BACKGROUND: Periodontitis is characterized by alveolar bone resorption, aggravated by osteoblast dysfunction, and associated with intracellular oxidative stress linked to the nuclear factor erythroid 2-related factor 2 (NRF2) level. We evaluated the molecular mechanism of periodontitis onset and development and the role of NRF2 in osteogenic differentiation. METHODS: Primary murine mandibular osteoblasts were extracted and exposed to Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) or other stimuli. Reactive oxygen species (ROS) and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining were used to detect intracellular oxidative stress. Alkaline phosphatase staining and alizarin red S staining were used to detect the osteogenic differentiation of osteoblasts. Immunofluorescence and western blotting were used to determine the changes in the mitogen-activated protein kinase (MAPK) pathway and related molecule activities. Immunofluorescence colocalization and co-immunoprecipitation were performed to examine the nuclear translocation of NRF2 and its interaction with dual-specific phosphatase 1 (DUSP1) in cells. RESULTS: Ligated tissue samples showed higher alveolar bone resorption rate and lower NRF2 level than healthy periodontal tissue samples. Pg-LPS increased intracellular oxidative stress levels and inhibited osteogenic differentiation, whereas changes in NRF2 expression were correlated with changes in the oxidative stress and osteogenesis rate. NRF2 promoted the dephosphorylation of the MAPK pathway by nuclear translocation and the upregulation of DUSP1 expression, thus enhancing the osteogenic differentiation capacity of mandibular osteoblasts. The interaction between NRF2 and DUSP1 was observed. CONCLUSIONS: NRF2 and its nuclear translocation can regulate the osteogenic differentiation of mandibular osteoblasts under Pg-LPS conditions by interacting with DUSP1 in a process linked to the MAPK pathway. These findings form the basis of periodontitis treatment.
Animals ; NF-E2-Related Factor 2/physiology* ; Lipopolysaccharides/pharmacology* ; Osteoblasts/drug effects* ; Mice ; Porphyromonas gingivalis/chemistry* ; Cell Differentiation ; Osteogenesis ; Dual Specificity Phosphatase 1/metabolism* ; Mandible/cytology* ; Reactive Oxygen Species/metabolism* ; Oxidative Stress ; Periodontitis/metabolism* ; Cells, Cultured ; Male ; Cell Nucleus/metabolism*

This study investigated the regulatory potential of salidroside (SAL), a primary active compound in Rhodiola rosea L., on osteoclast differentiation by modulating the hypoxia-inducible factor 1-alpha (HIF-1a) pathway in osteoblasts. Luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay were employed to validate whether the receptor activator of nuclear factor-?B ligand (RANKL) is the downstream target gene of HIF-1a in osteoblasts. The study also utilized lipopolysaccharide (LPS)-induced mouse osteolysis to examine the impact of SAL on osteolysis in vivo. Furthermore, conditioned medium (CM) from SAL-pretreated osteoblasts was used to investigate the paracrine effects on osteoclastogenesis through the HIF-1a pathway. Hypoxic condition-induced overexpression of HIF-1a upregulated RANKL levels by binding to the RANKL promoter and enhancing transcription in osteoblastic cells. In vivo, SAL significantly alleviated bone tissue hypoxia and decreased the expression of HIF-1a by downregulating the expression of RANKL, vascular endothelial growth factor (VEGF), interleukin 6 (IL-6), and angiopoietin-like 4 (ANGPTL4). In the paracrine experiment, conditioned media from SAL-pretreated osteoblasts inhibited differentiation through the HIF-1a/RANKL, VEGF, IL-6, and ANGPTL4 pathways. RANKL emerges as the downstream target gene regulated by HIF-1a in osteoblasts. SAL significantly alleviates bone tissue hypoxia and bone loss in LPS-induced osteolysis through the HIF-1a/RANKL, VEGF, IL-6, and ANGPTL4 pathways. SAL inhibits osteoclast differentiation by regulating osteoblast paracrine secretion.
Animals ; Osteoblasts/cytology* ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics* ; Glucosides/administration & dosage* ; Cell Differentiation/drug effects* ; Phenols/administration & dosage* ; Mice ; Osteoclasts/metabolism* ; RANK Ligand/genetics* ; Rhodiola/chemistry* ; Osteogenesis/drug effects* ; Signal Transduction/drug effects* ; Interleukin-6/genetics* ; Male ; RAW 264.7 Cells ; Osteolysis/genetics* ; Humans ; Mice, Inbred C57BL

