To investigate the effect of miR-705 on osteogenic differentiation of mouse embryo osteoblast precursor (MC3T3-E1) cells.miR-705 mimics, inhibitors and negative control were transfected into MC3T3-E1 cells. Alkaline phosphates (ALP) staining were performed and quantified after 7 days of osteogenic medium induction. The mRNA and protein expression levels of runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) were detected by real-time RT-PCR and Western blot after 14 days of osteogenic induction. Alizarin red staining was performed and quantified in MC3T3-E1 cells after 21 days of osteogenic induction.After 7 days of osteogenic induction, ALP staining showed that overexpression of miR-705 significantly reduced ALP activity, whereas knockdown of miR-705 increased ALP activity (all<0.05). Consistently, after 14 days of osteogenic induction, mRNA and protein expressions of Runx2 and OCN were suppressed by overexpression of miR-705, whereas they were promoted by knockdown of miR-705 (all<0.05). After 21 days of osteogenic induction, alizarin red staining showed that overexpression of miR-705 significantly reduced the formation of mineralized node, the opposite results were found in miR-705 knockdown group (all<0.05).miR-705 can inhibit the osteogenic differentiation of MC3T3-E1 cells.
Alkaline Phosphatase ; drug effects ; genetics ; Animals ; Calcification, Physiologic ; drug effects ; genetics ; Cell Differentiation ; drug effects ; genetics ; Core Binding Factor Alpha 1 Subunit ; drug effects ; genetics ; Down-Regulation ; drug effects ; genetics ; Fetal Stem Cells ; Mice ; MicroRNAs ; pharmacology ; Osteoblasts ; drug effects ; Osteocalcin ; drug effects ; genetics ; Osteogenesis ; drug effects ; genetics

OBJECTIVETo observe effects of blood circulation promoting compounds combined with medicinal guides on content of bone glaprotein (BGP), bone morphogenetic protein-2 (BPM-2) and expression of BMP-2 mRNA in rabbits with femoral head necrosis, and explore its mechanism.

METHODSNinety-eight healthy Spragur-Dawley male rabbits were collected and weighted 2.2 to 2.8 kg. Eighty-four rabbits were built femoral head necrosis model by freezing left femoral head in liquid nitrogen, then randomly divided into 6 groups, 14 in each group. The 6 groups included model group,promoting blood circulation to remove meridian obstruction group,promoting blood circulation to remove meridian obstruction combined with achyranthes bidentata group,radix angelicae pubescentis, asarum group, and platycodon grandiflorum group,other 14 rabbits were sham operation group. While drug groups were administrated corresponding Chinese herb after molding,model group and shamp operation group were given saline. Recombinant human granulocyte-colony stimulating factor ( 30 microg x kg(-1) x d(-1))were injected into all rabbits for 7 days. Samples were taken on the second and fourth week,the content of BGP and BMP-2 were detected by enzyme-linked immunosorbent assay (ELSA) and radioimmunoassay (RIA), histopathological changes of left femoral head were observed by Hematoxylin and Eeosin staining (HE), and expression of BMP-2 mRNA were tested by fluorescence in situ hybridization.

RESULTSCompared with sham operation group, the rate of empty lacunae femoral head were obviously increased in model group, and the content of BGP were increased on the second week, and BMP-2 and BMP-2 mRNA were decreased on the fourth week. Compared with model group, the content of BGP, BMP-2 and BMP-2 mRNA were higher both of the second and fourth week in promoting blood circulation to remove meridian obstruction group. The rate of empty la- cunae femoral head were lower in achyranthes bidentata group, BGP, BMP-2 and BMP-2 mRNA were higher on the fourth week. The rate of empty lacunae femoral head were lower in platycodon grandiflorum group, and BGP were decreased on the second and fourth week, BMP-2 were lower on the second week ,while BMP-2 mRNA were decreased on the fourth week; the content of BMP-2 and BMP-2 mRNA were increased in radix angelicae pubescentis group on the second week; while there was no change in asarum group.

CONCLUSIONRadix angelicae pubescentis can increase the content of BGP, BMP-2 and expression of BMP-2 mRNA ,which is an effective mechanism of preventing femoral head necrosis.

