基于BMP2和Wnt信号通路的续断-杜仲药对协同促进成骨的作用及机制研究

Osteoporosis is a serious medical problem for its high morbidity and disability, and heavily affects the health and working ability of human being. Dipsacus asperoides and Eucommia ulmoides Oliv. as a drug pairs of Chinese medicine, has the effects of kidney-tonifying and skeleton-strengthening and was a effective Chinese medicine for treatment osteoporosis. Based on our previous studies, Dipsacus asperoides saponins and Eucommia ulmoides Oliv. lignans was the effective fractions of the drug pairs to prevent and treat osteoporosis. Asperosaponin VI (ASVI) could promote the osteogenic action through the BMP-2 signal pathway, however the Wnt/β-catenin signal pathway was the target of pinoresinol di-O-β-D-glucopyranoside (PDG). Based on previous research, this subject will study the synergistic effect of Eucommia ulmoides Oliv. and Dipsacus asperoides to promote the bone formation and explore the possible mechanisms behind the effect. ①The bone marrow derived mesenchymal stem cells and osteoblast was cultured to explore the synergistic effect in vitro. The ovariectomized rat model was employed to investigate the anti-osteoporosis effect of Eucommia ulmoides Oliv. and Dipsacus asperoides. Three-point bending test was employed to determine the effect of various treatments on bone strength. The bone microarchitecture was assessed by microCT and histomorphometry analysis. ② The bone marrow derived mesenchymal stem cells and osteoblast was cultured to explore the possible mechanism behind this effect. The mechanism behind the drug promoting bone formation were measured with real time PCR and western blot in BMP2 and Wnt/β-catenin signal pathways. Our project will be find the synergistic promoting bone formation effect by Dipsacus asperoides and Eucommia ulmoides Oliv. was through the BMP2 and Wnt/β-catenin signal pathways.

骨质疏松症（OP）发病率与致残率极高，严重危害人类健康。续断-杜仲相须药对具有补肾壮骨作用，治疗OP疗效显著；前期研究显示续断总皂苷和杜仲总木脂素是续断杜仲防治OP的有效组分，且发现续断皂苷VI和杜仲松脂醇二葡萄糖苷分别通过BMP2和Wnt信号通路激活成骨细胞发挥作用，两者有协同效果。据此，本课题拟深入研究续断和杜仲协同增效作用及对BMP2和Wnt信号通路调控机制：①考察其对BMSC及成骨细胞的成骨相关特性的协同调节作用；建立去势大鼠OP模型，考察其对骨密度、骨形态学、骨力学性能和骨形成的协同促进作用；②检测其对BMP2和Wnt信号通路及其下游关键基因及蛋白的调控作用；采用阻断剂干预BMP2和Wnt信号通路后，检测加药前后相关蛋白的变化，并再考察其对BMSC及成骨细胞作用。该研究预期可明确续断和杜仲通过BMP2和Wnt信号通路发挥协同增效促进骨形成作用，揭示续断和杜仲相须壮骨的机制。

建立去卵巢SD大鼠骨质疏松模型，续断、杜仲和续断-杜仲配伍均可显著提高去卵巢大鼠骨密度和骨量，保护骨小梁微观结构，促进骨形成，改善骨生物力学性能，续断-杜仲配伍较续断或杜仲单独给药效果更显著，具有统计学意义。离体培养大鼠骨髓间充质干细胞（BMSC）和原代成骨细胞（OB），续断和杜仲对骨髓间充质干细胞及成骨细胞的形态具有协同保护作用，续断和杜仲协同作用下对骨髓间充质干细胞及成骨细胞的增殖、分化、活力、矿化等成骨相关特性具有促进作用，两药配伍使用具有协同增效作用。续断和杜仲均可显著促进BMP2和Wnt信号通路的BMP2、P38、ERK、Wnt、β-catenin等相关基因和蛋白表达，续断和杜仲配伍的杜续组相关基因和蛋白表达更高，具有统计学意义，该作用可被BMP2信号通路阻断剂Noggin及Wnt/β-catenin信号通路阻断剂Dkk1所逆转。体内外实验结果表明，杜仲和续断在促进骨形成上具有协同增效作用，分子水平结果显示，杜仲和续断协同促进骨形成是通过调节BMP2和Wnt/β-catenin信号通路发挥作用的。.