Objective: To investigate the effect of icaritin on maturation and mineralization of mouse osteoblast MC3T3-E1 cells and its mechanism. Methods: The cultured MC3T3-E1 cells were divided into blank control group, CXC chemokine receptor type 4 (CXCR4) inhibitor (AMD3100) group, icaritin group, and icaritin plus AMD3100 group. The expression of CXCR4, stromal cell-derived factor 1 (SDF-1) and osteogenesis-related genes and proteins were detected by real-time RT-PCR and Western blotting after drug treatment for 24 h. The alkaline phosphatase (ALP) activity was determined with ALP kit on d3 and d6; calcium nodules were detected by alizarin red staining after drug treatment for 14 d. Results: Real time RT-PCR showed that compared with the blank control group, relative expressions of CXCR4, SDF-1 and osteogenesis-related genes in icaritin group were significantly increased (P<0.05 or P<0.01); After AMD3100 treatment, the relative expression of CXCR4 gene was decreased (P<0.05). Western blot showed that compared with the blank control group, relative expressions of CXCR4, SDF-1 and osteogenesis-related proteins in the icaritin group were significantly increased (all P<0.01), but were decreased after AMD3100 was added (all P<0.01). The ALP activity of icaritin group was significantly higher than that of blank control group (all P<0.01) on d3 and d6 after drug treatment, while the activity of ALP was significantly decreased after AMD3100 treatment (all P<0.01). At d14 after drug treatment, compared with the blank control group, the area of alizarin red staining was increased in the icaritin group, while it was significantly reduced after the addition of AMD3100. Conclusion: Icaritin may promote maturation and mineralization of mouse osteoblast MC3T3-E1 cells through CXCR4/SDF-1 signaling pathway.
3T3 Cells ; Animals ; Calcification, Physiologic ; drug effects ; Chemokine CXCL12 ; metabolism ; Flavonoids ; pharmacology ; Gene Expression Regulation, Developmental ; drug effects ; Mice ; Osteoblasts ; cytology ; drug effects ; Receptors, CXCR4 ; metabolism ; Signal Transduction ; drug effects

There is an increasing interest in phytoestrogens due to their potential medical usage in hormone replacement therapy (HRT). The present study was designed to investigate the in vitro effects of estrogen-like activities of two widespread coumarins, osthole and imperatorin, using the MCF-7 cell proliferation assay and their alkaline phosphatase (ALP) activities in osteoblasts Saos-2 cells. The two compounds were found to strongly stimulate the proliferation of MCF-7 cells. The estrogen receptor-regulated ERα, progesterone receptor (PR) and PS2 mRNA levels were increased by treatment with osthole and imperatorin. All these effects were significantly inhibited by the specific estrogen receptor antagonist ICI182, 780. Cell cycle analysis revealed that their proliferation stimulatory effect was associated with a marked increase in the number of MCF-7 cells in S phase, which was similar to that observed with estradiol. It was also observed that they significantly increased ALP activity, which was reversed by ICI182,780. These results suggested that osthole and imperatorin could stimulate osteoblastic activity by displaying estrogenic properties or through the ER pathway. In conclusion, osthole and imperatorin may represent new pharmacological tools for the treatment of osteoporosis.
Alkaline Phosphatase ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cnidium ; chemistry ; Coumarins ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Furocoumarins ; pharmacology ; Humans ; MCF-7 Cells ; Osteoblasts ; cytology ; drug effects ; enzymology ; Phytoestrogens ; pharmacology ; Receptors, Estrogen ; genetics ; metabolism

