Objective To assess the coordinated regulation and the molecular mechanisms of 17β-estradiol and 1,25-dihydroxyvitamin D3 [ 1,25-( OH ) 2 D3 ] on the proliferation and differentiation of osteoblastic MC3T3-E1 cells.Methods MC3T3-E1 cells were cultured in phenol-red free α-MEM medium supplemented with 10％ FBS,MTT assay was performed to determine the effects of 17β-estradiol and 1,25-( OH )2 D3 on MC3T3-E1 cells proliferation.After cells were treated with different agents,cell cycle related genes [ cyclin E,proliferation cell nuclear antigen ( PCNA ),and cyclin-dependent kinase inhibitor 2b ( Cdkn2b ) ] and markers of osteoblastic differentiation [ type Ⅰ collagen ( COL Ⅰ ),alkaline phosphatase ( ALP),osteopontin ( OPN ) ] were detected with SYBR green-based quantitative PCR.ALP activity was detected with BCIP/NBT method.Results 17β-estradiol could promote proliferation of MC3T3-E1 cells,which was accordant to its ability to increase cyclin E and PCNA and to inhibit Cdkn2b mRNA expression in MC3T3-E1 cells.However,1,25-( OH)2D3 had no effect on the proliferation of MC3T3-E1 cells and also did not enhance the proliferation of MC3T3-E1 cells stimulated by 17β-estradiol.On the other hand,17β-estradiol promoted the gene expression of differentiation markers Col Ⅰ,ALP,and OPN,and 1,25-(OH) 2 D3 synergistically increased the expression of these genes with 17 β-estradiol.Conclusion As two of the most important hormones which regulate bone metabolism,estrogen and vitamin D may coordinately promote osteoblast differentiation,but may not regulate osteoblasts proliferation synergistically.

Objective To explore the detailed underlying molecular and signaling mechanisms in the effects of icariin on bone formation by an in vitro cell model. Methods The proliferation of MC3T3-E1 osteoblast-like cells was evaluated by MTT, and gene expression of cell cycle related proteins in MC3T3-E1 cells after icariin treatment was detected by real-time PCR. The phosphorylation of MAPK signals, including ERK, P38, and JNK was determined by Western blot, and then the inhibitors of MAPK signals were used to treat cells with icariin alone or together to determine the role of MAPKs in the process of icariin treatment on MC3T3-E1 cell proliferation. Alkaline phosphatase and Alizarin red staining were used to detect the formation of mineralization nodules, and gene expressions of alkaline phosphatase, type Ⅰ collagen, and osteocalcin in osteoblasts after being treated by icariin were evaluated by real-time PCR. ICI182780, and nilutamide was used to decide the participation of estrogen and androgen receptor signals in the process of icariin treatment on the differentiation and mineralization of MC3T3-E1 cells. Results Treatment with icariin promoted MC3T3-E1 cell growth in a time- and dose-dependent manner. This treatment also revealed that icariin increased the expression of mRNAs encoding both cyclin E and PCNA, positive regulators of cell growth, but decreased levels of mRNAs encoding Cdkn2b, a negative regulator of cell cycle progression. When MC3T3-E1 cells were cultured in a differentiated condition, icariin enhanced mineralized nodule formation and increased the expression of mRNAs encoding alkaline phosphatase, type Ⅰ collagen, and osteocalcin. Treatment with icariin significantly induced phosphorylation of both ERK and JNK and this phosphorylated effect occurred very rapidly within 5 minutes and reached peak at 15 minutes. Furthermore, the stimulated effects of icariin on proliferation and gene expression of cyclin E, PCNA, and Cdkn2b in MC3T3-E1 cells were dramatically attenuated by treatment with both U0126 and SP600125, inhibitors of MAPKs. Interestingly, such stimulating effects of icariin were at least partly reduced by treatment with ICI182780, an inhibitor of estrogen receptor. Icariin induced mineralized nodule formation and gene expression of alkaline phosphatase, type Ⅰ collagen, and osteocalcin in MC3T3-E1 cells were also partly reduced when the cells were treated with ICI182780. Conclusions Our findings indicate that the anabolic effect of icariin on bone formation is, at least partly, mediated through the MAPK signaling pathway in order to modulate osteoblast proliferation and differentiation.

