The Experiment Study Of The Effects Of Icariin On Smad1,4,5in MC3T3-E1Cell In Vitro

Objective:To explore the effect of icariin on the expression of Smadl,4,5mRNA and protein Smad1,4,5in preosteoblast MC3T3-E1cells in vitro.Methods:MC3T3-E1cells were divided into four groups according to stimulus concentrations of icariin, those were0、10-9、10-8、10-7M groups. Each dose group cells were planted in6-well plate and the plantation number of MC3T3-E1cells was1X105in each well. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to find out the mRNA expression level about Smad1、Smad5、 Smad4and actin. Western blot technique was adopted to detect the protein expression level of them. MC3T3-E1cells were cultured in6-well plate which was put in with cover glasses preliminarily and the initial cellular plantation number was1X105in each well.After72hrs cultured, expression of Smad1/5or pSmad1protein was localized and confirmed by immunohistochemistry staining or immunofluorescence. The relevant data for RT-PCR and Western blot results were dealt with SPSS13.0software.Results:RT-PCR exhibited that Smad1,4,5mRNA expressed no statistical significance at24hrs in all the groups(Smad1:F=0.064,P=0.961; Smad5:F=1.216,P=0.351;Smad4:F=0.051,P=0.022).AndSmad1,5expressed scontinuously as time went ahead in10-9M groups and they also expressed in10-8M groups or10-7M groups when cells were cultured till48hrs and72hrs. But the mRNA expression of Smadl were found a little in0M control groups at48hrs but Smad5mRNA was not detected expression at the time. At72hrs, both of them were not observed expression in0M groups. The mean net optical density ratios between Smad1or Smad5mRNA and actin mRNA were shown statistical significance (48hrs:Smad1,F=332.435,P=0.000; Smad5,F=373.487,P=0.000and72hrs: Smad1,F=132.356,P=0.000; Smad5,F=102.384,P=0.000) between the effective stimulus dose groups of icariin and Ong/ml groups at48hrs or72hrs. At24hrs.Western blot demonstrated that the mean net optical density ratios between Smad1protein and actin protein were shown statistical significance (72hrs: F=16.985, P=0.001) between any one of the three effective stimulus dose groups and0M groups at72hrs. The mean net optical density ratios of both Smad5protein and actin protein were shown statistical significance(24hrs:F=3.762, P=0.060;48hrs: F=14.579, P=0.001;72hrs:F=27.762, P=0.000)at24、48、72hrs. Phosphorylated protein of Smad1,5,4was detected to have statistical significance at48、72hrs (48hrs: F=6.407, P=0.016;72hrs:F=4.631, P=0.037).Contrasted to difference emergence of Smad1,5,4mRNA after48hrs, disparity of protein Smad1,4or Smad5between three effective ICA stimulus dose groups and0M groups emerged respectively after24hrs or at72hrs. Non-equal pace between mRNA and prtein level for Smad1,4,5may show existence of some kind post-trancriptional regulating mechanism for Smad1,4,5signal. Variance of Smad1,5,4protein emerged after48hrs, earlier to Smad1,4, later than Smad5, and the phenomenon could show protein Smad5probabily has the expressed prior status in secondary Smads signal system.Immunohistochemistry staining displayed that10-9、10-8and10-7Mgroups could promote expression of Smad1,4,54proteins in cytoplasm than OMgroup.Conclusion:Icariin is able to up-regulate the expression level of Smadl1、45mRNA and protein Smad1、4、5through which to be probably stimulate MC3T3-E1cell differentiation.