Animals ; Blood Circulation ; drug effects ; Bone Morphogenetic Protein 2 ; analysis ; genetics ; Femur Head Necrosis ; drug therapy ; pathology ; physiopathology ; Male ; Meridians ; Osteocalcin ; blood ; Osteogenesis ; drug effects ; Rabbits

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

To investigate the effect and possible mechanism of echinacoside-containing serum on the osteogenic differentiation in rat bone marrow mesenchymal stem cells. Rat bone marrow mesenchymal stem cells were cultivated by the whole bone marrow adherence method. The 3rd generation of cells were divided into 3 groups: the blank control group, the classic osteogenic-induced group and the 10% echinacoside-containing serum group. The expression of alkaline phosphatase and osteocalcin were detected by ELISA. The ex- pression of ZHX, protein was detected by Western blot technique. RT-PCR technique was used to detect the expression of ZHX₃mRNA. According to the result, the expressions of the alkaline phosphatase and osteocalcin in the classic osteogenic-induced group and the 10% echinacoside-containing serum group were significantly higher than that of the blank control group (P <0. 01). And expressions of the alkaline phosphatase activity and osteocalcin in the 10% echinacoside-containing serum group were significantly higher than that in the classic osteogenic-induced group (P < 0.01). Meanwhile, the classic osteogenic-induced group and the 10% echinacoside-containing serum group showed obviously higher ZHX₃ protain and mRNA expression than that of the black control group, with significant differences (P < 0.01); the 10% echinacoside-containing serum group showed obviously higher ZHX₃ protain and mRNA expression than that of the classic osteogenic-induced group, with a significant difference (P < 0.01). In conclusion, 10% echinacoside-containing serum can promote the differentiation of the bone marrow mesenchymal stem cells cultured in vitro. Its mechanism may be correlated with the increase in the ZHX₃expression.
Animals ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Female ; Glycosides ; blood ; pharmacology ; Homeodomain Proteins ; genetics ; metabolism ; Male ; Mesenchymal Stromal Cells ; cytology ; drug effects ; metabolism ; Osteogenesis ; drug effects ; Rats ; Rats, Sprague-Dawley ; Serum ; chemistry ; Transcription Factors ; genetics ; metabolism

Once pulp necrosis or apical periodontitis occurs on immature teeth, the weak root and open root apex are challenging to clinicians. Berberine (BBR) is a potential medicine for bone disorders, therefore, we proposed to apply BBR in root canals to enhance root repair in immature teeth. An in vivo model of immature teeth with apical periodontitis was established in rats, and root canals were filled with BBR, calcium hydroxide or sterilized saline for 3 weeks. The shape of the roots was analyzed by micro-computed tomography and histological staining. In vitro, BBR was introduced into stem cells from apical papilla (SCAPs). Osteogenic differentiation of stem cells from apical papilla was investigated by alkaline phosphatase activity, mineralization ability, and gene expression of osteogenic makers. The signaling pathway, which regulated the osteogenesis of SCAPs was evaluated by quantitative real time PCR, Western blot analysis, and immunofluorescence. In rats treated with BBR, more tissue was formed, with longer roots, thicker root walls, and smaller apex diameters. In addition, we found that BBR promoted SCAPs osteogenesis in a time-dependent and concentration-dependent manner. BBR induced the expression of β-catenin and enhanced β-catenin entering into the nucleus, to up-regulate more runt-related nuclear factor 2 downstream. BBR enhanced root repair in immature teeth with apical periodontitis by activating the canonical Wnt/β-catenin pathway in SCAPs.
Animals ; Berberine ; pharmacology ; Cell Differentiation ; drug effects ; Dental Papilla ; Male ; Osteogenesis ; drug effects ; Periapical Periodontitis ; therapy ; Rats ; Stem Cells ; cytology ; drug effects ; metabolism ; Wnt Signaling Pathway ; drug effects ; Wnt3A Protein ; genetics ; metabolism ; X-Ray Microtomography

As a member of the AFF (AF4/FMR2) family, AFF4 is a transcription elongation factor that is a component of the super elongation complex. AFF4 serves as a scaffolding protein that connects transcription factors and promotes gene transcription through elongation and chromatin remodelling. Here, we investigated the effect of AFF4 on human dental follicle cells (DFCs) in osteogenic differentiation. In this study, we found that small interfering RNA-mediated depletion of AFF4 resulted in decreased alkaline phosphatase (ALP) activity and impaired mineralization. In addition, the expression of osteogenic-related genes (DLX5, SP7, RUNX2 and BGLAP) was significantly downregulated. In contrast, lentivirus-mediated overexpression of AFF4 significantly enhanced the osteogenic potential of human DFCs. Mechanistically, we found that both the mRNA and protein levels of ALKBH1, a critical regulator of epigenetics, changed in accordance with AFF4 expression levels. Overexpression of ALKBH1 in AFF4-depleted DFCs partially rescued the impairment of osteogenic differentiation. Our data indicated that AFF4 promoted the osteogenic differentiation of DFCs by upregulating the transcription of ALKBH1.
Biomarkers ; metabolism ; Cell Differentiation ; Cells, Cultured ; Dental Sac ; drug effects ; metabolism ; Gene Expression Regulation ; Humans ; Osteogenesis ; genetics ; Repressor Proteins ; Transcription Factors ; genetics ; metabolism ; Transcriptional Elongation Factors ; metabolism

