoptosis, while the apoptosis inhibitor Z-VAD-FMK diminished cholesterol-induced autophagy. Moreover, cholesterol triggered reactive oxygen species (ROS) generation and activated the AKT/FOXO1 pathway, while the ROS scavenger NAC blocked cholesterol-induced activation of the AKT/FOXO1 pathway. NAC and the FOXO1 inhibitor AS1842856 rescued the apoptosis and autophagy induced by cholesterol. Finally, high cholesterol elevated the expression of cleaved caspase-3, Bax, LC3-II, and FOXO1 in vivo. CONCLUSION: The present study indicated that high cholesterol induced apoptosis and autophagy through ROS-activated AKT/FOXO1 signaling in TDSCs, providing new insights into the mechanism of hypercholesterolemia-induced tendinopathy. High cholesterol induces apoptosis and autophagy through the ROS-activated AKT/FOXO1 pathway in tendon-derived stem cells.


Assuntos
Hipercolesterolemia , Proteínas Proto-Oncogênicas c-akt , Animais , Apoptose , Autofagia , Linhagem Celular Tumoral , Colesterol , Feminino , Proteína Forkhead Box O1 , Camundongos , Proteínas do Tecido Nervoso , Proteínas Proto-Oncogênicas c-akt/genética , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio , Células-Tronco , Tendões
15.
Front Bioeng Biotechnol ; 8: 620629, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33569374

RESUMO

In the mammalian skeletal system, osteogenesis and angiogenesis are closely linked by type H vessels during bone regeneration and repair. Our previous studies confirmed the promotion of these processes by copper-containing metal (CCM) in vitro and in vivo. However, whether and how the coupling of angiogenesis and osteogenesis participates in the promotion of bone regeneration by CCM in vivo is unknown. In this study, M2a macrophages but not M2c macrophages were shown to be immunoregulated by CCM. A CCM, 316L-5Cu, was applied to drilling hole injuries of the tibia of C57/6 mice for comparison. We observed advanced formation of cortical bone and type H vessels beneath the new bone in the 316L-5Cu group 14 and 21 days postinjury. Moreover, the recruitment of CD206-positive M2a macrophages, which are regarded as the primary source of platelet-derived growth factor type BB (PDGF-BB), was significantly promoted at the injury site at days 14 and 21. Under the stimulation of CCM, mitochondria-derived reactive oxygen species were also found to be upregulated in CD206hi M2a macrophages in vitro, and this upregulation was correlated with the expression of PDGF-BB. In conclusion, our results indicate that CCM promotes the evolution of callus through the generation of type H vessels during the process of bone repair by upregulating the expression of PDGF-BB derived from M2a macrophages.

16.
Front Cell Dev Biol ; 8: 601188, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33384997

RESUMO

Antenatal corticosteroid therapy (ACT) has been shown to reduce morbidity and mortality rates in preterm delivery, but the fetus is more likely to face the risk of low bone mineralization and low fetal linear growth. However, the mechanism of ACT inducing low bone mineralization remains largely unknown. Pre-osteoclasts, which play an important role in angiogenesis and osteogenesis, are specifically regulating type H vessels (CD31hiEmcnhi) and vessel formation by secreting platelet-derived growth factor-BB (PDGF-BB). We find that the number of pre-osteoclasts and POC-secreted PDGF-BB is dramatically decreased in ACT mice, contributing to the reduction in type H vessels and bone mineralization during the mouse offspring. Quantitative analyses of micro-computed tomography show that the ACT mice have a significant reduction in the mass of trabecular bone relative to the control group. Mononuclear pre-osteoclasts in trabecular bone decreased in ACT mice, which leads to the amount of PDGF-BB reduced and attenuates type H vessel formation. After sorting the Rank+ osteoclast precursors using flow cytometry, we show that the enhancer of zeste homolog 2 (Ezh2) expression is decreased in Rank+ osteoclast precursors in ACT mice. Consistent with the flow data, by using small molecule Ezh2 inhibitor GSK126, we prove that Ezh2 is required for osteoclast differentiation. Downregulating the expression of Ezh2 in osteoclast precursors would reduce PDGF-BB production. Conditioned medium from osteoclast precursor cultures treated with GSK126 inhibited endothelial tube formation, whereas conditioned medium from vehicle group stimulated endothelial tube formation. These results indicate Ezh2 expression of osteoclast precursors is suppressed after ACT, which reduced the pre-osteoclast number and PDGF-BB secretion, thus inhibiting type H vessel formation and ACT-associated low bone mineralization.