The Wnt/beta-catenin pathway has a role in osteoblast differentiation and bone formation. We screened 100 plant extracts and identified an extract from Euodia sutchuenensis Dode (ESD) leaf and young branch as an effective activator of the Wnt/beta-catenin pathway. ESD extract increased beta-catenin levels and beta-catenin nuclear accumulation in murine primary osteoblasts. The ESD extract also increased mRNA levels of osteoblast markers, including RUNX2, BMP2 and COL1A1, and enhanced alkaline phosphatase (ALP) activity in murine primary osteoblasts. Both ESD extract-induced beta-catenin increment and ALP activation were abolished by beta-catenin knockdown, confirming that the Wnt/beta-catenin pathway functions in osteoblast differentiation. ESD extract enhanced terminal osteoblast differentiation as shown by staining with Alizarin Red S and significantly increased murine calvarial bone thickness. This study shows that ESD extract stimulates osteoblast differentiation via the Wnt/beta-catenin pathway and enhances murine calvarial bone formation ex vivo.
Animals ; Cell Differentiation/*drug effects ; Evodia/*chemistry ; HEK293 Cells ; Humans ; Mice ; Osteoblasts/cytology/*drug effects/*metabolism ; Osteogenesis/drug effects ; Plant Extracts/chemistry/*pharmacology ; Skull/anatomy & histology/drug effects/metabolism ; Wnt Signaling Pathway/*drug effects ; beta Catenin/genetics/metabolism

In order to investigate the roles of Wnt signal pathway in transformation of cardiac valvular myofibroblasts to the osteoblast-like phenotype, the primary cultured porcine aortic valve myofibroblasts were incubated with oxidized low density lipoprotein (ox-LDL, 50 mg/L), and divided into four groups according to the ox-LDL treatment time: control group, ox-LDL 24-h group, ox-LDL 48-h group, and ox-LDL 72-h group. Wnt signal pathway blocker Dickkopf-1 (DDK-1, 100 μg/L) was added in ox-LDL 72-h group. The expression of a-smooth muscle actin (α-SMA), bone morphogenetic protein 2 (BMP2), alkaline phosphatase (ALP), and osteogenic transcription factor Cbfa-1 was detected by Western blotting, and that of β-catenin, a key mediator of Wnt signal pathway by immunocytochemical staining method. The Wnt/β-catenin was observed and the transformation of myofibroblasts to the osteoblast-like phenotype was examined. The expression of α-SMA, BMP2, ALP and Cbfa-1 proteins in the control group was weaker than in the ox-LDL-treated groups. In ox-LDL-treated groups, the protein expression of a-SMA, BMP2, ALP, and Cbfa-1 was significantly increased in a time-dependent manner as compared with the control group, and there was significant difference among the three ox-LDL-treated groups (P<0.05 for all); β-catenin protein was also up-regulated in the ox-LDL-treated groups in a time-dependent manner as compared with the control group (P<0.05), and its transfer from cytoplasm to nucleus and accumulation in the nucleus were increased in the same fashion (P<0.05). After addition of DKK-1, the expression of α-SMA, bone-related proteins and β-catenin protein was significantly reduced as compared with ox-LDL 72-h group (P<0.05). The Wnt/ β-catenin signaling pathway may play an important role in transformation of valvular myofibroblasts to the osteoblast-like phenotype.
Actins ; metabolism ; Animals ; Aortic Valve ; cytology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Gene Expression Regulation ; drug effects ; Intercellular Signaling Peptides and Proteins ; pharmacology ; Lipoproteins, LDL ; pharmacology ; Myofibroblasts ; drug effects ; Osteoblasts ; physiology ; Phenotype ; Swine ; Wnt Signaling Pathway ; drug effects ; beta Catenin ; metabolism

OBJECTIVETo evaluate the effects of ginsenoside Rg-1 on the proliferation and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and to explore the possible application on the alveolar bone regeneration.

METHODSTo determine the optimum concentration, the effects of ginsenoside Rg-1 ranging from 10 to 100 μmol/L were evaluated by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide, alkaline phosphatase activity and calcium deposition. Expressions of runt-related transcription factor 2, collagen alpha-2(I) chain, osteopontin, osteocalcin protein were examined using real-time polymerase chain reaction.

RESULTSCompared with the control group, a certain concentration (10 μmol/L) of the Rg-1 solution significantly enhanced the proliferation and osteogenic differentiation of hPDLSCs (P<0.05). However, concentrations that exceeds 100 μmol/L led to cytotoxicity whereas concentrations below 10 nmol/L showed no significant effect as compared with the control.

