20(s)-hydroxycholesterol And Simvastatin Synergistically Promote Osteoblast Viability And Differentiation Via Raf/MEK/ERK Signaling Pathway

Background:Simvastatin is widely used to prevent the synthesis of cholesterol for the treatment of hypercholesterolemia due to its safety,effectiveness and low cost[1],and an anabolic effect promoting bone regeneration at both the cellular and molecular levels[4,5].However,several studies reported an inhibitory effect of SVA on osteoblastic proliferation,which is not completely conducive to bone formation[2,3,6,8],such as dose-dependent problems,individual differences in species,different drug routes,attenuation,inhibition of stem cell proliferation,etc.At the same time,it also reduces the level of hydroxycholesterol that is conducive to osteogenesis.Hydroxycholesterol,a large family of 27-carbon oxygenated products of cholesterol that are formed in vivo by various types of cells,including osteoblasts,play an osteoinductive role in various osteoprogenitor cells[7,8].As the most potent osteogenic naturally occurring oxysterol,20(S)-hydroxycholesterol(20(S)OHC)enhances the osteoblastic differentiation of bone marrow stromal cells(BMSCs),inhibits their adipogenic differentiation and increases calcium sedimentation in vitro;and can stimulate the secretion of endogenous cell inducer BMP-2[7,8],these changes ultimately promote bone regeneration in vivo[9].Given the evidence suggesting that SVA and 20(S)OHC significantly affected the osteogenic process,Simvastatin can inhibit the proliferation of stem cells at the later stage and enhance the osteogenic efficacy by promoting osteogenic differentiation.Hydroxysterol can promote the early proliferation of stem cells and osteogenic differentiation at the same time.Simvastatin and 20(S)hydroxycholesterol have significant effects on the osteogenic efficacy,and their mechanisms are similar and complementary,and simultaneously activate the ERK/MAPKs cell signaling pathway closely related to osteogenesist-5-7,9,10].It is speculated that the addition of 20(S)OHC to SVA may further enhance the proliferation and osteogenic differentiation of osteoblasts and promote bone regeneration and bone healing by neutralizing the inhibitory effect of simvastatin on the proliferation of stem cells or synergistically modulating mechanisms involving the functional osteogenesis-related signaling pathways.ObjectiveTo investigate the effect of a combination of SVA and 20(S)OHC on the cell proliferation,apoptosis and osteogenic differentiation of Bone Marrow Stroma Stem Cells(BMSCs)in vitro.And to investigate the osteogenic effect and characteristics of SVA and 20(S)OHC composite bone graft(bio-oss)in repairing rabbit skull defect.Moreover,the regulatory mechanism of SVA and 20(S)OHC on BMCSs cell activity and osteogenic differentiation was examined.MethodsEffects of a combination of SVA and 20(S)OHC on proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells.BMSCs of rats were cultured in four SVA cultures at different concentrations for 7 days,and the mRNA levels of related gene(ALP,OCN,BMP)were detected by MTT PCR.The effect of 20(S)OHC and SVA on the proliferation and apoptosis of BMSCs in each group was evaluated by MTT and membrane linked protein V apoptosis assay.Alizarin red staining was performed after 3,7 and 14 days of cell culture,and the mRNA levels of related genes(ALP,OCN,BMP)were detected by PCR.Animal experiments on the repair of skull defects by composite bone graft(bio-oss)of SVA and 20(S)OHC.Fourteen healthy male rabbits(New Zealand white rabbits),aged 6 months and weighing 2.5-3kg,were selected and divided into four groups according to the experimental scheme.The animals in each group were sacrificed 4 or 6 weeks after the operation,and the skull specimens of rabbits were taken out for gross morphology and imaging study of the characteristics of new bone formation.The 4-weeks specimens were decalcified,immuno histochemical staining with HE staining(bmp-2)and Masson staining were used to detect the defect repair.Study on the regulation mechanism of SVA and 20(S)OHC on BMSCs osteogenic efficacy via Raf/MEK/ERK pathway.Western blot was used to detect the expression levels of proteins associated with the Ras-MAPK signaling pathway.U0126 inhibitor was added or not added to the cell culture medium of each group,and the same amount of DMSO was used as the control group to set 8 groups.The culture was terminated after 3 days and 7 days.The effects of each group on BMSCs cell proliferation and apoptosis were assessed,and the mRNA levels of related genes(ALP,OCN,BMP-2,BMP-9)were detected.Results:1.In a dose-dependent manner,high concentrations of SVA(0.25 and 1.0μM)can enhance the expression of osteogenic genes but attenuate cell activity.After 5 μM 20(S)OHC was added,the inhibitory effect of SVA on BMSCs could be neutralized.2.Histological examination confirmed the synergistic effect of SVA and 20(S)OHC on bone regeneration in rabbit skull defect model.3.The addition of SVA and 20(S)OHC at the same time significantly enhanced the ALP activity,calcium deposition and osteogenic genes(ALP、OCN 和 BMP-2)expression of bone marrow mesenchymal stem cells,and the osteogenic effect was inhibited by the inhibitor U0126,indicating that the Raf/MEK/ERK signaling pathway played an important role.Conclusions:This newly developed SVA/20(S)OHC formulation may be a significant and safe alternative for promoting osteogenic differentiation and enhancing bone regeneration.This study showed that the combined application of SVA/20(S)OHC activated the Raf/MEK/ERK signaling pathway to synergistically enhance the proliferation and osteogenic differentiation of osteoblasts.This newly developed SVA/20(S)OHC formulation can be used as a bone-inducing drug to promote bone healing and has a clinical application prospect of bone regeneration.