17.
Mol Med Rep ; 20(4): 3555-3564, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31432182

RESUMO

Calcium phosphate­based bone substitutes have been widely used for bone repair, augmentation and reconstruction in bone implant surgery. While some of these substitutes have shown excellent biological efficacy, there remains a need to improve the performance of the current calcium phosphate­based bone substitutes. Strontium ions (Sr) can promote new osteogenesis, inhibit osteoclast formation and increase osteoconductivity. However, the therapeutic effect and mechanism of strontium­containing α­calcium sulfate hemihydrate (Sr­CaS) remains unclear. The present study created bone injuries in rats and treated the injuries with Sr­CaS. Then Cell Counting Kit­8, soft agar colony formation, flow cytometry, Transwell and Alizarin Red staining assays were performed to assess the bone cells for their proliferation, growth, apoptosis, invasion, and osteogenic differentiation abilities. The bone reconstructive states were measured by the microCT method, hematoxylin and eosin staining and Masson staining. Bone­related factors were analyzed by the reverse transcription­quantitative PCR assay; transforming growth factor (TGF)­ß, mothers against decapentaplegic homolog (Smad)2/3 and ß­catenin expression was measured by western blot analysis and osteocalcin (OCN) expression was assessed by immunohistochemistry. Sr­CaS did not significantly affect the proliferation and apoptosis of bone marrow stem cells (BMSCs), but did accelerate the migration and osteogenic differentiation of BMSCs in vitro. Sr­CaS promoted bone repair and significantly increased the values for bone mineral density, bone volume fraction, and trabecular thickness, but decreased trabecular spacing in vivo in a concentration­-dependent manner. In addition, Sr­CaS dramatically upregulated the expression levels of genes associated with osteogenic differentiation (Runt­related transcription factor 2, Osterix, ALP, OCN and bone sialoprotein) both in vitro and in vivo. Sr­CaS also increased Smad2/3, TGF­ß and phosphorylated­ß­catenin protein expression in vitro and in vivo. These results indicated that materials that contain 5 or 10% Sr can improve bone defects by regulating the TGF­ß/Smad signaling pathway.


Assuntos
Substitutos Ósseos/uso terapêutico , Sulfato de Cálcio/uso terapêutico , Osteogênese/efeitos dos fármacos , Proteínas Smad/metabolismo , Estrôncio/uso terapêutico , Fator de Crescimento Transformador beta/metabolismo , Animais , Osso e Ossos/lesões , Células Cultivadas , Masculino , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos
18.
FASEB J ; 33(10): 11137-11147, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31298955

