| BACKGROUNDOsteoporosis damage is a metabolic diseases which is characterized by a low bone mass and bone tissue micro-structure, and leads to bone fragility and susceptibility to fracture. Osteoporosis is not only the cause the elderly pain and skeletal deformities and fractures mainly, but also the low quality of life, high morbidity and high mortality of the disease, which poses a heavy financial burden to the society and the family. Osteoporosis patients in China ranks first in the world, and now there are about90million, accounting for7.1%of the total population, and with the increasing trend of the process of China’s aging society, the incidence of osteoporosis was. Therefore, the prevention and treatment of the disease has become a serious problem facing China’s public health career.The prevention and treatment of osteoporosis drugs mainly through two ways:to promote bone formation and to inhibit bone resorption. Promotion of bone formation drugs include fluoride agents and parathyroid hormone1-34; inhibition bone absorption of drugs contain estrogen, selective estrogen receptor modifiers, calcitonin and bisphosphonates, etc. Other than above mentioned drugs, strontium ranelate, which has been approved by the European Union in October2004, is the first listed drug with a "dual role". Since strontium ranelate has been listed in China in2009, it gets more and more attention, and represents a new direction of development in the treatment of osteoporosis, but its mechanism about bone formation and bone absorption is not yet fully elucidated, hindering its widespread use. So the research about relevant mechanism become the focus of current studies.Although scholars from around the world carry out many experiments on strontium salt at different levels, but the molecular mechanism of bone formation has not yet been fully elucidated. As we all know, the osteoblasts are differentiated from bone marrow mesenchymal stem cells (BMSCs). The current studies focuse on exploring the possible signaling pathways though which the strontium salt effect bone marrow mesenchymal stem cells. In order to prove that the molecular mechanism of bone formation of the strontium salt, several experiments have been carried out at the cellular level, for example, in Yang F’s experiment, human bone marrow mesenchymal stem cells were exposed tostrontium salt, and the results suggested Wnt pathway was involved in strontium salt induced differentiation of human bone marrow mesenchymal stem cells osteoblasts. Peng S found that the strontium salt can promote BMSCs osteogenic differentiation by activating the Ras/MAPK signaling pathway downstream transcription factor Runx2activity. BMP2/Smad pathway is an important pathway of osteoblasts. BMP2is one of the the osteoblast differentiation most important extracellular signaling molecules in promoting bone formation and inducing bone marrow mesenchymal stem cells differentiate. BMP2/Smad signaling pathway is a necessary of bone marrow mesenchymal cells to differentiate into osteoblasts and bone extracellular matrix synthesis and secretion, but whether the pathway effect when the strontium salt promotes bone marrow mesenchymal cells osteogenic differentiation, at home and abroad which is still a lack of relevant research. The experimental group early experiments have proved:the strontium salt upregulates the expression of BMP2,which mediated the role of bone marrow mesenchymal cells differentiation into osteoblasts. Our experiment will further observed BMP-2/Smad pathway how to effect when the strontium salt promotes bone marrow mesenchymal cells to differentiate into osteoblasts process.Research Methods1.Rat BMSCs isolation, purification and cultureTaking4-week-old SD rats, male or female, firstly,the rats were sacrificed by cervical dislocation.Then they were soaked in75%alcohol disinfection10min. After,we separate on both sides of the femur and tibia of the rats under sterile conditions, and cut the epiphyseal end of the femur and tibia, to expose bone marrow cavity, and then use the DMEM-F12culture medium,which contains10%fetal bovine serum,100U/ml penicillin and100μg/ml streptomycin),continually flushing the marrow cavity of20-30times, until the bone marrow cavity looks like white, and collected DMEM-F12culture medium containing cells. Centrifugal cells1000rpm/min for5min, the supernatant was removed.Adding3ml DMEM-F12culture medium to resuspend cells,we put cells for seeding in25cm2culture flasks, placed in37℃,5%CO2incubator culture. After24h or48h, the medium was changed to remove the suspension of blood cells and nonadherent cells to cells for the first time. After every2or3day for once liquid, we observe cells growth and pollution-free in inverted phase contrast microscope. The cells were passaged when their growth occupy nearly90%. 