Effects Of Icaritin On Differentiation And Differentiation-related Signaling Pathways Of MC3T3-E1Subclone 14 Cells In Vitro

Objective:Epimedium grandiflorum Morr,a traditional Chinese herb,was effective to treatment many diseases of bone.Icariin,a principal flavonoid glycoside in Epimedium grandiflorum Morr,is metabolized to Icaritin in vivo.Icaritin may have beneficial effects on bone mass.but its role and mechanism in osteoblasts is unclear.This study is to observe the effects of icaritin on the proliferation and differentiation of MC3T3-E1Subclonel4,a pre-osteoblasts cell line,and observe the effects on ER, p38MAPK and BMPs/Smads signaling pathways which were closely related to differentiation of osteoblasts.To define targets of icaritin on osteoblasts,which can provide cellular and molecular evidences for Epimedium grandiflorum Morr treating osteoporosis and other bone metabolic diseases.Methods:1.MC3T3-E1Subclonel4 was cultured and its biological activity was observed.2.MTT method was used to observe cytotoxicity of Icaritin,and to determine the experimental concentration of Icaritin in the Medium.3.WST-8 method and BrdU method were used to observe cell activity and proliferation of MC3T3-E1Subclonel4 cells after treating with different concentration of Icaritin.4.pNPP method was used to observe ALP activity of MC3T3-E1Subclonel4 cells after treating with different concentration of Icaritin.ELISA kit was used to observe TypeⅠcollagen and osteocalcin activity of MC3T3-E1Subclonel4 cells after treating with different concentration of Icaritin.Alizarin dyeing was used to observe mineralization of MC3T3-E1Subclonel4 cells after treating with different concentration of Icaritin.5.After ER Signaling pathways was blocked by ICI182780,p38MAPK Signaling pathways was blocked by SB203580 respectively,ALP,TypeⅠcollagen and osteocalcin activity of MC3T3-E 1Subclonel4 cell were observed after treating with Icaritin.6.Real-time PCR was used to observe the mRNA relative levels of BMP-2,4,7 after treating with Icaritin.After BMPs/Smads Signaling pathways was blocked by noggin,ALP,Type I collagen and osteocalcin activity of MC3T3-E1Subclonel4 cells were observed after treating with Icaritin.7.Western-blot was used to observe p38 and Smadl/5/8 protein phosphorylation after ER Signaling pathways was blocked by ICI182780 after treating with Icaritin.Results:1.MC3T3-E1Subclonel4 cells was induced successfully into osteoblasts in medium of inducing conditions.2.IC50 of Icaritin's concentration in medium in the cytotoxicity test was 10^0.154 mol/L.Less than the concentration of 10^-5.93mol/L,Icaritin can't inhibit the growth of MC3T3-E 1Subclone 14 cells.3.Cell activity and Proliferation index of MC3T3-E1Subclonel4 cells of Icaritin (0.01μM,0.1μM,1μM) groups have no different with control group after 48 and 72hours(P＞0.05).4.ALP,TypeⅠcollagen and osteocalcin activity oflcaritin(0.01μM,0.1μM,1μM) groups were higher than control group after 48 or 72hours(P＜0.01or 0.05). Mineralized nodus weren higher control group after 10 or 14days(P＜0.01).5.ALP,TypeⅠcollagen and osteocalcin activity of MC3T3-E1Subclone 14 cell of experimental group(Icaritin+blocker) were less than Icaritin group after ER Signaling pathways was blocked by ICI182780,p38MAPK Signaling pathways was blocked by SB203580 respectively(P＜0.01).6.The mRNA levels of BMP-2 and BMP- 4 of Icaritin group was higher than control group(P＜0.01 or 0.05),but the mRNA levels of BMP-7 of all groups have no significant difference(P＞0.05).7.ALP,TypeⅠcollagen and osteocalcin activity of MC3T3-E1Subclonel4 cell of experimental group(Icaritin+blocker) were less than Icaritin group after BMPs/Smads Signaling pathways was blocked by noggin(P＜0.01 or 0.05).8.p38 and Smadl/5/8 protein phosphorylation of icaritin -induced decreased significantly after ER Signaling pathways was blocked by ICI182780.Conclusions:1.Icaritin(0.01μM,0.1μM,1μM) can't increase activity and proliferation index of MC3T3-E 1Subclone 14 cells.2.Icaritin(0.01μM,0.1μM,1μM) can increase differentiation and mineralization of MC3T3-E1Subclone 14 cells.3.Icaritin's role in the promotion of MC3T3-E1Subclonel4 cells differentiation is closely related to ER,p38MAPK and BMPs/Smads Signaling pathways.4.In the differentiation process,ER Signaling pathways may be take place upstream of p38MAPK and BMPs/Smads Signaling pathways.5.Icaritin can increase differentiation of MC3T3-E 1Subclone 14 cells may be for its estrogen-like activity.