Strontium Ranelate Promotes The Osteogenic Differentiation Of Rat Bone Mesenchymal Stem Cells Through The TGF-β1/Smad Signaling Pathway

BACKGROUNDOsteoporosis is a kind of systemic systemic bone disease which is characterized by low bone mass, bone micro-structural damage, leading to bone fragility and prone to fracture. There’s a high incidence in the elderly population and postmenopausal women, which has serious impact on quality of life. The incidence of osteoporosis is very high, according to statistics, about60%to80%of the old who reached60years or older suffer from osteoporosis, osteoporosis has become a global health problem, The prevention and treatment about osteoporosis has become a serious problem facing China’s public health career. Currently, drugs for the treatment of osteoporosis can be divided into1.an anti-resorptive drugs, such as calcium, calcitonin, vitamin D, estrogen, bisphosphonate, etc.2, To increase bone formation drugs, such as fluoride, parathyroid hormone.3.Drugs can not only promote bone formation but also inhibit bone resorption dual, such as strontium ranelate. Strontium ranelate has a dual role that can promote bone formation while inhibiting bone resorption compareing to the traditional treatment of osteoporosis drugs. The research about Strontium ranelate’s anti-osteoporosis has become a hot.Strontium ranelate is the first listed drug has a "dual role" which has been approved by the European Union in October2004.It can not only promote bone formation but also inhibit bone resorption dual, strontium salt represents new direction of development in the history of the osteoporosis’s treatment.The bone mesenchymal stem cells, BMSCs are pluripotent adult stem cells, which have the ability to differentiate into osteoblasts. The process of BMSCs differentiating to the osteoblast is regulated by a variety of signaling pathway, including TGF-P (transforming growth factor β1, transforming growth factor β1)/of BMPs (bone morphogenetic proteins), Wnt, MAPK (mitogen-activated protein kinase) and hedgehog (hedgehog protein signaling pathway).Transforming growth factor-β (TGF-P) plays an important role in bone formation and resorption in balance, it can promote bone marrow mesenchymal stem cell differentiation into bone and inhibite osteoclast differentiation.Its role on bone remodeling is becoming a focus for researchers. Smads proteins directly involved in signal transduction of the TGF-βsuperfamily.Smads tranceduce TGF-P signaling from the extracellular to the nucleus as TGF-βdownstream signaling protein molecules.It is initiating factor of TGF-βsignaling pathway. Runx2also known as the core binding factor α1(Cbfα1) is a multi-functional transcription factor protein, which plays an important role in the osteoblast differentiation and bone formation. A variety of factors such as TGF-P can be as Runx2raw adjusting factor regulating Runx2activity.Our previous studies have confirmed Sr can promote BMSCs differentiate to osteoblast by up-regulating TGF-β1and Runx2expression. Therefore, this experiment further clarified whether Sr promote BMSCs differentiate to osteoblast through TGF-β1/Smad signaling pathways on the basis of our previous studies. METHODS1. Acquisition of the rat BMSCsA4-week-old male SD rat was first killed by cervical dislocation, then was soaed in75%alcohol for5-10minutes. the bilateral femur and tibia was removed and dissected free of soft tissues under the sterile conditions, the proximal femur and the distal tibia were cut,then the bone marrow cavity was exposed. Then the marrow cavity was flushed with DMEM sugar complete medium containing with10%fetal bovine serum,100U/ml penicillin and100μg/ml streptomycin and the cells were collected and then centrifuged at1000rpm for5minutes,single cell suspension was made, Cells were seeded in25cm2culture flasks at1-3x105cells/cm2concentration,cultured in the incubator at37℃,5%CO2and80%relative humidity. the half medium was changed After24hours,Since then the medium was changed every2-3days. Morphological observation of the cells was conducted by inverted phase contrast microscope. Cells were passaged when they were closed to80%-90%,since then passaged once every3-4day.2. Osteogenic differentiation of BMSCsThe third to fifth generation BMSCs were selected as the object of study. Cells were seeded in a petri dish or culture plate according to different experimental needs,and were cultured with osteogenic solution(DMEM sugar medium with10%fetal bovine serum,100U/ml penicillin,100ug/ml streptomycin,10-8mol/L dexamethasone,10mmol/L β-glycerophosphate and50μg/ml ascorbic acid).3. P-Smad2, Smad2and Runx2protein expression levels were detected by Western blottingthe third generation cells were selected, seeded in60mm petri dish, when cells were collected, first washed with cold PBS twice, the cell lysate was added, cracked