OBJECTIVETo explore the mechanism of Bushen Qiangji Granule (, BSQJ) in restraining the osteogenic differentiation of ankylosing spondylitis (AS) fifibroblasts.

METHODSHip joint capsules were obtained from AS patients (n=10) receiving total hip replacement and healthy hip joint capsules from patients with hip fracture (n=10) receiving surgery as a control. Finite fifibroblast lines were established from these tissue samples to observe the effect of BSQJ on suppressing osteogenic differentiation of fifibroblasts. The expression of osteogenic marker gene corebinding factor a1 (Cbfa1) and Smad family proteins were examined by Western blot and real-time quantitative polymerase chain reaction (qPCR).

RESULTSThe mRNA expression level of Cbfa1 was significantly higher in AS fibroblasts than that in normal fibroblasts and the expression of pSmad1, pSmad5, Smad4 and Cbfa1 in AS fibroblasts was also higher, demonstrating the activation of the BMP/Smads signal pathway in AS fifibroblasts. BSQJ-medicated serum not only restrained the mRNA and protein expression levels of Cbfa1 and inhibited protein expression level of Smad4 but also decreased the expression quantities of pSmad1 and pSmad5.

CONCLUSIONSBSQJ can inhibit osteogenic differentiation of AS fifibroblasts in vitro by suppressing the activation of the BMP/Smads signal pathway. This may be the important molecular mechanism of BSQJ in regulating AS ossifification.

Adult ; Bone Morphogenetic Proteins ; metabolism ; Cell Differentiation ; drug effects ; Core Binding Factor Alpha 1 Subunit ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Fibroblasts ; drug effects ; metabolism ; pathology ; Humans ; Middle Aged ; Osteogenesis ; drug effects ; genetics ; Phosphorylation ; drug effects ; RNA, Messenger ; genetics ; metabolism ; Serum ; metabolism ; Signal Transduction ; drug effects ; Smad Proteins ; metabolism ; Spondylitis, Ankylosing ; genetics ; pathology ; Young Adult

OBJECTIVETo investigated the effects of isopsoralen on bone marrow stromal stem cells (BMSCs) differentiate and proliferate in vitro.

METHODThe stratum of mononuclear cells were separated and collected from the rat bone marrow sample by the all bone marrow cell culture methods. The cells were cultured in DMEM contained 10% fetal bovine serum. The culture medium was changed after three days. Nine days later, cells were treatment by isopsoralen with the concentration 1 x 10(-5), 1 x 10(-4), 1 x10(-6), 1 x 10(-7) mol x L(-1). MTT method was used for the proliferated analyzing. Under the induced condition, the alkaline phosphatase (ALP) activity, calcium salt sediment yield and osteocalcin were measured at the 4, 8, 12, 16 d. At the fifteenth day, histochemistry dyeing for calcified tubercle and ALP was proceeded. Total RNA was isolated and the gene expression of bFGF, IGF-1, Osterix and Runx-2 were investigated by Real Time PCR.

RESULTThe BMSCs proliferation refrained by isopsoralen with dose dependent. But it significantly enhanced osteogenesis, which was represented by the promotion of the ALP activity, calcium salt sediment yield, osteocalcin, and calcified tubercle amount. Besides, it also enhanced the mRNA level of bFGF, IGF-1, Osterix and Runx-2.

CONCLUSIONThe isopsoralen with the concentration 1 x 10(-5) mol x L(-1) can promote BMSCs differentiation to osteoblasts. It demonstrated the isopsoralen can prevent antiosteoporotic, which is an active part of the traditional Chinese medicine psoralea corylifolia.

