Strontium Ranelate Promotes Osteogenic Differentiation Of Rat Bone Mesenchymal Stem Cells Through Hedgehog/Gli1 Signaling Pathway

BackgroundOsteoporosis (OP) is a systemic metabolic bone disease with low bone mass and bone micro-structural damage, resulting in increased bone fragility and being more likely to fracture (World Health Organization, WHO). As people live longer and the arrival of aging society, osteoporosis becomes an important human health problem. The serious consequence can lead to the occurrence of osteoporotic fracture, which is called brittle fracture. It means slight wound or daily activities may lead to fracture, resulting in increased death rate and disability rate, which seriously ruduces the quality of life. Moreover, the treatment of osteoporotic fracture is expensive, which may burden the patients and society. Osteoporotic fractures are preventable. Therefore, it is very important to popularize knowledge of osteoporosis and do a good job in the early precaution, diagnosis and treatment.Strontium ranelate (Sr) is a new generation of anti osteoporosis drug, which can act on both osteoclasts and osteoblasts, leading to inhibition of bone resorption and stimulation of bone formation. Many clinical studies indicate that strontium ranelate can significantly improve bone density and bone microstructure, and reduce the incidence of vertebral fractures and non vertebral fractures. At present, there have been some studies that support Sr can promote bone formation at the cellular level. For example, Choudhary found that Sr promoted osteogenic differentiation of bone mesenchymal stem cells through cyclooxygenase-2 and prostaglandin E2. Peng et al found strontium salt can promote osteoblastic differentiation of MSCs by activating Ras/MAPK signaling pathway and the transcription factor Runx2 (an important transcription factor for osteogenic differentiation) activity. Fromigue considerd that the activation of nuclear factor of activated T cells C1 (NFATc1) and the downstream Wnt signal pathway played an important role in promoting osteogenic differentiation of BMSCs induced by Sr.Our previous study found that Sr could induce BMSCs to differentiate into osteoblasts by activating the transforming growth factor beta 1 (TGF-beta 1)/smad2, bone morphogenetic protein 2 (BMP2)/smad1/5/8, Sonic Hedgehog (Shh) and the transcription factor Runx2. Hedghog pathway is a popular signaling pathway in recent years. Many studies showed that it played an important role in osteogenesis, and regulated the formation and development of vertebrates. However, whether Sr can induce the osteogenic differentiation of BMSCs through its downstream signaling molecule Gli is not yet completely clear. Therefore, we further study the function of Hedgehog/Gli pathway in promoting the osteogenic differentiation of BMSCs induced by Sr. Research methods 1 Rats BMSCs separation, purification and cultureA 4-week-old male SD rats was killed by cervical dislocation and immersed in 75% alcohol for 5 min, then isolated from both sides of the femur and the tibia under aseptic conditions. After eliminated the surrounding muscle, fascia and cut off the proximal femur and distal tibia, we repeatedly flushed the marrow cavity with DMEM/F12 culture medium containing 10% fetal bovine serum, then collected the cells, cemtrifuged it at 1000 r/min for 5 min, removed the supernatant, and added 5 ml DMEM/F12 medium into the cells. The cells were inoculated in the 25 cm2 culture bottle and placed in the incubator with 37℃ and 5% CO2. After 24 hours, change the medium for the first time and then it was changed every 2～3 days. The cells were observed under a microscope and passaged when it grew nearly 80%～90%.2 Osteogenic differentiation of BMSCsSelected the 3-5 generations of BMSCs as the research object. According to the different needs of the experiment, the cells were grown in culture dishes or culture plates, when the cells were fusioned to 80%～90%, the osteoinductive liquid (containing 10% fetal bovine serum, 1×10-8mol/L dexamethasone,10 mmol/L beta sodium glycerophosphate and 50 mg/L ascorbic acid in DMEM/F12 medium) could be added to the cells.3 The Glil and Runx2 expression were detected by Western blot.The third generation cells were inoculated in 60 mm culture dishes. Each group was given a different treatment according to different experimental objective. Then the cells were washed with cold PBS for two times, added 60-80ul cell lysate, placed in a refrigerator at 4℃ for 30 min, and centrifugated at 12000 r/min for 10 min. The supernatant was collected, and proteins were quantified by BCA assay kit. Total proteins were separated by SDS-PAGE, and were transferred to the PVDF membrane. After blocking the proteins with the 5% skim milk for 1 h, Glil antibody (1:200) or Runx2 antibody (1:1000) was added at 4℃ overnight. Then the membranes were washed with TBST for 3 times (each time 5 min), incubated with the secondary antibody(1:4000) at room temperature for 1.5 h, and then were washed with TBST again. Make the color using the ECL angent, exposure in the darkroom, scan by gel imaging system, and finally analyse the results.4 Detection of ALP activitySelected the third generation BMSCs, and then inoculated it in the 24 well plates. After the experimental group received different treatments, we detected the ALP activity by ELISA at the first 7 days. Lysate was added for 30～40min after the supernatant was removed. Then we collected the liquid with a pipette, and detected it at a wavelength of 492 nm in microplate reader according to kit instructions.5 Alizarin red staining of the calcium nodulesThe third generation cells were inoculated in 6-well plates (pre-placed with 24 mm* 24 mm coverslip). According