at4℃for20-30min, the cells were scraped with precooled cell scraper on ice and moved into1.5ml EP tubein, centrifuged12000r/min for10min, the supernatant was transferred to a new tube in the EP. The protein concentration was determined with Application BCA protein quantitation kit.The protein was transferred to PVDF membrane9V30min after vertical SDS-PAGE gel electrophoresis separation. Confined with5%skim milk for1h at room temperature then were added p-Smad2antibody (1:1500), Runx2antibody (1:500) and GAPDH (1:5000),4℃shaker incubated overnight. Horseradish peroxidase-labeled secondary antibody (1:2500) was added washed three times with TBS/T and incubated for1h, then washed3times in TBS/T, finally colored and exposured with the luminescent reagent ECL.The membrane was washed2-3h with TBS/T after exposured and Smad2(1:1000) antibody was added.The target striped gray value was analysed using the Smartview software.4.Small interfering There Smad2-specific siRNA and an non-target-siRNA were designed. Operate with gloves,using pipette tip,EP tube and reagents without RNA enzyme. SiRNA lyophilized powder was dubbed the20μM storage solution with sterile ddH2O after instantaneous centrifugation, Aliquot to save to avoid repeated freezing and thawing. The day before transfection, the appropriate number of cells were seeded to the culture plate or dish, the cells were cultured with double anti-free medium and were placed in37℃C,5%CO2incubator overnight, make sure the cell density reached60%-70%when transfected. The siRNA-Lipo2000mixture was first prepared before transfected:first siRNA stock solution, Lipo2000were diluted with Opti-MEM serum-free medium, gently mixed5min at room temperature and two mixture were mixed gently and incubated at room temperature for20min, the siRNA-Lipo2000mixture was added to the cell culture medium with double anti-free medium, and mixed, the mixture was discard after6h, to replace fresh medium contain without dual anti,then treated after cultivated2-3d. 5. Real-time PCR method for the determination of Runx2expressionSelect the third generation cells, seeded in25mm Petri dishes with the density of1×105/ml approximately,each dish inoculated with cell suspension of1mL,each experimental group was given different treatment,respectively.Total RNA was extracted at different time points for the gene expression determination.6. Detection of alkaline phosphatase(ALP) activityThe third generation cells were selected. Cells were inoculated in24-well plates,each group was given different treatment factors, and the ALP activity was detected by enzyme linked immunosorbent assay on the7th day. Cells were cracked fully with0.2%Triton X-100and freezing and thawing method, then were washed with PBS two times after cracked, centrifuged at12000rpm for10minutes, the supernatant was taken for detection according to the kit instructions, the results were measured with micreplate at490nm wavelength.7. Alizarin red staining for calcium nodulesThe third generation cells were selected and seeded in24mm×24mm coverslip prepositioned in six-well plates,each group was given different treatment factors, and the samples were fixed, stained, and clarified after21days according to the alizarin red staining kit instructions, calcium nodules were observed under inverted microscope,four samples were taken from each group,each sample was randomly taken from a field of vision (×100), then to compare the differences between the groups.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,and Dunnett’s T3method was used for multiple comparisions for heterogeneity of variance. Significance was defined as P<0.05.RESULTS1. Strontium ranelate(Sr) enhances the expression of p-Smad2in BMSCsBMSCs were separately treated with0.1mmol/L,1mmol/L,3mmol/L,5mmol/L and10mmol/L Sr for1h, compared with the control group, the expression of p-Smad2within BMSCs was increased, the differences were statistically significant (P<0.01), the expression of p-Smad2among the groups was statistically significant (F=1261.798, P<0.01); including when Sr concentration of1mmol/L, the expression of p-Smad2’mean levels were largest,the differences were statistically significant (P<0.01) compared with the rest of the group,it can be considered the expression of p-Smad2reached the peak when the concentration of Sr was1mmol/L. When Sr’concentration was3mol/L,5mol/L and10mol/L, the expression of p-Smad2was gradually reduced, but still higher than that in control group (P<0.01). BMSCs were separately treated with1mmol/L Sr for15min,30min,60min,120min and360min, compared with the control group, the expression of p-Smad2were increased, the differences were statistically significant (P<0.01). the expression of p-Smad2among the groups was statistically significant (F=723.554, P<0.01), when BMSCs were treated with1mmol/L Sr for60min, the expression of p-Smad2’mean levels