Icariin inducting MC3T3-E1 cell proliferation and differentiation with the activation of BMP-2 intracellular signal pathway

Abstract

Objective To explore the regulation function of Icariine on the expression of bone morphogenetic protein signaling pathway Smad1,Smad5,Smad4 and to explore the mechanism of promoting MC3T3-E1 cell proliferation and differentiation.Methods MC3T3-E1 cells were treated by 0,10-9,10-8 and 10-7 mol/L Icariine respectively.After stimulated by Icariine 1 d,2 d and 3 d,MTT method and population diploid time were used to observe the cell proliferation,and the cell alkaline phosphatase (ALP) level was assayed.At 21 days later,the alizarin red staining was proceeded.At 1,2 and 3 days later,the RT-PCR was used to detect the mRNA expression level about Smad1,Smad5 and Smad4,and the Western blot was to detect the Smad1,5 and Smad4 protein.At 2 days later,the RT-PCR was used to detect the mRNA expression level about Runx2,BMP-2 and osteoprotegerin (OPG),and the Western blot was used to analyze osteocalcin (OCN) protein level.Results After simulated by Icariine,the proliferation (MTT test),the ALP activity and mineralization of osteoblasts were increased,the cell population diploid was reduced (P＜0.05).At 1,2 and 3days later,the results of RT-PCR showed that Icariine continued increasing the mRNA level of Smad1,5 and Smad4 in 10-8 mol/L.At 2 days later,Smad1,Smad5 and Smad4 mRNA expression were obviously reduced in 0 mol/L group,and At 3 days later,Smad1,Smad5 and Smad4 mRNA expression were obviously reduced in 10-9 and 10-7 mol/L groups.At 2 days later,BMP-2,Runx2 and OPG mRNA were obviously increased in 10-8 mol/L group.The results of Western blot showed that OCN,Smad1,Smad5 and Smad4 protein were obviously up-regulated in 10-8 mol/L group.Conclusion Icariine occurs the expressions of BMP-2,Runx2,Smad1,Smad5 and Smad4 by stimulating the bone morphology of MC3T3-E1 cells directly,and promotes the osteogenic differentiation in the manner of expression level synchronous rising. Key words: Osteoblasts; Icariin; Smad proteins; Bone morphogenetic proteins