CONCLUSIONGinsenoside Rg-1 can enhance the proliferation and osteogenic differentiation of hPDLSCs at an optimal concentration of 10 μmol/L.

Adolescent ; Alkaline Phosphatase ; metabolism ; Biomarkers ; metabolism ; Calcification, Physiologic ; drug effects ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cell Separation ; Cell Shape ; drug effects ; Cells, Cultured ; Flow Cytometry ; Ginsenosides ; pharmacology ; Humans ; Osteoblasts ; drug effects ; metabolism ; Osteogenesis ; drug effects ; genetics ; Periodontal Ligament ; cytology ; Real-Time Polymerase Chain Reaction ; Stem Cells ; cytology ; drug effects ; enzymology ; Time Factors ; Young Adult

Aortic valve calcification (AVC) is a pathological process correlated with multiple disease causes and actively regulated by cardiac valve cells. In this study, porcine aortic valve myofibroblasts cultured in vitro were treated with 50 μg z L(-1) of pathological factor tumor necrosis factor α (TNF-α). Tanshinone II A (TSN) with the concentration of 50 mg x L(-1) and TNF-α were combined in incubating cells for 72 h (3 d) and 120 h (5 d). The Western blotting and Real-time PCR were adopted to detect the changes in smooth muscle α actin (α-SMA), bone morphogenetic protein 2 ( BMP2), alkaline phosphatase (ALP) in cells, and expressions of key effect proteins GSK-3β and β-catenin on Wnt/β-catenin signal pathway. According to the findings, TNF-α can significantly increase the expression of myofibroblasts α-SMA and add the transformation activity to them, with nearly no expression of BMP2, ALP and mRNA in the control group and the TSN group but significant increase in their expressions in the TNF-α group (P < 0.01), which showed osteoblast-like phenotype. Moreover, TNF-α down-regulated the expression of up-streaming regulator GSK-3β and mRNA expression (P < 0. 01) , notably increased the expression of key effect protein β-catenin, but with no significant difference in mRNA with the control group and the TSN group. The result demonstrated that TSN showed a certain inhibitory effect on TNF-α's pathological impact (P < 0.05) in a time-dependent manner. Inflammatory factor TNF-α may promote the transformation of aortic valvular myofibroblasts to osteoblast-like phenotype by activating Wnt/β-catenin signal pathway in aortic valvular myofibroblasts, so as to cause AVC. Tanshinone II A can have a preventive effect in AVC by activating GSK-3β proteins and regulating signal transduction of Wnt/β-catenin signal pathway.
Animals ; Aortic Valve ; cytology ; drug effects ; metabolism ; Cells, Cultured ; Diterpenes, Abietane ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Glycogen Synthase Kinase 3 ; genetics ; metabolism ; Glycogen Synthase Kinase 3 beta ; Myofibroblasts ; cytology ; drug effects ; metabolism ; Osteoblasts ; cytology ; drug effects ; metabolism ; Swine ; Tumor Necrosis Factor-alpha ; genetics ; metabolism ; beta Catenin ; genetics ; metabolism

OBJECTIVETo observe effects of serum Bushen Huoxue prescription(Chinese characters) on classic Wnt/β-catenin signaling pathways of osteoblasts, and explore mechanism of Bushen Huoxue prescription (Chinese characters) for preventing osteoporosis.

METHODSTwenty health female rats were randomly divided into two groups, including Bushen Huoxue (Chinese characters) group and saline group,10 in each group. Bushen Huoxue (Chinese characters) group and Saline group were gavaged Bushen Huoxue and saline every day for 1 week. Bushenhuoxue containing serum and saline containing serum were got according to methods of serum preparation of drug-containing. The osteoblasts was cultured with neonatal rat skull according to Enzyme Consumer Law, and was identified by Wright-Giemsa staining (R-J) and alkaline phosphatase staining (ALP). The third generation of osteoblasts was divided into three groups, including saline group, normal group,Bushen Huoxue (Chinese characters) group. Each group were added to 15% appropriate medium. ALP activity of osteoblasts and osteoblasts proliferation rate were tested, mineralized nodules were observed, the expression of β-catenin, Runx2, Osx mRNA of osteoblasts were tested by RT-PCR.

