Mechanisms Of Simvastatin Promoted Osteogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells In Rats

Background:Osteoporosis(OP)is a systemic osteoporosis caused by the destruction of bone structure and the decrease of bone mass due to a variety of factors,resulting in increased brittleness of bone tissue and prone to pathological fracture.Idiopathic osteoporosis,postmenopausal osteoporosis and senile osteoporosis are the three main types of primary osteoporosis.With the improvement of people’s quality of life,the average age has gradually increased,and the phenomenon of population aging has become a serious problem facing global public health.Data show that 60%of middle-aged and elderly people in China suffer from osteoporosis.Osteoporosis occurs in people over 65 years,which seriously affects the quality of life of patients and causes an overloaded economic burden on patients’ families.In bone metabolism,the imbalance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption causes a disorder of bone tissue remodeling,which is an important pathological process of osteoporosis.This process is controled by genetic(such as polymorphism)and environmental factors(diet,smoking,occupational exposure to environmental pollutants).Good living habits,drugs such as calcium and hormone replacement drugs can effectively prevent the formation of osteoporosis and reduce the risk of osteoporosis.However,disability and death caused by osteoporotic fractures are still important causes of reduced life and quality of life in the elderly.For the time being,the etiology of osteoporosis in the elderly remains unclear.Dysfunction of bone formation during bone reconstruction is the main pathological mechanism of osteoporosis.The imbalance of differentiation between osteoblasts and adipocytes leads to a decrease in the number of bone marrow mesenchymal stem cells(BMSCs),which leads to fracture healing disorders,which is one of the causes of senile osteoporosis.During bone formation,osteoblast precursor cells are mainly derived from bone marrow mesenchymal stem cells of the bone marrow,and the osteogenic differentiation capacity of BMSC cells is a pivotal factor affecting bone formation,bone quality and bone quantity.Therefore,to enhance the osteogenic differentiation potential of BMSC cells in patients with osteoporosis and reveal the molecular mechanisms of osteogenic differentiation of BMSC cells is the key for exploring the diagnosis and treatment of osteoporosis.Simvastatin(SIM)is one of crucial member of the statin family.A large number of studies have shown that simvastatin can inhibit osteoblast apoptosis and promote mesenchymal stem cells to differentiate into osteoblasts.The important role of simvastatin in regenerative medicine has received more and more attention.However,the way of simvastatin administration,dosage and drug response has not been clearly defined,and the mechanism of simvastatin-induced osteogenic differentiation of MSC cells are remains clear.The Wnt/β-catenin signaling pathway is highly activated in mesenchymal stem cells and osteoblasts,participates in the regulation of osteogenic differentiation of MSC cells,and plays an important role in the proliferation and terminal differentiation of osteoblast precursor cells.In addition,the activation of Wnt/β-catenin signaling pathway is necessary for osteoblast mineralization and preventing osteoblast apoptosis.Simvastatin has various functions including promoted the proliferation of mesenchymal stem cells and induced mesenchymal stem cells to differentiate into osteoblasts,and its mechanism is related to the activation of signaling pathways such as MAPK/Akt,TGFβ/Smad3,Hedgehog/Glil.However,the biological significance of the Wnt/β-catenin signaling pathway in BMSC cell proliferation and osteogenic differentiation induced by simvastatin is unknown.Whether the Wnt/β-catenin signaling pathway is involved in regulating simvastatin-induced osteogenic differentiation of BMSC cells has not been reported.In this study,we investigated the effects of simvastatin on osteogenic differentiation of BMSC cells in vitro and explored the role of Wnt/β-catenin signaling pathway in simvastatin-induced osteogenic differentiation of BMSC cells.Objective:1.To observe the effect of simvastatin on osteogenic differentiation of