Effect of Low-frequency Pulsed Electromagnetic Fields on Bone Formation in Rat Osteoblasts and Its Mechanism / 中国医学科学院学报

Objective To observe the effect of low-frequency pulsed electromagnetic fields(PEMFs) on bone formation in rat osteoblasts(ROBs) and explore the mechanism of action of the cyclic adenosine monophosphate(cAMP)/protein kinase A(PKA)/cyclic adenosine effect binding protein(CREB) signaling pathway.Methods The skulls of newborn Wistar rats were harvested,and the ROBs were obtained by multiple enzymatic digestion methods for subculture. After treatment with 50 Hz 0.6 mT PEMFs for 3,6,and 9 days,the alkaline phosphatase(ALP) concentration in ROBs was detected;after 0,15,30,60,90,and 120 min,the expression of bone formation-related factor(RUNX2),the protein expression of osteogenesis-associated transcription factor(OSX),the cAMP concentration,as well as the protein expressions of p-PKA,p-CREB,and CREB were detected. The p-CREB nuclear translocation was observed. After interference with IFT88 by RNA interference,the expressions of RUNX2,OSX,p-PKA,and p-CREB protein in ROBs were detected.Results After treatment with PEMFs for 3,6,and 9 days,the ALP activity values in ROBs were 24.356±4.911,37.688±2.151,and 39.922±5.486,respectively,which were significantly higher than 18.531±2.401(P=0.0121),33.675±4.366(P=0.0324),and 36.574±1.339(P=0.0134) in the control groups. RUNX2 and OSX activities in ROBs were significantly higher than untreated group after PEMFs treatment for 30(P=0.0042 and P=0.0058),60(P=0.0097 and P=0.0079),and 90 min(P=0.0083 and P=0.0098). After PEMFs treatment for 30(P=0.0012) and 60 min(P=0.0035),the cAMP concentrations in ROBs were significantly higher than that in untreated group. After PEMFs treatment for 15(P=0.0018),30(P=0.0087),90(P=0.0250),and 120 min(P=0.0350),the p-PKA levels in ROBs were significantly higher than that in the untreated group. After PEMFs treatment for 15(P=0.0075),30(P=0.0017),60(P=0.0074),and 90 min(P=0.0096),the level of p-CREB in the ROBs was significantly higher than in the untreated group. After PEMFs treatment of ROBs for 15 min,CREB phosphorylated and accumulated in the nuclei. PKA and p-PKA were co-localized with primary cilia and stained,and it was found that p-PKA was localized on the primary cilia. After the primary cilia was removed by RNA interference,the protein expression levels of p-PKA(F=78.602,P=0.0270),p-CREB(F=76.082,P=0.0089),RUNX2(F=41.064,P=0.0230) and OSX(F=57.524,P=0.0310) were significantly lower than those of the non-interfered group.Conclusion PEMFs promote bone formation in ROBs by activating the primary cilia-associated cAMP/PKA/CREB signaling pathway.

Bone Loss Induced by Simulated Microgravity, Ionizing Radiation and/or Ultradian Rhythms in the Hindlimbs of Rats / 生物医学与环境科学（英文）

OBJECTIVE@#To better understand the pathological causes of bone loss in a space environment, including microgravity, ionizing radiation, and ultradian rhythms.@*METHODS@#Sprague Dawley (SD) rats were randomly divided into a baseline group, a control group, a hindlimb suspension group, a radiation group, a ultradian rhythms group and a combined-three-factor group. After four weeks of hindlimb suspension followed by X-ray exposure and/or ultradian rhythms, biomechanical properties, bone mineral density, histological analysis, microstructure parameters, and bone turnover markers were detected to evaluate bone loss in hindlimbs of rats.@*RESULTS@#Simulated microgravity or combined-three factors treatment led to a significant decrease in the biomechanical properties of bones, reduction in bone mineral density, and deterioration of trabecular parameters. Ionizing radiation exposure also showed adverse impact while ultradian rhythms had no significant effect on these outcomes. Decrease in the concentration of the turnover markers bone alkaline phosphatase (bALP), osteocalcin (OCN), and tartrate-resistant acid phosphatase-5b (TRAP-5b) in serum was in line with the changes in trabecular parameters.@*CONCLUSION@#Simulated microgravity is the main contributor of bone loss. Radiation also results in deleterious effects but ultradian rhythms has no significant effect. Combined-three factors treatment do not exacerbate bone loss when compared to simulated microgravity treatment alone.

