Skeletal fluorosis is a chronically metabolic bone disease with extensive hyperostosis osteosclerosis caused by long time exposure to fluoride. Skeletal fluorosis brings about a series of abnormal changes of the extremity, such as joint pain, joint stiffness, bone deformity, etc. Differentiation and maturation of osteoblasts were regulated by osteoclasts via Sema4D/Plexin-B1 signaling pathway. Furthermore, the differentiation and maturation of osteoclasts are conducted by osteoblasts via RANKL/RANK/OPG pathway. Both of these processes form a feedback circuit which is a key link in skeletal fluorosis. In this study, an osteoblast-osteoclast co-culture model in vitro was developed to illustrate the mechanism of skeletal fluorosis. With the increase of fluoride concentration, the expression level of Sema4D was decreased and TGF-β1 was increased continuously. OPG/RANKL mRNA level, however, increased gradually. On the basis of that, the inhibition of Sema4D/Plexin-B1/RhoA/ROCK signaling pathway caused by fluoride promoted the level of TGF-β1 and activated the proliferation of osteoblasts. In addition, osteroprotegerin (OPG) secreted by osteoblasts was up-regulated by fluoride. The competitive combination of OPG and RANKL was strengthened and the combination of RANKL and RANK was hindered. And then the differentiation and maturation of osteoclasts were inhibited, and bone absorption was weakened, leading to skeletal fluorosis.
Animals ; Antigens, CD ; genetics ; metabolism ; Cell Proliferation ; drug effects ; Feedback, Physiological ; Fetus ; Fluorides ; pharmacology ; GTPase-Activating Proteins ; genetics ; metabolism ; Gene Expression Regulation, Developmental ; Osteoblasts ; drug effects ; metabolism ; pathology ; Osteoclasts ; drug effects ; metabolism ; pathology ; Osteogenesis ; drug effects ; genetics ; Osteoprotegerin ; genetics ; metabolism ; RANK Ligand ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Receptor Activator of Nuclear Factor-kappa B ; genetics ; metabolism ; Receptors, Cell Surface ; genetics ; metabolism ; Semaphorins ; genetics ; metabolism ; Signal Transduction ; Transforming Growth Factor beta1 ; genetics ; metabolism ; rho-Associated Kinases ; genetics ; metabolism ; rhoA GTP-Binding Protein ; genetics ; metabolism

Various kinds of schiff base metal complexes have been proven to induce apoptosis of tumor cells. However, it remains largely unknown whether schiff base zinc complexes induce apoptosis in human cancer cells. Here, we synthesized a novel schiff base zinc coordination compound (SBZCC) and investigated its effects on the growth, proliferation and apoptosis of human osteosarcoma MG-63 cells. A novel SBZCC was synthesized by chemical processes and used to treat MG-63 cells. The cell viability was determined by CCK-8 assay. The cell cycle progression, mitochondrial membrane potential and apoptotic cells were analyzed by flow cytometry. The apoptosis-related proteins levels were determined by immunoblotting. Treatment of MG-63 cells with SBZCC resulted in inhibition of cell proliferation and cell cycle arrest at G1 phase. Moreover, SBZCC significantly reduced the mitochondrial membrane potential and induced apoptosis, accompanied with increased Bax/Bcl-2 and FlasL/Fas expression as well as caspase-3/8/9 cleavage. Our results demonstrated that the synthesized novel SBZCC could inhibit the proliferation and induce apoptosis of MG-63 cells via activating both the mitochondrial and cell death receptor apoptosis pathways, suggesting that SBZCC is a promising agent for the development as anticancer drugs.
Antineoplastic Agents ; chemical synthesis ; pharmacology ; Apoptosis ; drug effects ; Caspase 3 ; genetics ; metabolism ; Caspase 8 ; genetics ; metabolism ; Caspase 9 ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Coordination Complexes ; chemical synthesis ; pharmacology ; Fas Ligand Protein ; genetics ; metabolism ; G1 Phase Cell Cycle Checkpoints ; drug effects ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondria ; drug effects ; metabolism ; pathology ; Osteoblasts ; drug effects ; metabolism ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; Schiff Bases ; chemistry ; Signal Transduction ; Zinc ; chemistry ; bcl-2-Associated X Protein ; genetics ; metabolism ; fas Receptor ; genetics ; metabolism

