OBJECTIVETo compare the ability of osthole (OST) and genistein (GEN) in enhancing bone peak bone mass of rats to prevent osteoporosis.

METHODSThirty-six female one-month-old SD rats of (125 +/- 3) g body weight were randomly divided into three groups, 12 rats in each group, one group was orally administered osthole at 9 mg x kg(-1) d(-1), one group was given genistein at 10 mg x kg(-1) d(-1) and another was given equal quantity of distilled water as the control. The body weight was monitored weekly and the bone mineral density (BMD) of total body was measured every month. All rats were sacrificed after three months, the femoral bone mineral density, the serum levels of osteocalcin (OC) and anti-tartaric acid phosphatase 5b (TRACP 5b) were measured by Elisa. The bone microarchitectures were analyzed with micro-CT and the bone biomechanics properties were tested with universal material machine.

RESULTSNo significant differences were observed between O-treated or GEN group and the control for the food-intake and body weight during three months. However, the rats treated with OST had significant higher BMD for both total body and femur than the control and GEN group. The O-treated rats also had higher level of serum OC and lower level of TRACP 5b. Besides, they owned bigger bone volume/tissue volume, trabecular thickness, trabecular number but smaller trabecular spacing. In the three point bending tests of femurs,they were found to have larger maximum load, the young's modulus and structural model index (SMI).

CONCLUSIONOrally administered osthole could efficiently increase the peak bone mass of rats,which provide new ideas for preventing osteoporosis.

Acid Phosphatase ; blood ; Animals ; Body Weight ; drug effects ; Bone Density ; drug effects ; Coumarins ; pharmacology ; Female ; Femur ; diagnostic imaging ; drug effects ; pathology ; Genistein ; pharmacology ; Isoenzymes ; blood ; Osteocalcin ; blood ; Radiography ; Rats ; Rats, Sprague-Dawley ; Tartrate-Resistant Acid Phosphatase

OBJECTIVETo investigate osthole effect on femoral tissue resorption activity of rat in vitro.

METHODSSix SD rats weighted (80 ± 5) g were used to isolate and culture femoral tissue (diaphyses and metaphysis) in vitro. The cultured tissue were devided into control group, estradiol group and osthole group. The femoral tissue was treated with final concentration of 1 x 10(-5) mol/L osthole and 1 x 10(-8) mol/L estradiol culture in vitro at 48 hours after cultured. Tartrate-resistant acid phosphatase (StrACP) activity, glucose and Lactic acid content, StrACP, MCSF (Macrophage colony stimulating factor) and CTSK (Cathepsin K) mRNA was detected by Real-Time RT-PCR were detected.

RESULTSConcetration of Alkaline phosphatase activity were 2226 and 2498 in 1 x 10(-5) mol/L osthole and 1 x 10(-8) mol/L estradiol respectively. As compared with control group, the activity of StrACP of 1 x 10(-5) mol/L osthole and 1 x 10(-8) mol/L estradiol were inhibited at 6, 9, 12 days (P < 0.05); under treatment of in l x 10(-5) mol/L osthole, the content of Lactic acid were increased and the content of glucose were decreased at 3, 6, 9 days (P < 0.05); StrACP, MCSF and CTSK mRNA expression level were inhibited at 6, 9 days (P < 0.05).

CONCLUSIONOsthole can inhibit bone resorption and raise the level of nutrition metabolism of femurs tissue.

Acid Phosphatase ; metabolism ; Animals ; Bone Resorption ; prevention & control ; Coumarins ; pharmacology ; Estradiol ; pharmacology ; Femur ; drug effects ; Glucose ; analysis ; Lactic Acid ; analysis ; Male ; Rats ; Rats, Sprague-Dawley

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

OBJECTIVETo study the effects of androgen on the expression of phosphacan and NG2 proteoglycan (NG2) and neurite regeneration in neonatal rats with hypoxic-ischemic brain damage (HIBD) and the potential mechanism underlying the protective effect of androgen against HIBD.