Low-frequency pulsed electromagnetic fields promotes rat osteoblast differentiation in vitro through cAMP/PKA signal pathway / 南方医科大学学报

<p><b>OBJECTIVE</b>To study whether low-frequency pulsed electromagnetic fields promotes the differentiation of cultured rat osteoblasts through the cAMP/PKA signal pathway.</p><p><b>METHODS</b>Rat calvarial osteoblasts isolated by enzyme digestion were exposed to 50 Hz 0.6 mT low-frequency pulsed electromagnetic field for varying lengths of time, and the concentration of cAMP and levels of phosphorylated PKA in the cells were assayed. In cells treated with DDA to inhibit the activity of adenylate cyclase, the changes of ALP activity and transcription of osteogenic gene were detected after exposure to low-frequency pulsed electromagnetic field. The changes of osteogenic gene transcription and protein expression were tested in the osteoblasts pretreated with KT5720 in response to low-frequency pulsed electromagnetic field exposure.</p><p><b>RESULTS</b>The intracellular cAMP concentration in the cells increased significantly at 20 min during exposure to low-frequency pulsed electromagnetic field, began to decrease at 40 min during the exposure, and increased again after a 2-h exposure; the same pattern of variation was also observed in p-PKA level. Application of DDA and KT5720 pretreatment both suppressed the increase in ALP activity and osteogenic gene transcription induced by electromagnetic field exposure.</p><p><b>CONCLUSION</b>Low- frequency pulsed electromagnetic field exposure improves the differentiation of cultured rat osteoblasts by activating cAMP/PKA signal pathway.</p>

Inhibition of osthole for resorption of rats femur tissue in vitro / 中国骨伤

<p><b>OBJECTIVE</b>To investigate osthole effect on femoral tissue resorption activity of rat in vitro.</p><p><b>METHODS</b>Six 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.</p><p><b>RESULTS</b>Concetration 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).</p><p><b>CONCLUSION</b>Osthole can inhibit bone resorption and raise the level of nutrition metabolism of femurs tissue.</p>

Icariin enhances differentiation and maturation of rat calvarial osteoblasts in collagen hydrogel three-dimensional culture / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the effects of icariin on the differentiation and maturation of rat calvarial osteoblasts(ROB) in collagen hydrogel three-dimensional culture.</p><p><b>METHODS</b>ROB were obtained by enzyme digestion from the segregated neonatal SD rats skull and were embedded in 2 mg/mL rat tail collagen for three-dimensional culture. The growth state of ROB was observed by FDA/PI staining, HE staining and scanning electron microscopy. ROB were treated with icariin at the concentration of 1 × 10⁻⁴, 1 × 10⁻⁵, 1 × 10⁻⁶ and 1 × 10⁻⁷ mol/L respectively. The activity of alkaline phosphatase(ALP) was detected after 3, 6, 9 d of icariin treatment. Three-dimensional cultured ROB were treated with optimal concentration icariin for 12, 24, 36, 48 h and total RNA was extracted and the mRNA expressions of bone morphogenetic protein-2 (BMP-2), Runt-related transcription factor 2 (RUNX-2) and Osterix were detected by real time RT-PCR. The protein expression of BMP-2, RUNX-2 and Osterix were examined by Western-blotting.</p><p><b>RESULTS</b>ROB were cultured in collagen hydrogel successfully. FDA/PI staining, HE staining, and scanning electron microscopy showed that ROB adhered with collagen tightly and distributed homogeneously. Icariin at final concentration of 1 × 10⁻⁵, 1 × 10⁻⁶ and 1×10⁻⁷ mol/L all enhanced the activity of ALP of collagen hydrogel three-dimensional cultured ROB, and 1 × 10⁻⁶ mol/L was the optimal concentration. Besides, icariin (1 × 10⁻⁶ mol/L) increased mRNA and protein expression of BMP-2、RUNX-2 and Osterix compared to control group.</p><p><b>CONCLUSION</b>Icariin can enhance the expression of osteogenic markers of ROB in collagen hydrogel three-dimensional culture significantly.</p>

Establishment of osteoblast primary cilia model removed by chloral hyrate / 中国骨伤

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Three 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.</p><p><b>RESULTS</b>Compared 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).</p><p><b>CONCLUSION</b>4mM 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.</p>

Icariin promote maturation of osteoblasts in vitro by an estrogen-independent mechanism / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the estrogenic activity of icariin and genistein with estrogen-dependent human breast cancer (MCF-7) cells.</p><p><b>METHOD</b>MCF-7 cells were incubated with media containing 5% charcoal dextran-treated FBS in phenol red-free media for 48 h. CCK-8 kit was used to study the impact of defferent concentration of icariin and genistein on MCF-7 proliferation in vitro. Optimal concentration icariin and genistein were added into medium and total RNA was isolated after 12, 24, 36, 48 h. The gene expression of ERalpha, ERbeta, PS2, and PR were investigated by Real-time RT-PCR Total protein was also isolated and secretion of ERalpha, ERbeta, PS2, and PR were examined by Western blot.</p><p><b>RESULT</b>10 micromol x L(-1) icariin and genistein could promote the proliferation of MCF-7 evidently. However, the ability of genistein to promote the proliferation was better than icariin. With the concentration of 10 micromol x L(-1), genistein group had a stronger expression of ERa, PS2 and PR mRNA levels than icariin while ERbetaexpression had no significant difference in two group. The same effects were detected by western blotting.</p><p><b>CONCLUSION</b>Both genistein and icariin have a strong estrogen-like effect, but the estrogenic activity of genistein is stronger than icariin. It showed that the activity of icariin is stron-ger than genistein to promote ROB maturation. So it must be that icariin promotes the maturation of osteoblasts in vitro by a estogen-independent mechanism.</p>

