生物素标记法钓取淫羊藿素受体及其下游促进骨形成信号转导途径的研究

Icaritin,the most effective metabolites of icariin isolated from traditional Chinese medicinal plants,has potential clinical application for its strong osteogenic activity. However,the mechanism of action, especially the receiving structure on cell surface and the receptor for icaritin, remain uncler despite the possible role as a phytoestrogen and reported effects on BMP, MAPK and WNT pathways.Basing on the research progress of non-genomic estrogen receptor pathways and the results obtained in our recent experiments, we here hypothesized that icaritin may bind with estrogen receptors or non-estrogen receptors located on plasma membrane and initiate osteogenic differentiation in the dependence of primary cilia,the recently identified non-motile sensory organelles on the surface of most mammalian cells.To verify our hypothesis and clarify the full-length signal transduction pathways, we are going to chemically label icaritin with biotin, which has high affinity with streptavidin, and then treat the bone cells with biotinylated icaritin (Bio-I), the conjugate of icartin and biotin. Since the greatly increased hydrophilicity, Bio-I would be impermeable in membrane and its distribution on bone cells would be demonstrated by immunofluorescence using fluorescein conjugated streptavidin. The icaritin-binding proteins would be fished out from the plasma membrane fractions in application of the high affinity between biotin and streptavidin conjugated with magnetic beads. The fished proteins would be identified by mass spectrum and the proteins functioning as receptors may be the receptors of icaritin. Their distribution and the combined interacting proteins would be determined by immunofluorescence under laser confocal microscope. All the above works, together with the related publications and our previous experimental results, would help to speculate the downstream signal transduction cascades. Finally, the speculated pathways would be verified by blocking with antagonists or RNA interference step by step.We have established the synthesis method for biotinylated icaritin and successfully showed the distribution of biotinylated icariin with immunofluorescence.As a result, icaritin was found to be located in the primary cilium, and its bone-formation promoting effect dispearred when the primary cilia of rat calvarial osteoblasts was removed by RNA interterference.This excting results,combined with our finding on the associations between increased intracellular cAMP, cGMP, NO and Ca2+ concentration and icaritin-induced osteogenic differentiation,make us believe that we would discover more than one signal transduction pathway which begin from the arrival of icaritin at bone cell surface and ending at the initiation of osteogenic differentiation. All we do would be extremely helpful to the development of new anti-osteoporosis drugs from the natural products including flavonoids.

淫羊藿素是一种具有强烈促进骨形成活性的中草药单体，应用前景广阔，但除作为植物雌激素和对BMP、MAPK、WNT等信号途径的影响外，其作用机制尚不明确，尤其缺乏作用靶点的研究报道。我们基于前期研究结果推测，淫羊藿素通过与位于细胞膜的雌激素受体或非雌激素受体结合而启动下游信号级联反应，在此过程中初级纤毛发挥了不可或缺的作用。为证明上述假说并阐明信号转导途径，本项目拟合成生物素化淫羊藿素并利用其不能自由通过细胞膜但仍具有促骨活性的特点开展以下研究：⒈进行免疫荧光示踪；⒉将生物素化淫羊藿素磁珠应用于免疫共沉淀，从质膜蛋白中钓出淫羊藿素受体及其复合蛋白；⒊进行质谱鉴定并根据鉴定结果和文献报道推测出促骨信号转导途径；⒋用逐步阻断法验证并确立至少一条完整的信号转导途径；⒌明确初级纤毛在该途径中的作用及机制。本项目已建立生物素化淫羊藿素的合成方法，研究结果将促进以天然产物为先导化合物的抗骨质疏松新药研发。