METHODSOne hundred and twenty neonatal Sprague-Dawley rats were randomly divided into three groups: sham-operated, HIBD and androgen treatment. HIBD was induced by the ligation of left common carotid artery and hypoxia exposure. The androgen treatment group rats were injected with testosterone propionate (25 mg/kg) immediately after HIBD. Phosphacan and NG2 expression in the cortex and the hippocampus was detected with the immunohistochemical method 24 and 72 hrs and 7 and 10 days after hypoxia-ischemia (HI). The ultrastructure and neurite regeneration of neurons in the cortex and the hippocampus were observed under a transmission electron microscope.

RESULTSThe neurite regeneration was obvious in the sham-operated group, but seldom in the HIBD group. The androgen treatment group showed increased neurite regeneration compared with the HIBD group. There were fewer phosphacan and NG2 positive cells in the cortex and the hippocampus in the sham-operated group. Phosphacan and NG2 expression in the cortex and the hippocampus was observed at 24 hrs, increased at 72 hrs, and peaked at 7 days after HI in the HIBD group and remained at a higher expression 10 days after HI than in the sham-operated group. The levels of phosphacan and NG2 expression in the cortex and the hippocampus in the androgen treatment group were significantly reduced compared with those in the HIBD group 24 and 72 hrs and 7 and 10 days after HI (P<0.01).

CONCLUSIONSPhosphacan and NG2 may be important inhibitory factors for neurite regeneration following HIBD in neonatal rats. The neuroprotection of androgen against neonatal HIBD is produced possibly through an inhibition of phosphacan and NG2 expression.

Animals ; Animals, Newborn ; Antigens ; analysis ; Brain Chemistry ; drug effects ; Female ; Hypoxia-Ischemia, Brain ; physiopathology ; Immunohistochemistry ; Male ; Microscopy, Electron, Transmission ; Nerve Regeneration ; drug effects ; Neurites ; physiology ; ultrastructure ; Proteoglycans ; analysis ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptor-Like Protein Tyrosine Phosphatases, Class 5 ; analysis ; Testosterone Propionate ; pharmacology

This study is to investigate effects of genistein on rat femoral bone metabolic in vitro. Rat femoral tissues was isolated and randomly divided into two groups including control group and genistein (1 x 10(-5) mol x(-1)) group. Determinations of alkaline phosphatase (ALP) activity, calcium content and osteoprotegerin (OPG), type I-collagen (Collagen-I), RANKL, Runx-2 and bone morphogenetic protein (BMP-2) mRNA expression were done by real-time PCR. The results showed that 1 x 10(-5) mol x L(-1) genistein could increase the activity of ALP and contents of Ca, regulate bone metabolism activity of OPG, RANKL, BMP-2, Collagen-I and Runx-2 mRNA expression level. Genistein can significantly modulate bone metabolism related gene expression level of rat femoral tissue in vitro, and can increase calcium content and the activity of ALP.
Alkaline Phosphatase ; metabolism ; Animals ; Bone Morphogenetic Protein 2 ; genetics ; metabolism ; Calcium ; metabolism ; Collagen Type I ; genetics ; metabolism ; Core Binding Factor Alpha 1 Subunit ; genetics ; metabolism ; Enzyme Activation ; drug effects ; Femur ; metabolism ; Gene Expression Regulation ; Genistein ; pharmacology ; Osteoprotegerin ; genetics ; metabolism ; Phytoestrogens ; pharmacology ; RANK Ligand ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction

OBJECTIVETo investigate the effects of static magnetic fields (SMFs) with different exposure time on the maturation of rat osteoblasts in vitro and the expression of the estrogen receptor (ER) gene.

METHODSThe calvarial osteoblasts were isolated from newborn rats by enzyme digestion and randomly divided into 9 groups after one passage based on the exposure time of the SMFs[0 (control), 0.5 h, 1.0 h, 1.5 h, 2.0 h, 2.5 h, 3.0 h, 3.5 h, and 4.0 h]. The intensity was 3.9 mT in all SMFs. Those without SMFs exposure were used as the controls. The oeteoblasts were observed under the contrast phase microscope on a daily basis. After 48 h, cell proliferation was assayed by MTT method. The osteocalcin contents were measured after exposure to SMFs for 3 d, 6 d, 9 d, and 12 d. ERΑ and ERΒ mRNA expressions were measured by real-time PCR after SMFs treatment for 0 h, 24 h, 48 h, and 72 h.

