The Experimental Study Of β-ecdysterone PromotingProliferation And Differentiation Of MC3T3-E1 Cells Through The Wnt/β-catenin Signaling Pathway

Objective:To study the effect of β-ecdysterone on the proliferation and differentiation of MC3T3-E1 cells and its potential molecular mechanism,so as to provide theoretical basis for the application of β-ecdysterone in the treatment of osteoporosis.Methods:The osteogenic mineralization potential of MC3T3-E1 cells was identified by observing the general condition and morphological characteristics of MC3T3-E1 cells as well as alizarin red S staining.CCK-8 method was used to detect the effect of β-ecdysterone on the proliferation of MC3T3-E1 cells at different concentrations(0,0.01,0.1,1,10 umol/L).Alkaline phosphatase activity was determined by alkaline phosphatase detection kit.Real-Time qPCR was used to detect the changes of osteogenesis-related markers in gene expression level.Western Blot was used to detect the changes of osteogenesis-related markers and Wnt/β-catenin signaling pathway related factors in protein expression level.After pretreated with DKK1,MC3T3-E1 cells were stained with alizarin red S,and the protein expression levels of osteogenesis-related markers and Wnt/β-catenin signaling pathway related factors were detected by Western Blot.Results:The osteogenic mineralization potential of MC3T3-E1 cells was identified by alizarin red S staining and observing the general status and morphological characteristics of the cells.CCK-8 assay showed thatβ-ecdysterone at various concentrations could promote the proliferation of MC3T3-E1 cells(P<0.05).The determination of alkaline phosphatase also indicated that it could improve the activity of the enzyme(P<0.05).β-ecdysterone at different concentrations upregulated the relative mRNA expression of ALP,COL I,OCN and Runx2 in MC3T3-E1 cells(P<0.05),but there was no significant difference in mRNA expression of OCN between 0.1μmol/L β-ecdysterone group and 1μmol/L group(P>0.05).β-ecdysterone at different concentrations upregulated the relative protein expression levels of ALP,COL I,OCN,Wnt3 a and β-catenin in MC3T3-E1 cells(P<0.05),and there was no significant difference in the relative expression level of OCN protein between 0.1μmol/L,1 μmol/L and 10 μmol/L groups(P>0.05),while there was no significant difference in Wnt3a protein expression level between 0.01 μmol/L group and 10 μmol/L group(P>0.05).There was no significant difference in the relative expression of β-catenin protein between 0.1 μmol/L group and 10 μmol/L group(P>0.05).At the same point in time,the number of mineralized calcium nodules in MC3T3-E1 cells decreased in DKK1 pretreatment group,and the relative expression level of β-catenin protein in MC3T3-Elcells pretreated with DKK1 decreased(P<0.05),while the relative expression level of Wnt3a protein had no significant difference(P>0.05).At the same time,the relative expression levels of ALP,COL I and OCN proteins were all down-regulated in different degrees(P<0.05).Conclusion:1.MC3T3-E1 cells have the potential of osteogenic differentiation.2.β-ecdysterone promoted the proliferation and differentiation ofMC3T3-E1 cells.3.The canonical Wnt/β-catenin signaling pathway is involved in the process of β-ecdysterone promoting the proliferation and differentiation of MC3T3-E1 cells.4.β-ecdysterone can be used as a potential drug for the treatment of osteoporosis.