RESUMO

So far, there has been no effective cure for osteoporotic cortical bone, the most significant change in long bone structure during aging and the main cause of bone fragility fractures, because its underlying molecular and cellular mechanisms remain largely unknown. We used 3- and 15-mo-old mice as well as 15-mo-old mice treated with vehicle and gefitinib to evaluate structural, cellular, and molecular changes in cortical bone. We found that the senescence of osteoprogenitors was increased, whereas the expression of phosphorylated epidermal growth factor receptor (EGFR) on the endosteal surface of cortical bone down-regulated in middle-aged 15-mo-old mice compared with young 3-mo-old mice. Further decreasing EGFR signaling by gefitinib treatment in middle-aged mice resulted in promoted senescence of osteoprogenitors and accelerated cortical bone degeneration. Moreover, inhibiting EGFR signaling suppressed the expression of enhancer of zeste homolog 2 (Ezh2), the repressor of cell senescence-inducer genes, through ERK1/2 pathway, thereby promoting senescence in osteoprogenitors. Down-regulated EGFR signaling plays a physiologically significant role during aging by reducing Ezh2 expression, leading to the senescence of osteoprogenitors and the decline in bone formation on the endosteal surface of cortical bone.-Liu, G., Xie, Y., Su, J., Qin, H., Wu, H., Li, K., Yu, B., Zhang, X. The role of EGFR signaling in age-related osteoporosis in mouse cortical bone.


Assuntos
Osso Cortical/metabolismo , Receptores ErbB/metabolismo , Osteoporose/metabolismo , Transdução de Sinais/fisiologia , Envelhecimento/metabolismo , Animais , Senescência Celular/fisiologia , Regulação para Baixo/fisiologia , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Feminino , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Osteogênese/fisiologia
19.
Clin Sci (Lond) ; 133(12): 1297-1308, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31175224

RESUMO

Bone loss in Staphylococcus aureus (S. aureus) osteomyelitis poses a serious challenge to orthopedic treatment. The present study aimed to elucidate how S. aureus infection in bone might induce bone loss. The C57BL/6 mice were injected with S. aureus (106 CFU/ml, 100 µl) or with the same amount of vehicle (control) via the tail vein. Microcomputed tomography (microCT) analysis showed bone loss progressing from week 1 to week 5 after infection, accompanied by a decreased number of osteocalcin-positive stained osteoblasts and the suppressed mRNA expression of Runx2 and osteocalcin. Transcriptome profiles of GSE30119 were downloaded and analyzed to determine the differences in expression of inflammatory factors between patients with S. aureus infected osteomyelitis and healthy controls, the data showed significantly higher mRNA expression of granulocyte colony-stimulating factor (G-CSF) in the whole blood from patients with S. aureus infection. Enzyme-linked immunosorbent assay (ELISA) analysis confirmed an increased level of G-CSF in the bone marrow and serum from S. aureus infected mice, which might have been due to the increased amount of F4/80+ macrophages. Interestingly, G-CSF neutralizing antibody treatment significantly rescued the bone loss after S. aureus infection, as evidenced by its roles in improving BV/TV and preserving osteocalcin- and osterix-positive stained cells. Importantly, we found that G-CSF level was significantly up-regulated in the serum from osteomyelitis patients infected by S. aureus Together, S. aureus infection might suppress the function of osteoblastic cells and induce progressive bone loss by up-regulating the level G-CSF, suggesting a therapeutic potential for G-CSF neutralization in combating bone loss in S. aureus osteomyelitis.


Assuntos
Remodelação Óssea , Fator Estimulador de Colônias de Granulócitos/metabolismo , Osteoblastos/metabolismo , Osteomielite/metabolismo , Infecções Estafilocócicas/metabolismo , Tíbia/metabolismo , Adulto , Animais , Anticorpos Neutralizantes/farmacologia , Remodelação Óssea/efeitos dos fármacos , Estudos de Casos e Controles , Modelos Animais de Doenças , Feminino , Fator Estimulador de Colônias de Granulócitos/antagonistas & inibidores , Fator Estimulador de Colônias de Granulócitos/imunologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , Osteoblastos/efeitos dos fármacos , Osteoblastos/microbiologia , Osteomielite/diagnóstico por imagem , Osteomielite/tratamento farmacológico , Osteomielite/microbiologia , Transdução de Sinais , Infecções Estafilocócicas/diagnóstico por imagem , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Tíbia/diagnóstico por imagem , Tíbia/efeitos dos fármacos , Tíbia/microbiologia , Fatores de Tempo , Microtomografia por Raio-X
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