Aortic valve calcification is a common disease in the elderly, but its cellular and molecular mechanisms are not clear. In order to verify the hypothesis that Wnt/β-catenin signaling pathway is involved in the process of calcification of aortic valve, porcine aortic valve interstitial cells (VICs) were isolated, cultured and stimulated with oxidized low density lipoprotein (ox-LDL) for 48 h to induce the differentiation of VICs into osteoblast-like cells. The key proteins and genes of Wnt/β-catenin signaling pathway, such as glycogen synthase kinase 3β (GSK-3β) and β-catenin, were detected by using Western blotting and real-time polymerase chain reaction (PCR). The results showed that the VICs managed to differentiate into osteoblast-like cells after the stimulation with ox-LDL and the levels of proteins and genes of GSK-3β and β-catenin were increased significantly in VICs after stimulation for 48 h (P<0.05). It is suggested that Wnt/β-catenin signaling pathway may play a key role in the differentiation of VICs into osteoblast-like cells and make great contribution to aortic valve calcification.
Alkaline Phosphatase ; genetics ; metabolism ; Animals ; Aortic Valve ; metabolism ; pathology ; Aortic Valve Stenosis ; Blotting, Western ; Bone Morphogenetic Protein 2 ; genetics ; metabolism ; Calcinosis ; Cell Differentiation ; drug effects ; genetics ; Cells, Cultured ; Gene Expression ; drug effects ; Glycogen Synthase Kinase 3 ; genetics ; metabolism ; Lipoproteins, LDL ; pharmacology ; Osteoblasts ; drug effects ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Swine ; Wnt Signaling Pathway ; genetics ; physiology ; beta Catenin ; genetics ; metabolism

OBJECTIVETo culture osteoblast in vitro and evaluate CCL3 receptor CCR1 expression in patients with multiple myeloma (MM).

METHODSBone marrow osteoblasts from MM patients were cultured in vitro with dexamethasone, β-sodium glycerophosphate and vitamin C, which were identified by alkaline phosphatase staining, Von Kossa's staining. The CCL3 receptor expression was evaluated by flow cytometry. The morphology and quantity of osteoblast were observed after exposure to CCL3.

RESULTSBone marrow osteoblasts from MM patients could be cultured in vitro and be identified by positive staining of alkaline phosphatase and Von Kossa's. MM-derived osteoblasts expressed higher levels of CCR1 (74.48 ± 7.31)%, compared with normal controls (48.35 ± 8.81)%. Calcium deposition of osteoblasts after exposure to CCL3 was less than that of controls.

CONCLUSIONBone marrow osteoblasts could be cultured in vitro from MM Patients. CCL3 may contribute to the development of myeloma bone disease.

Adult ; Aged ; Cells, Cultured ; Chemokine CCL3 ; pharmacology ; Female ; Humans ; Male ; Middle Aged ; Multiple Myeloma ; pathology ; Osteoblasts ; cytology ; drug effects ; metabolism ; Receptors, CCR1 ; metabolism ; Young Adult

OBJECTIVETo investigate the effects of sodium copper chlorophyllin (SCC) on the proliferation, differentiation and immunomodulatory function of mesenchymal stem cells (MSCs) from mice with aplastic anemia.

METHODSA mouse model of aplastic anemia was established by exposure of BALB/c mice to sublethal doses of 5.0 Gy Co60 γ radiation, followed by transplantation of 2×10(6) lymph node cells from DBA/2 donor mice within 4 h after radiation. Aplastic anemic BALB/c mice were randomly divided into six groups: the treated groups, which received 25, 50, or 100 mg/kg/day SCC, respectively; a positive control group treated with cyclosporine A (CsA); and an untreated model control group (model group); while, the non-irradiated mice as the normal control group. SCC or CsA were administered by gastrogavage for 20 days, starting on day 4 after irradiation. Peripheral blood cells were counted and colony-forming fibroblasts (CFU-F) in the bone marrow were assayed. The ability of MSCs to form calcium nodes after culture in osteoinductive medium was also observed. The immunosuppressive effect of MSCs on T lymphocytes was analyzed by enzyme-linked immunosorbent assay and flow cytometry, to evaluate the efficacy of SCC in mice with aplastic anemia.

RESULTSPeripheral blood white cell and platelet counts were increased by medium and high SCC doses, compared with the untreated control. CFU-Fs were also increased compared with the untreated control, and the numbers of calcium nodes in MSCs in osteoinductive medium were elevated in response to SCC treatment. The percentage of Forkhead box protein 3 (FOXP3(+)) T cells was increased in T cell-MSC cocultures, and the cytokine transforming growth factor β1 was up-regulated in SCC-treated groups.

