淫羊藿素通过雌激素受体和骨形态发生蛋白信号诱导MC3T3-E1 subclone 14细胞分化

Icaritin induces differentiation of MC3T3-E1 subclone 14 cells via estrogen receptor and BMP/Smad signaling pathway in vitro

目的:观察淫羊藿素(ICT)对MC3T3-E1 subclone 14前体成骨细胞株增殖、分化的影响,以及雌激素受体(ER)和骨形态发生蛋白(BMP)信号在分化中的作用。方法:WST-8、BrdU法检测ICT对MC3T3-E1subclone 14细胞活力和增殖的影响;ICI182780阻断ER受体信号后,检测ICT和noggin对MC3T3-E1 subclone 14细胞碱性磷酸酶(ALP)活性、I型胶原(Col I)和骨钙素(BGP)的影响;实时荧光PCR检测ICT对BMP-2、4、7 mR-NA表达的影响;Western blotting检测ER受体信号阻断后,ICT对Smad1/5/8蛋白磷酸化的影响。结果:ICT(0.1μmol/L、1μmol/L)可以提高MC3T3-E1 subclone 14细胞ALP、Col I、BGP和矿化结节数量(P<0.01或P<0.05),表明ICT有促分化的作用,但对细胞活力和增殖指数无明显影响;阻断ER受体信号后,ICT促分化作用明显下降(P<0.01);ICT可以提高BMP-2、4 mRNA的表达(P<0.01),但对BMP-7 mRNA无作用(P>0.01);阻断ER信号后,ICT促Smad1/5/8磷酸化明显减弱(P<0.01)。进一步阻断BMP/Smad信号可以抑制ICT促分化的作用(P<0.01)。结论:ICT可以通过ER受体激活BMP/Smad信号通路,进而促进MC3T3-E1 subclone 14细胞的分化。

AIM： To observe the effects of icaritin（ICT） on the proliferation and differentiation of MC3T3-E1 subclone 14 cells（a pre-osteoblast cell line） and to observe the role of estrogen receptor（ER） and bone morphogenetic protein（BMP）/Smads signaling pathways in the differentiation of the cells.METHODS： The methods of WST-8 and BrdU were used to observe the viability and proliferation of MC3T3-E1 subclone 14 cells after treatment with different concentrations of ICT.The effects of ICT and noggin on the levels of alkaline phosphatase（ALP）,type I collagen（Col I） and bone Gla protein（BGP） in MC3T3-E1 subclone 14 cells were observed after ER was blocked by ICI182780.The relative mRNA levels of BMPs（2,4,7） were detected by real-time PCR.The protein phosphorylation of Smad1/5/8 was determined by Western blotting after ER signaling pathway was blocked by ICI182780.RESULTS： ICT at concentrations of 0.1 μmol/L and 1 μmol/L increased the levels of ALP,Col I and BGP,and the numbers of mineralized nodules in MC3T3-E1 subclone 14 cells,indicating that ICT-promoted the differentiation,but did not affect the cell viability and proliferation.After the ER receptor signaling was blocked,ICT-promoted differentiation was significantly decreased.ICT improved the mRNA expression of BMP-2,4,but did not affect the mRNA expression of BMP-7.After the ER receptor signaling was blocked,ICT-promoted phosphorylation of Smad1/5/8 was significantly decreased.Blockage of BMP/Smad signaling inhibited the effect of ICT on the differentiation.CONCLUSION： Icaritin induces the differentiation of MC3T3-E1 subclone 14 cells by activating BMP/Smad signaling pathway through ER.