| ObjectiveIn this experiment,we explore the effect of icariin(ICA)on osterogenesis by regulating Eph B4-Ephrin B2 signaling pathway in vitro.P.gingivalis was used to stimulate MC3T3-E1 cells and Ephrin B2-Fc protein was added to simulate the co-culture environment of osteoclast and osteoblast.The effect of ICA on the osteogenisis of MC3T3-E1 cells through the regulation of Eph B4-Ephrin B2 signaling pathway can provide some theoretical basis for the treatment of periodontitis.MethodsMC3T3-E1 cells were cultured in vitro,and treated with Ephrin B2-Fc protein with different concentrations(2,4,6,8 μg/m L).CCK-8 was used to detect the proliferation activity of MC3T3-E1 cells,the expression of Eph B4,ALP,OCN and OPN were detected by Western blot,and the optimum concentration of Ephrin B2-Fc was screened.The effects of ICA with different concentrations(0.01,0.1,1,10,100 μmol/L)on the proliferation of MC3T3-E1 cells were detected by CCK-8.MC3T3-E1 cells were stimulated by P.gingivalis(MOI=100),and treated with ICA at different concentrations(0.1,1,10 μmol/L).The formation of mineralized nodules was observed by Alizarin red staining;the ALP activity of the cells was evaluated by ALP activity assay;the expressions of Eph B4,ALP,OCN and OPN were detected by Western blot.Ephrin B2-Fc protein was added to simulate the co-culture system between osteoclasts and osteoblasts.Alizarin red staining,ALP activity detection and Western blot detection were performed to observe the effects of ICA on osteogenic differentiation.In addition,to further investigate the effect of ICA on the osteogenic differentiation of MC3T3-E1 cells through the Eph B4-Ephrin B2 signaling pathway,the Eph B4 gene was silenced and Western blot was used to detect the expression of EphB4,ALP,OCN,and OPN proteins.ResultsMC3T3-E1 cells were treated with Ephrin B2-Fc with different concentrations(2,4,6,8 μg/m L).CCK-8 results showed that 4 μg/m L Ephrin B2-Fc had the most obvious effect on proliferation of MC3T3-E1 cells(P<0.01).Compared with control group,4 μg/m L Ephrin B2-Fc significantly promotes the expression of Eph B4,ALP,OCN and OPN in MC3T3-E1 cells(P<0.01).Therefore,4 μg/m L Ephrin B2-Fc was selected for the follow-up experiment.After ICA treatment,CCK-8 results showed that 0.1,1,10 μmol/L ICA could promote the proliferation of MC3T3-E1 cells at 24 h,48 h and 72 h compared with control group,and 10 μmol/L ICA had the most obvious effect on the proliferation of MC3T3-E1 cells(P<0.001).MC3T3-E1 cells were stimulated by P.gingivalis(MOI=100).The results of Alizarin red staining showed that compared with P.gingivalis group,the mineralized nodules of MC3T3-E1 cells in 10 μmol/L ICA group increased significantly.Compared with P.gingivalis group,the activity of ALP in 10 μmol/L ICA group increased significantly(P<0.01),and the expression of Eph B4,ALP,OCN,OPN protein increased significantly(P<0.01).After the addition of Ephrin B2-Fc protein,Alizarin red staining showed that compared with control group and Ephrin B2-Fc group,the mineralized nodules of MC3T3-E1 cells in ICA group increased significantly.ALP activity and expression of Eph B4,ALP,OCN,OPN in MC3T3-E1 cells in ICA group were significantly higher than those in other groups(P<0.01).After silencing the Eph B4 gene,Western blot results showed that compared with the ICA group,the expression levels of Eph B4,ALP,OCN and OPN in the si-Eph B4+ICA group were significantly decreased(P<0.01).Compared with the si-Eph B4 group,the expression levels of Eph B4,ALP,OCN,and OPN proteins in the si-Eph B4+ICA group were increased(P<0.05).ConclusionThe results showed that 10 μmol/L ICA could promote the proliferation and differentiation of MC3T3-E1 cells.ICA up-regulated the expression of Eph B4,ALP,OCN,OPN,promoted the positive signaling pathway of Eph B4-Ephrin B2,accelerated the osteogenic differentiation of MC3T3-E1 cells,and decreased the inhibitory effect of P.gingivalis on the proliferation and differentiation of MC3T3-E1 cells. |