2.Osteogenic differentiation of rat BMSCsThe third to fifth generation rat BMSCs were selected as the object of study, and were seeded in culture dishes or plates, and were cultured with osteogenic induction medium which contains10%fetal bovine serum,10-8mol/L dexamethasone,10mmol/L β-glycerophosphate sodium,50μg/ml ascorbic acid in DMEM-F12culture medium.3.Western blotting was used to detect phosphorylated Smadl/5/8and Runx2protein expression levelsThe third to fifth generation rat BMSCs were selected as the object of study, and were seeded in60mm Petri dishes.After experimental groups were given different treatment,respectively.Then protein was extracted and quantified by electrophoresis, transferred to a membrane and wrap antibody exposure. Last the results were analyzed by using the gel imaging system.4.small interfering RNA (SiRNA)The third to fifth generation rat BMSCs were selected as the object of study, and were seeded in60mm Petri dishes with double anti-free DMEM-F12culture medium, when cells grown to70%confluence and transfceted the Smadl SiRNA with Lipofectamine2000transfection, according to the manufaceturer’s instruction.48hour after transfection, experimental groups were given different treatment,respectively.Protein Smadl/5/8and phosphorylation of Smadl/5/8and Runx2levels were detected using Western blotting assay. Alkaline phosphatase activity and Qian prime red staining of calcium nodules were analyzed at day7and21after strontium salt treatment in rat BMSCs trasfected with SiRNA.5.Real-time PCR method for the determination of small interfering Smad1and Runx2gene expression in cellsThe third to fifth generation rat BMSCs were selected as the object of study, and were seeded in25mm Petri dishes, After experimental groups were given different treatment,respectively.RNA was isolated after the detection of gene expression.6.Alkaline phosphatase (ALP) activityThe third to fifth generation rat BMSCs were selected as the object of study, and were seeded in12-well plates, After experimental groups were given different treatment,respectively. And the ALP activity was detected by enzyme linked immunosorbent assay on the7th day. Cells were cracked fully with cell lysate full cleavage, centrifuged at12000rpm/min high-speed for10minutes, the supernatant was taken for detection according to the kit instructions, the results were measured with micreplate at492nm wavelength.7.Alizarin Red calcified nodules stainingThe third to fifth generation rat BMSCs were selected as the object of study, and were seeded in coverslips. After experimental groups were given different treatment,respectively. When cells were cultured for21days, calcium alizarin red staining reagents stained samples observed calcium nodules staining. Each group of three samples, each sample randomly selected three fields (x100), the differences between each group were compared.8. Statistical AnalysesAll measurement data were expressed as mean±SD.Statistical analyses were conducted with SSPSS13.0for Windows,comparing continuous variables of groups used One-way ANOVA. LSD method was used for multiple comparison for homoscedasticity,Significance was defined as P<0.05.RESULTS1. Sr raised BMSCs phosphorylated Smad1/5/8protein expressionRat BMSCs were treated with0.1mmol/L,1mmol/L,3mmol/L,5mmol/L, and 10mmol/L Sr treatment for1hour,0.1mmol/L of Sr begin to increase in the phosphorylation of p-Smad1/5/8expression, but with the control group,there was no statistically significance(P>0.05); when Sr concentration was1mmol/L, p-Smadl/5/8expression level was significantly increased compared with the control group, the difference was statistically significant (P<0.05). However, when Sr concentration was increased to3mmol/L,5mmol/L, and10mmol/L, p-Smadl/5/8expression no significant effect. Other hand, when the Sr concentration of0.1mmol/L,3mmol/L,5mmol/L and10mmol/L, compared with the control group, the expression of Smadl/5/8was no statistically significance (P>0.05); by lmmol/L Sr process of BMSCs30min,1hour,2hours, compared with the control group, p-Smadl/5/8expression within cells were significantly increased, which BMSCs were exposed Sr treatment for1hour, The expression of p-Smadl/5/8levels peaked (P<0.01), and BMSCs were exposed Sr treatment exposed for6hours, the expression of p-Smadl/5/8levels significantly reduced close to the level of the control group (P>0.05).2.BMP2antagonist noggin inhibits Sr-induced expression of phosphorylated Smadl/5/8protein expressionRat BMSCs were treated with Sr treatment for1hour, compared with the control group, intracellular p-Smadl/5/8expression