基于ERβ途径雌马酚对成骨细胞增殖的调控作用及其机理研究

It is new way to prevent postmenopausal osteoporosis with supplement of phytoestrogen by dietary pathway. Based on our early study that the equol ( Eq ) can estrogenic effect on osteoblasts ( OB ), which is one of the soybean isoflavone metabolite, we make the hypothesis that Eq regulate osteoblast proliferation by estrogen receptor pathways. In the research we would plan to establish castrated rats animal model and ER silent osteoblastic model. We would use some molecular biology technology method such as luciferase reporter gene assay and Chromatin immunoprecipitaion(CHIP). Firstly，we study Eq bind to and active ER after it come into cell. The activation ER would directly bind to target genes ERE by two dimeric forms. They would produce synergistic effect to recruitment cofactor by AF-1 and AF-2, which would regulate target gene OPG / RANKL expression effects on osteoblast proliferation. Secondly, part of Eq bind to cell membrane ER and active protein kinase cascade reaction. And then the JNK signaling pathway would be activation, it promotes cell cycle and cell proliferation. Perhaps it is a complement pathway about regulation of the proliferation of OB. Finally, we want to identify Eq has regulation effect on the bone metabolism by animal experiments. After we have finished this study we would provides the experiment basis to clearly know about the molecular mechanism of the prevention PMOP by Eq. There would be important guiding significance that how to supply soybean isoflavone by dietary pathway to prevent PMOP with different Eq metabolic phenotype.

通过膳食途径补充植物雌激素类化合物是预防绝经后骨质疏松症（PMOP）的新思路，本项目在前期研究发现大豆异黄酮代谢物雌马酚（Eq）对成骨细胞（OB）类雌激素作用的基础上提出Eq通过雌激素受体β途径调控OB增殖的假设。研究拟通过建立去势大鼠动物模型和ER?沉默成骨细胞模型，采用荧光素酶报告基因分析和CHIP等分子生物技术方法。首先研究Eq进入细胞与ER结合并活化，ER以二聚体形式直接与靶基因ERE结合，通过AF-1和AF-2 协同作用募集辅因子，进而调控靶基因OPG/RANKL表达影响OB增殖。其次部分Eq与细胞膜上ER?结合，激活蛋白激酶级联放大反应，活化JNK等信号通路促进细胞周期和细胞增殖，作为调控OB增殖补充途径。最后以动物实验验证Eq对OB增殖调节作用；本研究为进一步明确Eq预防PMOP分子机制提供实验依据，并对不同Eq代谢表型人群通过膳食途径补充大豆异黄酮防治PMOP有重要指导意义

绝经后骨质疏松症（postmenopausal osteoporosis，PMOP）是绝经后女性的常见疾病，由于传统雌激素替代疗法副作用明显，通过膳食途径补充植物雌激素化合物成为预防PMOP的新思路。本项目在前期研究发现大豆异黄酮代谢产物雌马酚（Eq）对大鼠成骨细胞类雌激素作用的基础上提出Eq通过雌激素受体beta（ERβ）途径调控成骨细胞增殖的假设。研究采用建立去卵巢后大鼠骨质疏松症模型，观察Eq对去卵巢大鼠骨代谢影响及其相关蛋白表达变化；同时以大鼠成骨细胞为研究对象，检测Eq对成骨细胞增殖影响及其相关蛋白表达变化，通过萤光素酶报告基因检测比较Eq对ERα和ERβ转录活性的影响，分析得出Eq对成骨细胞增殖作用的分子机制。研究发现，Eq能够改善去卵巢后大鼠的股骨形态结构，减少骨密度丢失，改善骨代谢，调节骨保护素OPG和破骨细胞因子RANKL平衡，进而维持骨转化的平衡。同时，Eq能够促进成骨细胞增殖和分化，减少成骨细胞凋亡，调节OPG/RNAK/RANKL的表达，并且这些作用均与ERβ密切相关，表明Eq是通过ERβ途径调节OPG/RANK/RANKL信号通路实现对成骨细胞的增殖调控，从而改善大鼠POMP的发生。本研究结果为进一步明确Eq预防POMP分子机制提供了实验依据，对POMP的预防和治疗有重要理论价值，并对不同Eq代谢表型人群通过膳食途径补充大豆异黄酮防治POMP有重要指导意义。