RESULTSThere was blue granules in cytoplasm, cell nucleus was flint with 1 to 3 nucleoli showed by R-J staining, morphology of osteoblasts were cultured. ALP staining showed cytoplasm with purple granules, the results showed that the cultured cell was osteoblasts. The content of ALP in Bushen Huoxue (Chinese characters)group was (6.272±0.131) ,appreciation rate was (0.81? 0.172), and could significantly improve differentiation and proliferation activity of osteoblasts compared with Saline group (P< 0.01). There were four different size orange nodules, the Maximun nodule was 1.0 x 1.0 cm in Bushen Huoxue (Chinese characters) group after Alizarin red staining, the results showed Bushen Huoxue (Chinese characters)group could obviously improve mineralization of osteoblasts. The expression of mRNA of β-catenin, Runx2 and Osx in Bushen Huoxue (Chinese characters)group were (1.782±0.944), (1.935±0.994) and (1.610±0.811) by RT-PCR,it was significantly increased compared with saline group (P<0.01), but there was no difference between Bushen Huoxue (Chinese characters)group and normal group (P>0.05).

CONCLUSIONBushen Huoxue (Chinese characters)group could obviously promote differentiation, proliferation and mineralization of osteoblasts through activation of Wnt, β-catenin signaling pathway. It suggested that the mechanism of action of Bushen Huoxue (O'f f Il.t)particle clould prevent osteoporosis through the activation of Wnt, β-catenin signaling pathway.

Animals ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Female ; Humans ; Osteoblasts ; cytology ; drug effects ; metabolism ; Osteoporosis ; drug therapy ; genetics ; metabolism ; physiopathology ; Rats ; Rats, Sprague-Dawley ; Wnt Proteins ; genetics ; metabolism ; Wnt Signaling Pathway ; drug effects ; beta Catenin ; genetics ; metabolism

OBJECTIVETo establish osteoblast model, primary cilla model was removed by chloral hyrate, observe effects of osteoblast primary cilla moved on enhancing ALP staining and calcified nodules staining in electromagnetic field.

METHODSThree 3-day-old male SD rats weighed between 6 and 9 g were killed, cranial osteoblast was drawed and adherencing cultured respectively. Cells were subcultured and randomly divided into 4 groups until reach to fusion states. The four groups included chloral hydrate non-involved group (control group), 2 mM, 4 mM and 8 mM chloral hydrate group, and cultured in 37 °C, 5% CO2 incubator for 72 h. Morphology of primary cilla was observed by laser confocal scanning microscope, and incidence of osteoblast primary cilia was analyzed by Image-Pro Plus 6.0 software. Cells in the correct concentration group which can removed cillia most effectively were selected and divided into 3 groups, including control group (C), Electromagnetic fields group (EMFs), and EMFs with 4 mM chloral hydrate group. DMEM nutrient solution contained 10%FBS were added into three groups and cultured for 9 days and formation of ALP were observed by histochemical staining of alkaline phosphatase. After 12 days' cultivation, formation of mineralization nodes was observed by alizarin red staining.

RESULTSCompared with control group and 2mM chloral hydrate group,4 mM chloral hydrate group could effectively remove osteoblast primary cilla (P<0.01). Removal of osteoblast primary cilla could weaken the formation of ALP and mineralization nodes in osteoblast in EMFS. Compared with EMFs group, the area of ALP and mineralization nodes in EMFs with 4 mM chloral hydrate group were decreased obviously (P<0.01).

CONCLUSION4mM chloral hydrate could effectively remove osteoblast primary cilia. Primary cilla participate in EMFs promoting formation of ALP and mineralization nodes in osteoblast and provide new ideas for exploring mechanism of EMFs promoting osteoblast maturation and mineralization.

Alkaline Phosphatase ; metabolism ; Animals ; Cell Culture Techniques ; instrumentation ; methods ; Cells, Cultured ; Chloral Hydrate ; pharmacology ; Cilia ; drug effects ; enzymology ; physiology ; Male ; Osteoblasts ; cytology ; enzymology ; Rats ; Rats, Sprague-Dawley