Effects Of Icaritin On Estrogen Receptor And MAPK Signaling Pathway In MC3T3-E1 Subclone14

Objective:To observe the effect of icaritin on prolifereation and differentiation of MC3T3-E1 Subclone14. To define targets of icaritin on osteoblasts via studying the effect of icaritin on estrogen receptor、MAPK signaling pathway and Runx2, which can provide cellular and molecμlar evidences for icaritin treating osteoporosis and the other bone metabolic diseases.Methods:1. CCK-8 method and flow cytometry were used to observe cell activity and prolifereation; Alizarin dyeing was used to observe osteoblast mineralization of MC3T3-E1 Subclone14 cells after treating with differenr concentration of Icaritin;Sirius red stain method was used to observe the expression of cell Collagen.2. ALP kit was used to observe ALP activity of MC3T3-E1 Subclone14 cells after treating with different concentration of Icaritin. ELISA kit was used to observe Type I Collagen and Osteopontin activity of MC3T3-E1 Subclone14 cells after treating with different concentration of Icaritin. Real-time PCR was used to observe the m RNA relative levels of ALP, Col I, OPN and Runx2 after treating with different concentration of Icaritin.3. Western-blot method was used to observe ERK1/2、P38MAPK、JNK protein phosphorylation after treating with different concentration of Icaritin.4. Real-time PCR was used to observe the m RNA relative levels of ALP, Col I, OPN after respectively ER signaling pathway, P38 MAPK signaling pathway or ERK1/2 signaling pathway was blocked by ICI182780, SB203580, PD98059 after treating with Icaritin.5. Western-blot method was used to observe ERK1/2 、 P38 MAPK protein phosphorylation after ER signaling pathway was blocked by ICI182780 after treating with Icaritin.6. Immunofluorescence method was used to observe ERK1/2、P38MAPK protein phosphorylation after ER signaling pathway was blocked by ICI182780 after treating with Icaritin.Results:1. CCK-8 kit was used to observe cell activity, ICT(0.1μM、1μM、10μM) can improve cell activity significantly(P<0.05), and 10μM group is the best(P<0.01). Flow cytometry was used to observe Proliferation index, it is no different with control group in ICT group(P>0.05)after 48 h and 72 h.2. ICT(0.1μM、1μM、10μM)can improve the expression of ALP m RNA and protein significantly, and 10μM group is the best(P<0.01); ICT(0.1μM、1μM、10μM) can improve the expression of Col I m RNA and protein significantly, and 10μM group is the best(P<0.01); ICT(0.1μM、1μM、10μM) can improve the expression of OPN m RNA and protein significantly, and 10μM group is the best(P<0.01); ICT(1μM、10μM) can improve the expression of Runx2 m RNA significantly, and 10μM group is the best(P<0.05).3. ICT(0.1μM、1μM、10μM)can improve the P38 MAPK protein phosphorylation significantly, and 10μM group is the best(P<0.01); ICT(1μM、10μM)can improve the ERK1/2 protein phosphorylation significantly, and 10μM group is the best( P<0.01);There is no difference with control group of JNK protein phosphorylation after treating with ICT(0.1μM、1μM、10μM).4. The expression of ALP、Col I and OPN m RNA of MC3T3-E1 Subclone14 cell of experimental group(Icaritin+blocker) were less than Icaritin group after ER receptor、P38MAPK or ERK1/2 signaling pathway was blocked by ICI182780、SB203580 or PD98059(P< 0.01 or 0.05).5. P38 MAPK and ERK1/2 protein phosphorylation of icaritin-induced decreased significantly after ER signaling pathways was blocked by ICI182780.Conclusions:1. Icaritin(0.1μM、1μM、10μM) can not improve cell proliferation index of MC3T3-E1 Subclone14,but Icaritin can improve cell activity.2. Icaritin(0.1μM、1μM、10μM) can promote differentiation、 maturity and mineralization of MC3T3-E1 Subclone14. The effect of 10μM group is the best.3. Icaritin(0.1μM、1μM、10μM)can promote phosphorylation of P38 MAPKand ERK1/2,it can not promote phosphorylation of JNK. The effect of 10μM group is the best.4. MC3T3-E1 Subclone14 differentiation induced by Icaritin(0.1μM、1μM、10μM)is related to ER, P38 MAPK and ERK1/2 signaling pathways.5. ER signaling pathways may be take place upstream of P38 MAPK and ERK1/2 signaling pathways.