CONCLUSIONThe results of this study suggest that SCC not only promotes the proliferation and differentiation of MSCs, but also improves their immunoregulatory capacity in mice with aplastic anemia.

Anemia, Aplastic ; blood ; pathology ; therapy ; Animals ; Anthraquinones ; metabolism ; Biomarkers ; metabolism ; Bone Marrow Cells ; drug effects ; pathology ; Calcium ; metabolism ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Chlorophyllides ; pharmacology ; Colony-Forming Units Assay ; Female ; Immunosuppression ; Leukocyte Count ; Male ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; cytology ; drug effects ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Inbred DBA ; Osteoblasts ; drug effects ; pathology ; Platelet Count ; T-Lymphocytes ; drug effects

OBJECTIVETo evaluate the roles or effects of oviductus ranae (OR) or oviductus ranae eggs (ORE) in preventing and treating postmenopausal osteoporosis.

METHODSIn vivo experiment: Sixty female adult Wistar rats were randomly divided into 5 groups of 12. To provide an osteoporosis model 4 groups of rats were ovariectomized (OVX), with the 5th being sham operated. Medication commenced 7 days after the operation and lasted continuously for 12 weeks. Sham operated and OVX groups were given equivalent volumes of 5% Tween-80. The other three groups intragastrically received conjugated estrogens (CE), OR or ORE of the corresponding doses. At the 12th week, serum estrogen, bone gla protein (BGP), serum calcium, phosphorus, and alkaline phosphatase (ALP) were assayed; bone mineral densities (BMD) were measured and bone scanning was conducted; uteri were weighed, and weight, volume and length of the femoral bones were determined; and cortical thickness of femoral heads and area of bone trabecula were measured by image analyzer. In vitro experiment: Eighty 10-month old SD rats, with equal numbers of males and females, were randomly divided into 8 groups. Osteoblasts were isolated from neonatal rat calvariae, and the cells were exposed to various concentrations of serum from OR and ORE groups to study the impact of these sera on osteoblastic proliferation, ALP activity and mineralization. Osteoclastic numbers were determined using tartrate resistant acid phosphatase (TRAP).

RESULTSIn vivo experiment: The body weight of the four OVX groups increased significantly (P<0.01). Uterine weight of the CE group was the highest (P<0.01); Compared with the model group, estrogen level, BMD, bone scanning/bone imaging index weight of the femoral bones, cortical thickness of femoral heads in the OR and ORE groups increased significantly (P<0.05, P<0.01); femoral volume in the ORE group increased significantly (P<0.05); and the content of osteocalcin, phosphorus, and ALP in serum decreased significantly (P<0.05, P<0.01). In vitro experiment: Sera from OR and ORE groups had notable effects on the proliferation of osteoblasts (P<0.05 and P<0.01, repsectively) and stimulated the formation of calcium nodes (P<0.05, P<0.01), while the enhancement of ALP activity in osteoblasts was significant (P<0.05, P<0.01). The number of TRAP-positive cells was significantly reduced as well (P<0.01).

CONCLUSIONSOR and its eggs could effectively suppress OVX-induced osteoporosis in rats, and increase bone turnover possibly by both an increase in osteoblastic activity and a decrease in osteoclastic activity. The present study provides evidence that OR and its eggs could be considered a complementary and alternative medicine for the treatment of postmenopausal osteoporosis.

Acid Phosphatase ; metabolism ; Alkaline Phosphatase ; metabolism ; Animals ; Biomarkers ; blood ; Body Weight ; drug effects ; Bone Density ; drug effects ; Bone and Bones ; metabolism ; Calcification, Physiologic ; drug effects ; Cell Count ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Female ; Femur ; drug effects ; metabolism ; pathology ; Isoenzymes ; metabolism ; Male ; Materia Medica ; pharmacology ; therapeutic use ; Organ Size ; drug effects ; Osteoblasts ; drug effects ; enzymology ; pathology ; Osteoclasts ; drug effects ; enzymology ; pathology ; Osteoporosis ; blood ; drug therapy ; metabolism ; physiopathology ; Ovariectomy ; Ovum ; metabolism ; Rats ; Rats, Wistar ; Tartrate-Resistant Acid Phosphatase ; Uterus ; drug effects ; pathology

