The Study Of Hyperoside Stimulates Osteoblast Proliferation And Differentiation Through The BMP And Wnt/β-catenin Signaling Pathways In MC3T3-E1 Cells

Objective:For the purpose to study the influence of hyperoside on osteoblast proliferation and differentiation in MC3T3-E1 cells and the relevant molecular mechanism that plays a role.Methods:Preparing different concentrations of hyperoside to act on MC3T3-E1 cells in five groups, which consist of the experimental group where the concentration of the hyperoside is separately 10μmol/L, 20μmol/L,40μmol/L and 80μmol/L and the blank control group without hyperoside. Measure the proliferation rate of MC3T3-E1 cells through the MTT method and detect the alkaline phosphatase(ALP) activities of MC3T3-E1 cells through the p-NPP method. Meanwhile detect the mineralized nodule formation of MC3T3- E1 cells through the Alizarin red staining method and use the RT- PCR to measure the expression of the following genes including ALP,Col-I, OPN, BMP2, Runx2, Osx,β-catenin and LRP5 that are related to the osteoblast differentiation in MC3T3-E1 cells. Also measure the expression of the following protein including BMP2 and β-catenin related to the osteoblast differentiation in MC3T3-E1 cells through the Western Blot method,and finally make a statistical analysis on the acceleration effect of the hyperoside on osteoblast proliferation and differentiation through the SPSS software.Result: The result through the MTT method shows that hyperoside with the concentration separately up to 10μmol/L, 20μmol/L, 40μmol/L and 80μmol/L will accelerate the proliferation of MC3T3-E1 cells after they have acted on the cells for24h-72 h. Also it will promote significantly the ALP activities of MC3T3-E1 cells, if the hyperoside has acted on the MC3T3-E1 cells for 3 days. Meanwhile, it will promote evidently the expression of the MC3T3- E1 during the mineralized nodule formation. This result proves the concentration-dependence, which is extremely strong when the concentration of the hyperoside is 40μmol/L. Also the result through the RT-PCR method reveals that hyperoside is able to promote the expression of the following genes including ALP, Col-I, OPN, BMP2, Runx2, Osx, β-catenin and LRP5 in MC3T3-E1 cells. In addition, the result through the Western Blot method indicatesthat hyperoside will promote the expression of BMP2 protein and β-catenin protein.Especially it indicates that the hyperoside with the concentration up to 40μmol/L has the most significant effect.Conclusion : Hyperoside will accelerate osteoblast proliferation and differentiation via both of the BMP and Wnt/β-catenin signaling pathways in MC3T3-E1 cells.