were largest,the differences were statistically significant (P<0.01) compared with the rest of the group,it can be considered the expression of p-Smad2reached the peak (P<0.01), the expression of p-Smad2when BMSCs were treated with1mmol/L Sr for120min and360min, the expression of p-Smad2level was gradually reduced, but still high in the control group (P<0.01).2.Strontium ranelate(Sr) enhances the expression of Runx2in BMSCsBMSCs were separately treated with1mmol/L Sr for1d,3d,and5d, compared with the control group, the expression of Runx2were increased, the differences were statistically significant (P<0.01). the expression of Runx2among the groups was statistically significant (F=58.106, P<0.01), when BMSCs were treated with1mmol/L Sr for5d, the expression of Runx2’mean levels were largest,the differences were statistically significant (P<0.01) compared with the rest of the group,it can be considered the expression of Runx2reached the peak (P<0.01), the expression of Runx2when BMSCs were treated with1mmol/L Sr for5d, the expression of Runx2was gradually reduced, but still high in the control group (P <0.01).3. Inhibitor of TGF-β1(SB431542) inhibits Sr-induced over-expression of p-Smad2in BMSCs of rats.BMSCs were treated with Sr(lmmol/L) for1h, compared with the control group, the expression of p-Smad2within BMSCs was significantly increased, the differences were statistically significant (P<0.01), BMSCs were pretreated with SB431542for lh,followed by the treatment of lmmol/L Sr for1h, the expression of p-Smad2was gradually reduced compared to Sr group, the differences were statistically significant (P<0.01).4. Selection of the effective Smad2siRNAThree Smad2small interfering RNA siRNA001, siRNA002siRNA003were designed, BMSCs were transfected with siRNA001, siRNA002siRNA003respectively, and then were treated with osteogenic liquid, the expression of the Smad2protein in group of small interfering (Western blotting method) and the Smad2mRNA (Real Time PCR) reduced compared to the control group, the difference was statistically significant (P<0.01), the expression of the Smad2protein in group of small interfering (Western blotting method) and the Smad2mRNA (Real Time PCR) in Smad2siRNA003transfection group was the least, the difference was statistically significant (P<0.01), So the interfere of Smad2siRNA003to the expression of the Smad2was the strongest.5. Smad2siRNA inhibits Sr-induced over-expression of p-Smad2in BMSCs of rats.BMSCs were treatment with1mmol/L Sr for1h, compared with the control group, the expression of p-Smad2were significantly increased, the difference was statistically significant (P<0.01), BMSCs were transfected with Smad2siRNA, followed by the treatment of1mmol/L Sr, the expression of p-Smad2was gradually reduced compared to Sr group, the differences were statistically significant (P <0.01).6. Smad2siRNA inhibits Sr-induced over-expression of Runx2in BMSCs of rats.BMSCs were treatment with1mmol/L Sr for1h, compared with the control group, the expression of Runx2were significantly increased, the difference was statistically significant (P<0.01), BMSCs were transfected with Smad2siRNA, followed by the treatment of1mmol/L Sr, the expression of the Smad2protein in group of small interfering (Western blotting method) and the Smad2mRNA (Real Time PCR) reduced compared to Sr group, the difference was statistically significant (P<0.01).7. Smad2siRNA inhibits Sr-induced the ALP activity in BMSCs of rats.BMSCs were treatment with1mmol/L Sr for1h, compared with the control group, the activty of ALP were significantly increased, the difference was statistically significant (P<0.01), BMSCs were transfected withSmad2siRNA,followed by the treatment of lmmol/L Sr, the activty of ALP was gradually reduced compared to Sr group, the differences were statistically significant (P<0.01).8. Smad2siRNA inhibits Sr-induced mineralization in BMSCs of rats.BMSCs were treatment with1mmol/L Sr for21d, compared with the control group, the calcium nodules number was significantly increased, the difference was statistically significant (P<0.01), BMSCs were transfected with Smad2siRNA, followed by the treatment of1mmol/L Sr for21d, the calcium nodules number was gradually reduced compared to Sr group, the differences were statistically significant (P<0.01).CONCLUSIONS1.In the process of Sr induced BMSCs to differentiate into osteoblasts, Sr raised the expression of P-Smad2and Runx2. TGF-β1specific inhibitor (SB431542) inhibits Sr-induced over-expression of p-Smad2in BMSCs of rats. Sr-induced over-expression of p-Smad2and Runx2in BMSCs of rats significately reduced by Smad2siRNA, and the ALP, calcium nodules number were also significantly reduced.2. It can be showed that the TGF-β1/Smad2/Runx2signaling pathway mediates Strontium ranelate promoting the osteogenic differentiation of rat bone mesenchymal stem cells.