rat bone marrow mesenchymal stem cells in vitro,and to explore the roles of Wnt/β-catenin signaling pathway in simvastatin-induced osteogenic differentiation of BMSC cells.2.Reveal the molecular mechanism of simvastatin-induced osteogenic differentiation of bone marrow mesenchymal stem cells,and provide a theoretical basis for targeted Wnt/β-catenin pathway agonists combined with simvastatin in the treatment of bone-related diseases.Methods:1.6-8 week SPF SD rats were sacrificed by dislocation,and BMSC cells were isolated by whole bone marrow adherent culture and enzymatic digestion for primary and passage cell culture;2.The expression of CD29,CD90,CD34 and CD45 on the surface of BMSC cells was detected by cytometry.The third generation BMSC cells were treated with simvastatin at different doses(0,0.01,0.03,0.1,0.3,1.0,3.0 nmol/L).CCK8 method,Annexin V/PI staining method and flow cytometry were used to determine the proliferation and apoptosis of BMSC cells at different time;3.BMSC cells were treated with by 0.3 nmol/L simvastatin and DMSO for indicated time,respectively.Alizarin red staining was performed on the 3rd,7th,14th,and 21th days after administration Alizarin red staining(ARS)and Alkaline phosphatase(ALP)staining.The absorbance at 570 nm of each group was measured by a UV spectrophotometer,and the calcium salt content,ALP activity,and area of ARS were computed;4.After BMSC cells were treated with simvastatin,and the cell culture supernatants were collected,and the concentrations of ALP and bone morphogenetic protein(MBP-2)were measured by an enzyme-linked immunosorbent assay(ELISA);BMSC cells were collected,and Real time PCR and Western blot were respectively used to detect the expression of of BMP-2,bone gla protein(Bglap),osteoblast-specific transcription factor(OSX),Runx2,osteopontin(OPN),osteopontin(OPN),osteocalcin(OCN),β-catenin,AKT,and JNK mRNA and protein;5.The third passage BMSC cells were transfected with luciferase-reporter plasmid,and then treated with 0.3 nmol/L simvastatin for 48 h,and the β-catenin promoter activity was measured by UV spectrophotometry;6.The 3rd passage BMSC cells were induced by 0.3 nmol l L simvastatin for 7 days,and the protein expression and nuclear transfer of β-catenin were detected by immunofluorescence(IF);7.The shβ-catenin/BMSC and shLuc/BMSC stable cell lines was constructed,and transfect sip-catenin and siNC into BMSC cells using the liposome 2000 reagent method,and then use 0.3 nmol/L simvastatin alone or combination with HLY87(Wnt/β-catenin pathway agonist)or DDK 1(Wnt/β-catenin pathway inhibitor)acting on shβ-catenin and shLuc-BMSC cells.Alizarin red staining was performed after 7 days of culture,and the expression ofβ-catenin,ALP and BMP-2 proteins were detected by using Western blot;UV spectrophotometry was used to determine ALP activity,and ELISA was used to detect ALP and BMP-2 contents in BMSC cell culture supernatants.Results:1.Isolation and identification of BMSC cellsThe results of flow cytometry showed that the positive rates of mesenchymal stem cell markers CD29 and CD90 on the surface of BMSC cells were(95.5 ±1.6)%and(97.2 ± 1.3)%,and the positive rates of hematopoietic stem cell markers CD34 and CD45 was(4.3 ± 1.5)%and(2.8 ± 0.8)%respectively,which consistent with the surface antigen phenotype of bone marrow mesenchymal stem cells,BMSC cells were successfully established in this study.2.Effects of simvastatin on BMSC cell proliferation and apoptosisThe results form the CCK8 and Annexin V/PI staining showed that simvastatin at 0.01-0.3 nmol/L did not impact the proliferation of BMSC cells;when the concentration of vastatin was>1 nmol/L,compared with the DMSO group(0 nmol/L),simvastatin significantly promoted BMSC cell proliferation in a time-and dose-dependent manner(P<0.05);Simvastatin at 0.3 nmol/L had no significant effect on apoptosis,while simvastatin at 1 nmol/L and 3 nmol/L slightly induced apoptosis(P>0.05).3.Effect of simvastatin on osteogenic differentiation of BMSC cellsAlizarin red staining:Compared with the control group(DMSO)cells,the orange-red mineralized nodules in the simvastatin-treated cells increased significantly in a time-dependent fashion(P<0.05);ALP staining:Compared with the control group,the red-brown particles in the cells of the simvastatin-treated group increased