RESULTSCompared with the controls, the cell proliferation was significantly enhanced in the 2.0-h, 2.5-h, and 3.0-h groups (P<0.05). After SMFs treatment for 6 d, 9 d and 12 d, the 2.5-h group had significantly higher osteocalcin content than the control group did (P<0.05). After SMFs treatment for 0 h and 72 h, elevated ERΑ mRNA expression and reduced ERΒ mRNA expression were observed.

CONCLUSIONExposure to SMFs, regardless of exposure time, is associated with enhanced cell proliferation, increased osteocalcin contents, and altered ERΑ and ERΒ mRNA expressions in opposite directions.

Animals ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Magnetic Fields ; Osteoblasts ; cytology ; metabolism ; Rats ; Receptors, Estrogen ; genetics ; metabolism

OBJECTIVETo investigated the effect of 50-Hz 3.6-mT sinusoidal electromagnetic fields (SEMFs) on the proliferation and differentiation of osteoblasts in vitro.

METHODSThe newborn rat calvarial osteoblasts were isolated by enzyme digestion and randomly divided into 6 groups after one passage. The treatment groups under 50-Hz 3.6-mT SEMFs and controls without SEMFs treatment. The cells were exposed in the SEMFs for 0.5 h, 1.0 h, 1.5 h, 2.0 h, and 2.5 h. They were observed under the contrast phase microscope each day. The calcified nodules were stained by alizarin red. The SEMFs were arranged in spiral appearance after 3 to 5 days.

RESULTSThe SEMFs showed characteristic distribution 3 to 5 days after SEMFs treatment. On the 9(th) day after treatment, the activity of alkaline phosphatase (ALP) significantly increased in the 0.5-h group, whereas the ALP histochemical straining results and the area of calcified nodules were consistent with ALP activity. In the 48-h and 96-h groups, the genetic expression levels of osteoprotegerin and collagen-1 were significantly higher than that in the control group; particularly, the mRNA expression increased in the 0.5-h group.

CONCLUSIONThe SEMFs at 50-Hz 3.6-mT could suppress the proliferation of osteoblasts maturation but stimulate the differentiation and maturation of osteoblasts in vitro.

Animals ; Cell Differentiation ; radiation effects ; Cell Proliferation ; radiation effects ; Cells, Cultured ; Electromagnetic Fields ; Male ; Osteoblasts ; cytology ; radiation effects ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effect of exposure to static magnetic fields (SMFs) of 3.9 mT on proliferation and differentiation of osteoblasts in vitro.

METHODSThe newborn rat calvarial osteoblasts were isolated by enzyme digestion and randomly divided into 9 groups after one passage. The intensity of the SMFs was 3.9 mT. The cells were exposed in the SMFs for 0 (control group), 0.5, 1.0, 1.5, 2.0, 2.5, 3, 3.5 and 4.0 h groups respectively. They were observed under the contrast phase microscope each day. After 48 h, cell proliferation was assayed by MTT method. The alkaline phosphatase (Alkaline Phosphatase, ALP) activities and calcium content were measured after 3, 6, 9, and 12 days exposed with SMFs. The ALP positive colonies were histochemically stained after 8 days and the calcified nodules were stained by Alizarin Bordeaux after 10 days; BMP-2, Runx-2 and Opg mRNA expression were measured after SMFs treatment in 0, 24, 48 and 72 h.

RESULTSContrast with control group, all SMFs groups enhanced cell proliferation (P < 0.01 or P < 0.05), and they promoted maturation and mineralization of the osteoblasts. The results showed that SMFs improved the ALP activity, promoted calcium content, boost BMP-2, Runx -2 and Opg mRNA expression.

CONCLUSIONThe cells exposed to the SMFs of 3.9 mT at 2.5 h apparently promote proliferation and differentiation of osteoblasts in vitro.