The effect of the anti-inflammatory flavonoid chrysin on osteogenesis was determined in preosteoblast MC3T3-E1 cells. Results demonstrated that chrysin could induce osteogenic differentiation in the absence of other osteogenic agents. Chrysin treatment promoted the expression of transcription factors (Runx2 and Osx) and bone formation marker genes (Col1A1, OCN, and OPN) as well as enhanced the formation of mineralized nodules. During osteogenic differentiation, chrysin preferentially activated ERK1/2, but not JNK nor the p38 MAPKs. Further experiments with inhibitors revealed the co-treatment of U0126, PD98059, or ICI182780 (a general ER antagonist) with chrysin effectively abrogated the chrysin-induced osteogenesis and ERK1/2 activation. Thus, the effect of chrysin on osteogenesis is ERK1/2-dependent and involves ER. Therefore, chrysin has the significant potential to enhance osteogenesis for osteoporosis prevention and treatment.
Animals ; Cell Differentiation ; drug effects ; Cell Line ; Enzyme Activation ; drug effects ; Flavonoids ; pharmacology ; therapeutic use ; Mice ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Mitogen-Activated Protein Kinases ; metabolism ; Osteoblasts ; drug effects ; pathology ; Osteogenesis ; drug effects ; Osteoporosis ; drug therapy ; pathology ; Phosphorylation ; drug effects ; Receptors, Estrogen ; metabolism

BACKGROUNDMost hydatid cysts with calcified walls are biologically and clinically silent and inactive. Transforming growth factor-beta 1 (TGF-β1) plays a critical role in the calcification process of cells. The aim of this study was to assess the effect of modulating TGF-β1 signaling on the calcification of hydatid cysts.

METHODSPericyst cells isolated from hepatic hydatid cysts were cultured with osteogenic media. These cells were assessed for alkaline phosphatase activity and mineralization capacity using Alizarin Red staining. Cells were also treated with recombinant human TGF-β1 and TGF-β inhibitor, and the expression profiles of osteoblast markers (RUNX2, osterix, and osteocalcin) were analyzed using Western blotting. The effects of inhibiting TGF-β1 signaling on calcification of pericyst walls were assessed using different doses of TGF-β inhibitor for 7 weeks in a preclinical disease model of liver cystic echinococcosis.

RESULTSCells within the pericyst displayed high levels of alkaline phosphatase activity and mineralized nodule formation, as induced by osteogenic media. These activities, as well as expression profiles of osteoblast markers (RUNX2, osterix, and osteocalcin) could be inhibited by addition of recombinant human TGF-β1 (rhTGF-β1) and enhanced by TGF-β inhibitor. In the animal model of cystic echinococcosis, inhibition of TGF-β1 signaling increased calcification of the pericyst wall, which was associated with decreased cyst load index and lower viability of protoscoleces.

CONCLUSIONSCells within the pericysts adopt an osteoblast-like phenotype and have osteogenic potential. Inhibition of TGF-β1 signaling increases hydatid cyst calcification. Pharmacological modulation of calcification in pericysts may be a new therapeutic target in the treatment of hydatid disease.

Animals ; Blotting, Western ; Calcification, Physiologic ; drug effects ; Cell Differentiation ; drug effects ; Core Binding Factor alpha Subunits ; metabolism ; Echinococcosis ; metabolism ; pathology ; Echinococcus granulosus ; pathogenicity ; Enzyme Inhibitors ; pharmacology ; Humans ; Male ; Mice ; Osteoblasts ; cytology ; Osteocalcin ; metabolism ; Recombinant Proteins ; pharmacology ; Sp7 Transcription Factor ; Transcription Factors ; metabolism ; Transforming Growth Factor beta1 ; antagonists & inhibitors ; metabolism