The role of primary cilium in signal transduction and its mechanism / 浙江大学学报·医学版

The primary cilium is a solitary and special organelle that emanates from the cell surface of most mammalian cells, which is anchored to the cell by mother centriole during the interphase and G0 of cell cycle. Recent studies have revealed that the primary cilium is a sensory organelle to receive extracellular signals and plays a key role in the signal transduction and pathogenesis of diseases. This review presents the structure and the forming process of the primary cilium during cell cycle. The signal transductions associated with primary cilium, including platelet-derived growth factor receptor αα, hedgehog, Wnt are discussed and the relevant researches in the future are proposed.

Comparison between icariin and genistein in osteogenic activity of marrow stromal cells / 中国中药杂志

<p><b>OBJECTIVE</b>To compare the effect of icariin and genistein in the osteogenic differentiation of rat bone marrow stromal cells (rBMSC).</p><p><b>METHOD</b>Rat marrow stromal cells were seperated in vitro, and the optimal concentration of genisten and icriin were screened. Genistein and icariin with the concentration of 1 x 10(-5) mol x L(-1) were adopted to intereven rBMSCs cultured in vitro. Alkaline phosphatase (ALP) was determined at 3, 6, 9, 12,15 d after intervention; calcified nodule was detected with alizarin red staining at 12 d; OXS, Runx-2, bone morphogenetic protein (BMP-2) and Collagen-I mRNA expression were observed with Real-time RT-PCR at 12, 24, 48, 72, 96 h.</p><p><b>RESULT</b>Genistein and icariin with the concentration of 1 x 10(-5) mol x L(-1) could increase the activity of ALP and the content of Ca, regulate OXS, BMP-2, Runx-2 and Collagen-I mRNA expression.</p><p><b>CONCLUSION</b>Icariin showed a stronger effect in improving the osteogenic differentiation of rat bone marrow stromal cells than genistein.</p>

Mechanisms of icariin in regulating bone formation of osteoblasts and bone resorption of osteoclasts / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the molecular mechanisms of icariin (ICA) in regulating the bone formation of osteoblasts and the bone resorption of osteoclasts.</p><p><b>METHODS</b>Primary osteoblast cell cultures were obtained from newborn rat calvarial. Calcified nodules were stained by alizarin red. The mRNA levels of osterix (OSX), runt-related transcription factor 2 (Runx-2), alkaline phosphatase (ALP), Collagen1, osteoprotegerin (OPG), and receptor activator of nuclear factor-ΚB ligand (RANKL) were analyzed by quantitative real-time RT-PCR, the protein levels of OPG, RANKL, and Collagen1 were examined by Western blotting, and the intracellular Ca(2+) concentration of osteoblasts was measured on a flow cytometer using the Cellquest program.</p><p><b>RESULTS</b>Compared with control group, ICA markedly promoted bone formation by significant up-regulating the gene expressions of OSX, Runx-2,ALP, and Collagen1, the protein expression of Collagen1(all P<0.01), and the Ca(2+) concentration. Furthermore, ICA remarkably inhibited bone resorption by significant up-regulating the mRNA and protein expressions of OPG as well as the OPG/RANKL ratio.</p><p><b>CONCLUSIONS</b>ICA could promote bone formation of osteoblasts through inducting the gene expressions of OSX,Runx-2, ALP and Collagen1, and the protein expressions of Collagen1, and by increasing the Ca (2+) concentration. Moreover, ICA could inhibit bone resorption of osteoclasts through regulating OPG/RANKL signal pathway.</p>

Effect of osthole on bone metabolism in rat femoral tissues in vitro / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the effect of osthole on bone metabolism in rat femoral tissues in vitro.</p><p><b>METHODS</b>The rat femoral tissues were isolated in vitro. The optimal concentrations of ostehole (1×10(-5) mol/L) and estradiol (1×10(-8) mol/L) (the positive control) were selected by alkaline phosphatase activity (ALP). The ALP and calcium levels were detected by commmerical regents, and the expressions of osteoprotegerin, receptor activator of nuclear factor-κB ligand, runx-related gene 2, and bone morphogenetic protein-2 mRNA were determined by real-time reverse transcription-polymerase chain reaction.</p><p><b>RESULT</b>The osthole (1×10(-5) mol/L) significantly increased the activity of ALP, calcium level as well as the expressions of osteoprotegerin, receptor activator of nuclear factor-κB ligand, runx-related gene-2 and bone morphogenetic protein-2 mRNA in rat femoral tissues in vitro.</p><p><b>CONCLUSION</b>Osthole can improve calcium level and ALP activity and regulate the bone metabolism-related genes in rat femoral tissues.</p>