Simvastatin Induces Middle/Late Stage Osteogenic Differentiation Of BMSCs Via The P38MAPK Pathway

Objectives Simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A(HMGCo A) reductase, has been known to reduce cholesterol biosynthesis. Recent experimental studies have suggested that simvastatin may also have Osteoinductive effects. In this study, we investigated the stimulatory effect of simvastatin on the osteogenic differentiation of Bone marrow mesenchymal stem cells(BMSCs) in middle/late stages. Under conditions of Osteoinductive environment, to explore the best time of p-p38 MAPK during Osteogenic Differentiation of BMSCs applied simvastatin.Methods Twenty SPF 4-week-old female SD rats, all the rats were killed by cervical dislocation and remove both bilateral femur and tibia under sterile conditions. Whole bone marrow culture method for primary and passaged cultures and took the second generation of cells were randomly divided into five groups according to the experimental needs. Control group(Control medium group, CM): complete culture medium; simvastatin group(Simvastatin medium group): complete medium containing 10-7mol/L simvastatin; osteogenic induction medium group(Osteoinduce medium group, OM): containing 10mmol/L β-glycerophosphate and 50μg/ml ascorbic acid osteogenic culture medium; simvastatin and osteogenic medium group(Simvastatin and Osteoinduce medium group, OM + SIM): a final concentration of 10-7 mol/L simvastatin and osteogenic medium. Blocker group(SB203580 + Simvastatin + Osteoinduce medium group group, SB + OM + SIM): first with p38 MAPK pathway inhibitor SB203580 intervention after 30 min, adding induction medium and a concentration of 10-7 mol/L simvastatin- induced. Select the CM group and the SIM group MTT assay section 2d, 3d, 4d, 5d and 6d cell activity; ALP ELISA of OM and SIM was measured after the administration 7d and 14 d. Protein expressions of OCN and GAPDH were detected by Western Blot after the administration 1h, 12 h and 24 h on the 21 d and 28 d. In the administration of 1h, 12 h and 24 h on 21 d and 28 d, OCN and GAPDH m RNA expression using RT-PCR assay. Protein expressions of pp38 MAPK and p38 MAPK were detected by Western Blot after the administration 12 h on 21 d and 28 d.Results 1 MTT assay: Compared with the control group, 10-7 mol/L simvastatin had no effect on cell viability. 2 ELISA: 7d ALP expression of OM group and OM+SIM groupwere higher than the 14d(P<0.05). 7d ALP expression of OM+SIM group were highest.(P<0.05). 3 Real-time PCR: In the OM and OM+SIM groups, expression of OCN levels were the highest in 12 h on 21 d and 28d(P<0.05).In the OM+SIM group expression of OCN levels were higher than OM group(P<0.05). 4 Western Blot analysis: ① OCN: In the OM and OM+SIM groups, expression of OCN levels were the highest in 12 h on 21 d and 28d(P<0.05).In the OM+SIM group expression of OCN levels were higher than OM group(P<0.05). ②p-p38/p38: In the OM+SIM+SB group, expression of p-p38/p38 levels were lower than the OM group and OM+SIM group(P<0.05). In the OM+SIM group, expression of p-p38/p38 levels were than the OM group(P<0.05).Conclusions 1 Simvastatin of 10-7 mol/L had no effect on cell viability. Simvastatin have the ability of inducing osteogenesis. 2 ALP expression is highest on 7d and simvastatin induces a significant induction of ALP. 3 Best effect time of simvastatin was administered after 12 h. 4 The expression levels of OCN and p-p38 are increased by simvastatin.