Parathyroid hormone-related protein (PTHrP) is synthesized by diverse tissues, and its processing produces several fragments, each with apparently distinct autocrine and paracrine bioactivities. In bone, PTHrP appears to modulate bone formation in part through promoting osteoblast differentiation. The putative effect of PTH-like and PTH-unrelated fragments of PTHrP on human mesenchymal stem cell (MSCs) is not well known. Human MSCs were treated with PTHrP (1-36) or PTHrP (107-139) or both (each at 10 nM) in osteogenic or adipogenic medium, from the start or after 6 days of exposure to the corresponding medium, and the expression of several osteoblastogenic and adipogenic markers was analyzed. PTHrP (1-36) inhibited adipogenesis in MSCs and favoured the expression of osteogenic early markers. The opposite was observed with treatment of MSCs with PTHrP (107-139). Moreover, inhibition of the adipogenic differentiation by PTHrP (1-36) prevailed in the presence of PTHrP (107-139). The PTH/PTHrP type 1 receptor (PTH1R) gene expression was maximum in the earlier and later stages of osteogenesis and adipogenesis, respectively. While PTHrP (107-139) did not modify the PTH1R overexpression during adipogenesis, PTHrP (1-36) did inhibit it; an effect which was partially affected by PTHrP (7-34), a PTH1R antagonist, at 1 microM. These findings demonstrate that both PTHrP domains can exert varying effects on human MSCs differentiation. PTHrP (107-139) showed a tendency to favor adipogenesis, while PTHrP (1-36) induced a mild osteogenic effect in these cells, and inhibited their adipocytic commitment. This further supports the potential anabolic action of the latter peptide in humans.
Adipogenesis/drug effects ; Alkaline Phosphatase/biosynthesis/genetics ; Antigens, Differentiation/biosynthesis/genetics ; Bone Marrow/pathology ; Cell Differentiation/drug effects ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit/biosynthesis/genetics ; Culture Media ; Gene Expression Regulation ; Humans ; Lipoprotein Lipase/biosynthesis/genetics ; Mesenchymal Stem Cells/*drug effects/metabolism/pathology ; Osteoblasts/drug effects/*metabolism/pathology ; Osteogenesis/drug effects ; PPAR gamma/biosynthesis/genetics ; Parathyroid Hormone/*pharmacology ; Peptide Fragments/*pharmacology ; Receptor, Parathyroid Hormone, Type 1/antagonists & inhibitors

OBJECTIVEOn the basis of YouGui drink ([Chinese characters: see text]) to act on the normal rats in vitro osteoblast proliferation and differentiation, further observe YooGui drink ([Chinese characters: see text]) inductive effect for steroid induced necrosis of femoral head rats in vitro on cultivation of osteoblast proliferation and differentiation.

METHODSPrednisolone acetate 49 mg/kg x d were injected into gluteal of 20 male SD rats (body weight in 100-120 g), continuously 6 days for model. After 7 days, under sterile conditions from the model rats bone marrow stromal cells were induced and cultured to osteoblast, and then randomly divided into 4 groups, respectively, add the high, medium and low concentration YouGui drink ([Chinese characters: see text]) containing serum (group R1, R2, R3) and control serum (group R4). After 72 h, osteoblast proliferation, alkaline phosphatase activities (APA) were detected, and expression level of osteoprotegerin (OPG) and osteoclast differentiation factor (RANKL) were detected by real-time fluorescence quantitative PCR.

RESULTSThe high and medium concentration YouGui drink ([Chinese characters: see text]) containing serum group compared with the control group (R1, R2 vs. R4), the rate of osteoblast proliferation was significantly high (P < 0.01), APA significantly increased (P < 0.01), relative expression level of osteoblast OPG mRNA significantly increased (P < 0.01), and with the concentration of a certain dose was positively correlated; RANKL mRNA significantly was lower (P < 0.01). The low concentration YouGui drink (t 91 t) containing serum group compared with the control group (R3 vs R4), there was no significantly different in the rate of osteoblast proliferation, ALP and relative expression level of osteoblast OPG mRNA, RANKL mRNA.

CONCLUSIONThe high concentration of YouGui drink ([Chinese characters: see text]) containing serum can obviously promote the proliferation and differentiation of steroid induced necrosis of femoral head rats in vitro osteoblast, and plays a clear role in promoting and enhance the relative expression of osteoblast OPG mRNA and an inhibitory effect on RANKL mRNA. The effect of YouGui drink ([Chinese characters: see text]) containing serum acting on induced in vitro cultured osteoblasts have relevance with dose, at a certain concentration range it can promote osteoblast differentiation and proliferation.

Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Drinking ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Femur Head Necrosis ; chemically induced ; drug therapy ; metabolism ; pathology ; Gene Expression Regulation ; drug effects ; Male ; Osteoblasts ; drug effects ; metabolism ; pathology ; Osteoprotegerin ; genetics ; RANK Ligand ; genetics ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Steroids ; pharmacology