significantly,and the ALP activity in the cell culture supernatants of the simvastatin-treated group was significantly higher than that of the control group(P<0.05);ELISA results showed that compared with the DMSO group,the concentrations of ALP and BMP-2 in the cell culture supernatants of the simvastatin-treated group increased significantly in a time-dependent fashion(P<0.05).4.Effects of simvastatin on mRNA and protein expression of osteogenic differentiation-related genesReal time PCR and Western blot results showed that the levels of BMP-2,Bglap,OSX,Runx2,OPN and OCN mRNA and protein expression in cells treated with simvastatin were significantly higher than those in the control group(P<0.05),which had a time-dependent effect.5.Effects of simvastatin on Wnt/β-catenin,AKT and JNK signaling pathwaysThe results from the Real time PCR and Western blot showed that simvastatin obviously enhanced the mRNA and protein expression of β-catenin in a time-dependent fashion;Simvastatin significantly activates the expression of phosphorylated AKT(pAKT)protein,while the levels of AKT,JNK mRNA and protein in DMSO and simvastatin-treated cells have no statistical difference(P<0.05).6.Molecular mechanisms of simvastatin-induced osteogenic differentiation of BMSC cellsThe results of the luciferase reporter gene showed that the relative luciferase activity in the cells treated with simvastatin was significantly higher than that in the control group(P<0.05);the results of immunofluorescence showed thatβ-catenin in the control group was mainly expressed in the nucleus,and a small amount was expressed in the cytoplasm.β-catenin in the cells of the simvastatin-treated group was only expressed in the nucleus,and the level ofβ-catenin in the nucleus was significant Higher than the DMSO group(P<0.0001).7.Role of Wnt/β-catenin signaling pathway in BMSC cell differentiation induced by simvastatinWestern blot results showed that compared with siNC cells,the level ofβ-catenin protein expression in siβ-catenin/BMSC cells was significantly reduced,and siβ-catenin significantly inhibited the expression of ALP and MBP-2 proteins;HLY78 significantly activated β-catenin protein expression,and SIM combined with HLY78 reactivated the expression of ALP and MBP-2 proteins;Alizarin red staining results showed that the area of alizarin red-positive mineralized calcium nodules in siβ-catenin/BMSC cells was significantly reduced(P<0.05);The area of alizarin red staining positive mineralized calcium,ALP activity,ALP and BMP-2 concentration in the siβ-catenin cells were significantly lower than those of siNC cells(P<0.05);and SIM combined with HLY78 reactivated ALP activity,promoted ALP and MBP-2 protein expression,restored alizarin red positive staining;The area of mineralized calcium nodules,ALP activity,ALP and BMP-2 protein expression in DDK1-treated cells were significantly reduced;HLY78 combined with SIM treatment significantly increased the area of mineralized calcium nodules,reactivated ALP activity,and promoted the expression of osteogenic differentiation-related proteins.Conclusion:1.Low dose of simvastatin(0.3 nmol/L)can induce BMSC cells to differentiate into osteoblasts,promote the expressions of osteogenic differentiation-related genes mRNA and proteins;Simvastatin also can enhance the ALP activity,increase the ALP staining and Alizarin red staining,stimulate the secretion of ALP and BMP-2 in BMSC cells.2.shRNA-mediated low expression of β-catenin or Wnt/β-catenin pathway inhibitor DDK1 can inhibit osteogenic differentiation of BMSC cells,while targeting Wnt/β-catenin pathway agonist HLY78 promotes osteogenic differentiation of BMSC cells.3.Simvastatin may be binded to β-catenin transcription initiation site to activate β-catenin promoter activity and promote β-catenin expression and nuclear transport.4.Wnt/β-catenin signaling pathway is necessary for simvastatin to induce osteogenic differentiation of BMSC cells.Simvastatin promotes osteogenic differentiation of BMSC cells at least in part by activating Wnt/β-catenin signaling pathway.5.The molecular mechanism of simvastatin-induced osteogenic differentiation of BMSC cells at least in part dependents on the Wnt/β-catenin signaling pathway(which may be related to activation of the AKT signaling pathway),and not on the JNK pathway.