to the different experimental purposes, the experimental groups were given different treatments for 21d. Then the samples were fixed and colored with alizarin red staining agents, and observed under inverted microscope. Each group chose 3 samples, each sample randomly selected a field of view (x 100), and finally compared the difference between the groups.6 Small interfering RNA technologyDesigned three Glil-siRNAs and one negative siRNA (non-target siRNA). The pearhead, EP pipe and reagent should be non RNA enzyme. ddH2O was added to the siRNA freeze-dried powder (Storage solution was 20 umol/L), and stored it in-20℃ refrigerator. One day before the transfection, inoculated the appropriate number of cells to the culture dishes or culture plates, placed in the incubator with 37℃ and 5% CO2. When the cell density reached 60%～70%, it can be transfected with the siRNA. SiRNA-Lipo2000 mixed liquid should be prepared in advance. Diluted the siRNA storage solution and Lipo2000 with Opti-MEM respectively, put them at room temperature for 5 min, then mixed them, and put them at room temperature for 20 min. Finally, added the siRNA-Lipo2000 mixed liquid into culture medium without antibiotics. After 6 hours, discarded the liquid mixture, replaced it with fresh medium, and given it different treatment according to the different experiment purpose.7 Statistical processingData were expressed as mean±standard deviation (x±s) to indicate, using SPSS 13.0 statistical software for statistical analysis, comparison between groups using single-factor analysis of variance (One-Way ANOVA), and pairwise comparison using LSD test. P<0.05 was said that the difference was statistically significant..Results1 Sr could upregulate the Glil expression of BMSCsDifferent concentrations of Sr (0.1,1,3,5 mmol/L) treatment at BMSCs for 7 d, Glil expression were significantly higher than those in the control group (P< 0.01), and the expression level of Glil reached the highest value in 3 mmol/L Sr. In the concentration of 5 mmol/L, the over-expression of Gli1 induced by Sr was decreased, but there was still statistically significant compared with the control group (P< 0.01). While in the 10 mmol/L concentration of Sr group, the expression of Gli1 was no significant difference compared with the control group (P> 0.05). Using 3 mmol/L Sr for 3 days, the expression of Gli1 was no significant difference compared with the control group (P> 0.05), however, exposed to 3 mmol/L Sr for 5 d,7 d or 14 d, the Glil expression were significantly higher than those in the control group (P< 0.05 or P< 0.01), and the Glil expression reached the highest value in 7 d.2 Hedgehog receptor antagonist Cy inhibited the Sr induced upregulation of Gli1 expression of BMSCs.Exposed to 3 mmol/L Sr for 7 d, the Gli1 expression was significantly increased compared with the control group (P< 0.01). While using 10 mol/L Cy and 3 mmol/L Sr at the same time for 7d, the expression of Gli1 decreased significantly compared with the Sr group (P< 0.05).10 umol/L Cy itself had no obvious effect on the basal expression of Gli1.3 Screening the effective Glil-siRNACompared with the non-target siRNA group, Glil-siRNA001 could not reduce the expression of Glil (P> 0.05).However, both Glil-siRNA002 and Glil-siRNA003 could decreased the Gli1 expression (P< 0.05), in which the effect of Glil-siRNA002 was the best (P< 0.01).4 Glil-siRNA inhibited the Sr induced upregulation of Glil expression of BMSCs.Exposed to 3 mmol/L Sr for 7 d, the expression of Glil increased obviously compared with the control group (P< 0.01). After transfected with Gli1-siRNA or non-target siRNA respectively, and then exposed to 3 mmol/L Sr for 7 d, Glil-siRNA can inhibited the Sr induced upregulation of Gli1 expression compared with the Sr group (P< 0.01), while there was no effect on the non-target siRNA group (P> 0.05).5 Glil-siRNA inhibited the Sr induced upregulation of Runx2 expression of BMSCs.Exposed to 3 mmol/L Sr for 7 d, the expression of Runx2 increased obviously compared with the control group (P< 0.01). After transfected with Gli1-siRNA or non-target siRNA respectively, and then exposed to 3 mmol/L Sr for 7 d, Glil-siRNA can inhibited the Sr induced upregulation of Runx2 expression compared with the Sr group (P< 0.01), while there was no effect on the non-target siRNA group (P> 0.05).6 Glil-siRNA antagonized the promoting effect on the ALP activity induced by Sr.Exposed to 3 mmol/L Sr for 7 d, ALP activity significantly increased compared with the control group (P<0.01).After transfected with Glil-siRNA or non-target siRNA respectively, and then exposed to 3 mmol/L Sr for 7 d, Glil-siRNA can inhibited the promoting effect on the ALP activity induced by Sr compared with the Sr group (P< 0.01), while there was no effect on the non-target siRNA group (P> 0.05).7 Glil-siRNA antagonizedthe promoting effect on the formation of calcium nodules induced by Sr.Exposed to 3 mmol/L Sr for 21 d could significantly increased the number of calcium nodules. After transfected with Glil-siRNA or non-target siRNA respectively, and then exposed to 3 mmol/L Sr for 21 d, Glil-siRNA can inhibited the the promoting effect on the formation of calcium nodules induced by Sr compared with the Sr group (P< 0.01), while there was no effect on the non-target siRNA group (P> 0.05).Conclusion1 Sr could upregulate the Gli1 expression by activating the Hedgehog receptor.2 Glil plays an important role in the promotion of BMSCs osteogenic differentiationinduced by Sr, including the increase of Runx2 expression, the promotion the ALP activity and the formation of calcium nodules.3 Activation of Hedgehog/Gli1 pathway may be an important mechanism of Sr to promote osteogenic differentiation of BMSCs.