大豆异黄酮染料木素的异戊烯基化以组织选择性的雌激素样作用方式抗骨质疏松症的机制研究

Osteoporosis, obtaining more attentions by people, is a worldwide health problem. Currently, the drug research and development for the prevention and treatment of osteoporosis focus on the potential natural resources from Chinese medicinal materials. The applicant’s previous studies clarified that 8-prenylgenistein (8-PG) is the effective constituent in Erythrina variegata L for anti-osteoporosis, moreover, the underlying mechanism may be attributed to the potential regulation of 8-PG on the estrogenic signaling pathway in osteoblasts. Thus, it is proposed that the extended studies as the following two respects need to be further performed by using animal and cell models: 1) in vivo selectivity of estrogenic effects of 8-PG in ovariectomized mice and immature female mice; 2) molecular mechanism of 8-PG in activating estrogen-like signaling in osteoblasts. The above-mentioned studies will demonstrate not only the in vivo osteoprotective efficacy of 8-PG, but also its pharmacological mechanism from the view of tissue selectivity of estrogenic effects. This project will provide experimental basis for clarifying the molecular mechanism of 8-PG in regulating estrogenic signaling pathway of osteoblasts, and for the research and development of selective estrogen receptor modulator, and also provide strong scientific evidences for exploring lead compounds with anti-osteoporosis effect, investigating structure-activity relationship, predicting function, designing drug and modifying structure.

骨质疏松症是一个世界范围的越来越引起人们重视的健康问题。目前防治骨质疏松药物研究，主要是以潜在的中药资源为研究重点。申请人前期研究明确了8-异戊烯基染料木素（8-PG）是刺桐抗骨质疏松症的有效物质，并且，调控成骨细胞的雌激素样信号通路可能是其潜在的作用机制，从而提出通过动物、细胞模型，继续以下两方面的延续性研究：1) 8-PG在去卵巢小鼠、未成熟雌性小鼠体内选择性的雌激素样作用；2) 8-PG激活成骨细胞雌激素样信号的分子机制。以上研究不仅阐明8-PG体内抗骨质疏松症的有效性，还从组织选择性的雌激素样作用角度阐明其药理机制。本研究为明确8-PG对成骨细胞雌激素信号通路的分子调控机制、为选择性雌激素受体调节剂的研发提供实验基础，为抗骨质疏松先导化合物、构效关系研究、功能预测、药物设计与结构修饰提供坚实科学依据。

去卵巢（OVX）大鼠经服8-PG后，松质骨的骨密度明显提高，皮质骨的生物力学得到有效改善。8-PG显著修复OVX 大鼠的骨小梁网织结构，提高松质骨的面积百分比。在未成熟雌性小鼠短期给药实验中，发现与正常对照组相比（子宫系数，1.12 ± 0.13 mg/g; 阴道系数，2.32 ± 0.18 mg/g），染料木素明显提高小鼠子宫系数（1.98 ± 0.21 mg/g）、阴道系数（3.83 ± 0.11 mg/g），同时，染料木素诱导阴道上皮细胞角质化、子宫、阴道组织肥大伴随组织内膜细胞增生。8PG对未成熟雌性小鼠性周期、子宫、阴道的组织形态没有产生明显影响。分子检测分析发现，染料木素能够上调未成熟雌性小鼠子宫、阴道ER-α、PCNA的蛋白表达水平，但是8PG没有展示类似染料木素这种调控作用。染料木素与8PG在所有受试浓度范围内均能提高MG63细胞的ERE依赖型荧光素酶的活性，在各对应的检测浓度中，染料木素和8PG对MG63细胞ERE活性的提升强度相当，两者之间的作用强度无统计学差异。8PG在10-12~10-10M浓度范围内能显著提高MG63细胞中ALP的活性，但染料木素在10-12~10-6M浓度范围内对MG63细胞中ALP的活性无明显影响。本课题研究顺利完成了对所提出科学问题的初步地实验探索，有望为防治骨质疏松症提供一类具有特色化学结构（具有异戊烯基侧链修饰）的黄酮类先导化合物；此外，对已报道具有潜在抗骨质疏松活性的黄酮类化合物，提示对其进行化学结构改造，有可能增强原母体化合物的药理活性。关于8-异戊烯基染料木素体内的生物利用度、吸收、分布等成药性方面也是后续需要重点开展的研究内容。