Animals ; Bone Morphogenetic Protein 2 ; genetics ; Calcium ; metabolism ; Cell Differentiation ; radiation effects ; Cell Proliferation ; radiation effects ; Core Binding Factor Alpha 1 Subunit ; genetics ; Magnetic Fields ; Osteoblasts ; physiology ; radiation effects ; Osteoprotegerin ; genetics ; Rats ; Rats, Sprague-Dawley ; Time Factors

This study is to investigate the effect of 8-prenylnaringenin (8-PNG) on osteoclastogensis of bone marrow cells and bone resorption activity of osteoclasts. Osteoclasts were separated from long bone marrow of newborn rabbits and cultured in alpha-MEM containing 10% FBS. 8-PNG was added into culture media at 1 x 10(-7), 1 x 10(-6), 1 x 10(-5) mol xL(-1), separately. 17beta-Estradiol (E2, 1 x 10(-7) mol x L(-7)) was used as positive control. T RAP staining and TRAP activity measurement were performed after 5 days, and the bone resorption pits were analyzed after 7 days. Annexin V staining for the detection of apoptotic osteoclasts was performed after 2, 4, 8, 12, 24, 36 and 48 h separately. The mRNA expression level of TRAP and cathepsin K (CTSK) was measured by real-time RT-PCR. 8-PNG significantly reduced the number of osteoclasts which was TRAP staining positive and with more than three nucleus, the area and number of bone resorption pits decreased obviously in 8-PNG-supplemented groups. The apoptosis rate peaked earlier in the 8-PNG-supplemented groups and the mRNA expression level of TRAP and CTSK decreased significantly. All these inhibitory effects were in a dose dependent manner, the highest effect was obtained by 1 x 10(-5) mol x L(-1) 8-PNG. 8-PNG inhibits bone resorption activity of osteoclasts by inducing osteoclast apoptosis and inhibiting the gene expression and enzyme activity including TRAP and CTSK, and restrains bone marrow cells to osteoclast differentiation.
Acid Phosphatase ; genetics ; metabolism ; Animals ; Apoptosis ; drug effects ; Bone Marrow Cells ; cytology ; Bone Resorption ; Cathepsin K ; genetics ; metabolism ; Cells, Cultured ; Dose-Response Relationship, Drug ; Flavanones ; administration & dosage ; pharmacology ; Isoenzymes ; genetics ; metabolism ; Osteoclasts ; cytology ; metabolism ; RNA, Messenger ; metabolism ; Rabbits ; Tartrate-Resistant Acid Phosphatase

OBJECTIVEThe purpose of this study was to clone and analyze mutation in the eda-A1 gene for hypohidrotic ectodermal dysplasia (HED), and to construct a new recombined eukaryotic expression vector (mutant M, wild W) as a basis for further study on the genetic function.

METHODSAfter total mRNA was extracted from peripheral blood lymphocytes from the HED affect patient and control, eda-A1 gene was amplified by reverse transcription polymerase chain reaction (RT-PCR) with a pair of specific primers containing the constriction enzyme sites of BamH I and Hind III. When the vector pcDNA3.1(-) and eda-A1 (M/W) were digested by BamH I and Hind III respectively, eda-A1 (M/W) fragment was then ligated to vector pcDNA3.1 (-) and the new vector was named as pcDNA3.1 (-)-eda-A1-M/W.

RESULTSeda-A1 gene was successfully cloned and a novel missence mutation was identified, which changes the codon 306 from glutamine to proline. PCR, restrictive endonuclease analysis and DNA sequencing were then performed to identify the recombinant eukaryotic expression vector pcDNA3.1 (-)-eda-A1-M/W, and the results were surely confirmed.

CONCLUSIONOur result indicates that the novel missense mutation in eda is associated with the isolated tooth agenesis and provide preliminary explanation for the abnormal clinical phenotype at a molecular structural level. And also, the recombinant eukaryotic expression vector pcDNA3.1 (-)-eda-A1-M/W was successfully constructed, which will be thereafter taken use of further study on eda gene in odontogenesis.

Ectodermal Dysplasia 1, Anhidrotic ; Genetic Vectors ; Humans ; Mutation ; Odontogenesis ; RNA, Messenger ; Sequence Analysis, DNA