Alkaline Phosphatase ; metabolism ; Animals ; Bone Marrow Cells ; cytology ; drug effects ; metabolism ; Cell Differentiation ; drug effects ; genetics ; Cell Proliferation ; drug effects ; Core Binding Factor Alpha 1 Subunit ; genetics ; Fibroblast Growth Factors ; genetics ; Furocoumarins ; pharmacology ; Gene Expression Regulation ; drug effects ; Insulin-Like Growth Factor I ; genetics ; Male ; Mesenchymal Stromal Cells ; cytology ; drug effects ; metabolism ; Osteocalcin ; metabolism ; Osteogenesis ; drug effects ; RNA, Messenger ; genetics ; Rats ; Stromal Cells ; cytology ; drug effects ; metabolism ; Transcription Factors ; genetics

Through protein-protein BLAST of homologous sequences in different species in NCBI database and preliminary simulating molecular docking and molecular dynamics by computer software discovery studio 3.1, three amino acids R25K26K27 of natural human parathyroid hormone (1-34) with Q25E26L27 were mutated and the biological activity of the mutant peptide was evaluated. Result showed that: root mean superposition deviation RMSD value between PTH (1-34)-(RKK-QEL) and PTH (1-34) peptide main chain was 2.509 3, indicating that the differences between the two main chain structural conformation was relatively small; the interaction energy between PTH (1-34)-(RKK-QEL) and its receptor protein PTH1R had been enhanced by 7.5% compared to nature PTH (1-34), from -554.083 kcal x mol(-1) to -599.253 kcal x mol(-1); the number of hydrogen bonds was increased from 32 to 38; PTH (1-34)-(RKK-QEL) can significantly stimulate the RANKL gene expression (P < 0.01) while inhibiting the OPG gene expression (P < 0.01) in UAMS-32P cells; in the co-culture system of UAMS-32P cells and mouse primary femur bone marrow cells, PTH (1-34)-(RKK-QEL) stimulated the formation of osteoclasts (P < 0.01) and had a higher biological activity than PTH (1-34) standard reagents.
Animals ; Bone Marrow Cells ; cytology ; metabolism ; Coculture Techniques ; Mice ; Mutant Proteins ; genetics ; pharmacology ; Mutation ; Osteoclasts ; cytology ; drug effects ; Osteogenesis ; drug effects ; Osteoprotegerin ; genetics ; metabolism ; RANK Ligand ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Receptor, Parathyroid Hormone, Type 1 ; metabolism ; Teriparatide ; pharmacology

PURPOSE: Imbalances between osteogenic and adipogenic differentiation leads to diseases such as osteoporosis. The aim of our study was to demonstrate the differences in extracellular signal-regulated kinase (ERK) phosphorylation during both adipogenesis and osteogenesis of human bone marrow-derived stem cells (BMSCs). MATERIALS AND METHODS: Using troglitazone, GW9662 and U0126, we investigated their role in hBMSC differentiation to adipogenic and osteogenic fates. RESULTS: ERK1/2 inhibition by U0126 suppressed proliferator-activated receptor (PPAR)gamma expression and lipid accumulation, while it decreased the mRNA expression of adipogenic genes (lipoprotein lipase, PPARgamma, and adipocyte protein) and osteogenic genes (type I collagen and osteopontin). ERK phosphorylation was transient and decreased during adipogenesis, whereas it occurred steadily during osteogenesis. Troglitazone, a PPARgamma agonist, induced adipogenesis by inhibiting ERK phosphorylation even in an osteogenic medium, suggesting that ERK signaling needs to be shut off in order to proceed with adipose cell commitment. Cell proliferation was greatly increased in osteogenesis but was not changed during adipogenesis, indicating that ERK might play different roles in cellular proliferation and differentiation between the two committed cell types. CONCLUSION: The duration and magnitude of ERK activation might be a crucial factor for the balance between adipogenesis and osteogenesis in human bone marrow-derived stem cells.
Adipogenesis/*drug effects/genetics ; Adult ; Anilides/pharmacology ; Bone Marrow Cells/*cytology/drug effects/metabolism ; Butadienes/pharmacology ; Cell Differentiation/drug effects ; Cells, Cultured ; Chromans/pharmacology ; Extracellular Signal-Regulated MAP Kinases/*metabolism ; Female ; Humans ; Male ; Middle Aged ; Nitriles/pharmacology ; Osteogenesis/*drug effects/genetics ; PPAR gamma/agonists/antagonists & inhibitors ; Phosphorylation/drug effects ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cells/*cytology/drug effects/*metabolism ; Thiazolidinediones/pharmacology