was significantly increased (P<0.01), Preconditioning with100ng/ml noggin for2hours, can significantly block Sr-induced expression of phosphorylated Smadl/5/8protein expression(P<0.01), that suggested BMP-2was a upstream of Smadl/5/8,and mediated Sr induced the activation Smad1/5/8in rat BMSCs.3. Sr increases Runx2protein expression in BMSCsRat BMSCs were treated with1mmol/L,3mmol/L,5mmol/L of Sr treatment for6hours, compared with the control group, intracellular Runx2expression were significantly elevated, when Sr concentration was1mmol/L, Runx2expression level of the highest value. When Sr concentration was5mmol/L, the increased expression of Runx2decreased, but was still significantly higher than the control group (P<0.05). After1mmol/L Sr process of BMSCs3hours,6hours,1day,3days, compared with the control group, Runx2expression within cells were significantly increased (P <0.01), which deal with6hour, the expression of Runx2levels peaked.4. Smadl SiRNA konckdown reduce Smadl/5/8protein expressionRat BMSCs were transfected with the Smadl SiRNA01, Smadl SiRNA02, Smadl SiRNA03, respectively, for48hours, compared with the control, Smadl SiRNA01and Smadl SiRNA02and Smadl SiRNA03groups, Smadl/5/8expression was significantly decreased(P<0.05), which Smadl/5/8expression was at least in Smad1SiRNA02group(P<0.01), so subsequent experimental,we selected Smadl SiRNA02as effective siRNA.5.Smadl SiRNA konckdown decresed the Sr-induced upregulation of phosphorylated Smadl/5/8protein expressionRat BMSCs wereexposured in1mmol/L Sr treatment for1hour, and then compared with separate control group, p-Smadl/5/8expression within cells was significantly higher (P<0.01); Rat BMSCs were transfected with the Smadl SiRNA for48hours,that significantly decresed the Sr-induced upregulation of phosphorylated Smadl/5/8protein expression (P<0.01)6.Smadl SiRNA konckdown decresed the Sr-induced upregulation of Runx2protein expressionRat BMSCs were exposured in1mmol/L Sr treatment for6hours, and then compared with separate control group, Runx2expression within cells was significantly higher (P<0.01); Rat BMSCs were transfected with the Smadl SiRNA for48hours,that significantly decresed the Sr-induced upregulation of Runx2protein expression (P<0.01).It suggested that the Smadl/5/8pathway mediated Sr-induced upregulation of Runx2protein expression.7. Smadl SiRNA konckdown reduce Smadl gene expressionRat BMSCs were transfected with the Smadl SiRNA for24hours, and exposured in lmmol/L Sr treatment for2hours, and then compared with separate control group, Smadl gene expression in the Sr group was no significant difference (P>0.05); compared with separate control group, the Si-smadl+Sr group, Smadl gene expression was significantly decreased (P<0.01).8.Smadl SiRNA konckdown reduce the Sr-induced upregulation of Runx2gene expressionRat BMSCs were transfected with the Si-control, Smadl SiRNA, respectively, for24hours, and exposured in1mmol/L Sr treatment for2hours, and then compared with the control group, Runx2gene expression in the Sr group was significantly increased (P<0.01); compared with Sr group,the Si-smadl+Sr group, Runx2gene expression was significantly decreased (P<0.01).9. Smadl SiRNA konckdown inhibited Sr-induced activity of ALP in BMSCsRat BMSCs were exposured in lmmol/L Sr treatment for7days, and then compared with separate control group,ALP activity in the cells was significantly increased (P<0.01); Rat BMSCs were transfected with the Smadl SiRNA for48hours, inhibiting Sr-induced activity of ALP in BMSCs (P<0.05).10.Smad1SiRNA konckdown inhibited Sr-induced mineralization in BMSCsRat BMSCs were transfected with the Si-control and exposured in lmmol/L Sr treatment for21days, and then compared with separate control group,the number of intracellular calcium nodules increased significantly; Rat BMSCs were transfected with the Smadl SiRNA for48hours, cells significantly reduced the number of intracellular calcium nodules. Separate Smadl SiRNA transfection group calcium nodules didn’t decreased significantly.CONCLUSIONSIn hte process of BMSCs differentiating into osteoblasts, Sr raised phosphorylated Smad1/5/8and Runx2expression, and BMP-2antagonist noggin antagonistic Sr-intrduced upregulation of expression of phosphorylated Smad1/5/8; lowered the expression of phosphorylated Smad1/5/8inhibited Sr-induced promoting bone formation,the performances were the reduction of Runx2expression and the inhibitation of activity of ALP, and a significant reduction in the number of calcified nodules within the cells,The present results suggested that BMP-2/Smad pathway is involved in Sr-induced osteoblasts differentiation of rat BMSCs. |
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