Objective:To investigate the relationship of hypertension with osteoporosis(OP)and bone mineral density(BMD) in elderly type 2 diabetic women.Methods: A total of 179 elderly type 2 diabetic women were categorized into hypertension group(n=124)and non-hypertension group(n=55).Lumbar and hip BMD were measured by dual-energy X-ray absorptiometry.Prevalences of osteoporosis were compared between the two groups using Chi-square test,and BMD was compared using covariance analysis.The correlation between the osteoporosis and risk factors was analyzed using binary logistic regression method.Results: The prevalences of osteoporosis(total OP,lumber OP and hip OP) in non-hypertension group were higher than that of the hypertension group(Pt=0.037,Pl=0.042 and Ph=0.051,respectively).Lumbar and hip BMD in the hypertension group was higher than that in the non-hypertension group,and there were significant differences in L3 and L4 BMDs between the two groups(P3=0.040,P4=0.021);however,the difference disappeared after adjustment for body mass index(BMD).After adjustment for C peptide,the P value also increased,but not as obvious as after adjustment for BMI.Binary logistic regression analysis showed that OP was associated with age,BMI and osteocalcin,but not with hypertension.Conclusion: OP is associated with the age,BMI,and osteocalcin,but not with hypertension in elderly type 2 diabetic women.

Objective To investigate the effects of icarrin on the activity and protein expression of core binding factor otl(Cbfa1) in rat osteoblasts cultured in vitro,and to explore whether mitogen-activated protein kinase (MAPK) pathway is involved in this process.Methods Calvarial osteoblasts were obtained from newborn (<24 h) SD rats by trypsin-coUagenase digestion method.The second generation osteoblasts were cultured in the medium containing icariin (10 ng/ml) or estradiol (10-8 mol/L) with or without extracellular-signal regulated kinase (ERK) inhibitor (UO126) or p38MAPK inhibitor (SB203580).Nuclear protein was extracted from osteoblasts.And then the activity of Cbfa1 was detected by ELISA.The amounts of Cbfa1 protein were detected by Western blot.Results Calvarial osteoblasts were obtained successfully and were used in this study after indentified by alkaline phosphatase and mineralized nodus staining.Cbfa1 expression and the activity in osteoblasts were up-regulated by both icariin and estradiol (P<0.05).The effects were partly inhibited by addition of U0126or SB203580 (P<0.05).Conclusions Either icarrin or estradiol can stimulate the proliferation and maturation of cultured osteoblasts in vitro via up-regulating the activity and expression of Cbfal.The MAPK signal pathway inhibitor seems to partly decrease Cbfa1 activity.It suggests that MAPK pathway may be involved in the transduction of icariin's impact on proliferation and mineralization of osteoblasts.

Skeleton has been proven to be an endocrine organ. Osteoblasts,the bone-forming cells in the skeleton,can secret osteocalcin which regulates global energy metabolism and male fertility in its undercarboxylated form and lipocalin 2 which influences appetite through binding to MC4R in the hypothalamus. Being a highly metabolic cell,osteoblast needs lots of energy through utilizing glucose and fatty acid to support its proliferation,differentiation, and mineralization. Recent data have shown the consumption change of fuels by osteoblasts, which contributes to global energy metabolism. Wnt/β-catenin signaling in osteoblasts is very important for bone formation,and disruption or constitutive activation of this pathway through gene editing may influence global energy metabolism. Through interacting with Wnt/β-catenin signaling,parathyroid hormone and sclerostin monoclonal antibody perform their anti-osteoporosis effect,and affect glucose homeostasis,insulin sensitivity,lipid metabolism and browning of peripheral adipose tissue. Though great progress has been made to understand the regulation of energy metabolism by bone during recent years,a lot of questions still remain to be answered.

Objective To study the changes of bone mineral density(BMD)and bone turnover in postmenopausal osteoporotic patients treated with salmon calcitonin nasal spray. Methods Sixty-seven postmenopausal osteoporotic patients were enrolled in our trial. All of them received calcium and vitamin D; 37patients were treated with salmon calcitonin nasal spray for 12 months and the other 30 patients received calcium and vitamin D only. Dual-energy X-ray absorptiometry(DEXA)and measurements of a series of bone turnover indices were performed before and after medication for 6 and 12 months. Results After treatment with salmon calcitonin nasal spray for6 months, BMD in lumbar spine 2-4 increased but no change occurred in femoral neck. However, after treatment for 12 months, BMD in both lumbar spine 2-4 and femoral neck increased. In the control group, BMD in lumbar spine 2-4 decreased after treatment for 6 and 12 months, but BMD in femoral neck decreased only after 12months. Comparing with the control group, after treatment with salmon calcitonin nasal spray, BMD in lumbar spine 2-4 and femoral neck were increased obviously. The level of TRACP-5b and NTX/Cr decreased after treatment with salmon calcitonin nasal spray for6 months and 12 months, while BALP increased only after treatment for 12 months. In the control group, BALP decreased after treatment for 12 months. The level of 25-(OH)vitamin D increased after treatment for 6 months and 12 months in both groups. Conclusions Long-term treatment with salmon calcitonin nasal spray prevents bone loss and may increase bone mass.

Fracture risk is higher in the patients with type 2 diabetes mellitus (T2DM) comparing to non-diabetic subjects,but bone mineral density (BMD) in type 2 diabetic patients may be increased,normal or decreased compared to non-diabetic population.Thus,BMD is not an accurate index for deciding to start antiosteoporotic treatment in type 2 diabetic patients with high fracture risk,and the more accurate assessment tools which can reflect the fracture risk in type 2 diabetic patients are needed.Studies have shown that the fracture risk in type 2 diabetic patients is increased with the decreasing of BMD,but the fracture usually happened with relative high BMD.Trabecular bone score is lower in type 2 diabetic patients than that in non-diabetic patients.High-resolution peripheral quantitative computed tomography (HR-pQCT) accurately reflects the bone microstructure of T2DM.FRAX may get more accurate fracture risk in type 2 diabetic patients by replacing rheumatoid arthritis with T2DM.DeFRA is a new algorithm derived from FRAX,which can evaluate fracture risk more accurately than FRAX in type 2 diabetic patients.Skeletal muscle mass is decreased in type 2 diabetic patients.Pentosidine,as one of advanced glycation end products,is related to the fracture risk in type 2 diabetic patients.Based on these data,this paper will review the assessments which may be used to evaluate the fracture risk in type 2 diabetic patients.

Objective To compare the clinical efficacy and safety between recombinant human parathyroid hormone ( rhPTH) ( 1 -34) and elcatonin in the treatment of postmenopausal women with osteoporosis in China.Methods This 6 month, multicenter, randomized and controlled study enrolled 205 postmenopausal women with osteoporosis.They were randomized to receive either rhPTH (1 -34) 20 μg (200 U) daily or elcatonin 20 U weekly.Lumbar spine (L1-4 ) and femoral neck bone mineral density (BMD) and biochemical markers of bone turnover were measured. In the meantime adverse events were recorded. Results The results showed that both rhPTH ( 1 -34) and elcatonin increased L1-4 BMD significantly at the endpoint of the study, but femoral neck BMD did not change significantly.From baseline to endpoint, BMD of L1-4 and femoral neck in the rhPTH( 1-34) group increased by 5.51% (P <0.01) and 0.65% (P >0.05), but BMD of L1-4 and femoral neck in elcatonin group increased by 1.55% (P <0.05) and 0.11% (P>0.05).Moreover, the rhPTH(1-34) group had better improvement in L1-4 BMD than the elcatonin group at 3, 6 months, but there was no difference of BMD in these two groups with regard to femoral neck.There were greater mean increases of the bone markers in the rhPTH( 1-34) group than those in the elcatonin group at 3, 6 months [serum bone-specific alkaline phosphatase ( BSAP) 36.79% vs 0.31% ; 92.42% vs -0.17% ; the ratio of urine N-telopeptide of type I collagen and creatinine (NTX/Cr) 48.91% vs -5.32% ; 68.82% vs - 10.86%].Both kinds of treatment were well tolerated and there were no differences between the two groups in the rates of adverse events and serious adverse events.Conclusion It is concluded that rhPTH (1 -34) has more positive effects on bone formation than elcatonin as shown by the greater increments of L1-4 BMD and bone formation markers and the less occurrence of adverse events as well as no significant change in hepatic, renal or hemopoietic function.