淫羊藿女贞子“平补阴阳，益肾壮骨”调控骨重建稳态治疗原发性骨质疏松的作用及配伍机理研究

The unbalance of bone reconstruction steady-state is the basic pathogenesis of the primary osteoporosis (POP). The maintenance of bone reconstruction balance depends on the complex relationship network, consisting of many regulatory factors secreted by osteoblasts (OB) or osteoclasts (OC). According to TCM theories, OB and bone formation dominated by OB belongs to the category of yang. In contrast, OC and bone resorption dominated by OC belongs to the category of yin. Based on the common understanding for POP in Chinese and Western medicine and the pre-work, two kinds of POP models (the senile and postmenopausal osteoporosis models) are used, combined with the cell level in this project. Focuses on bone reconstruction steady-state, detection techniques, including Micro-CT, bone tissue pathology, bone density and bone biomechanics are applied to evaluate drug efficacy. The serum levels of bone turnover markers will be tested by ELISA. Many regulatory factors Secreted by OB (including RANKL, M-CSF, Wnt5a, OPG) or OC (including TGF-β, IGF, BMPs, Atp6v0d2) will be tested by immunohistochemistry, western blot, real-time PCR. The application of neural network modeling is to evaluate the dynamic intervention effects on the development process of POP. Regulatory factors network will be constructed according to the experimental results. The project aims is to studies the differences of targets or pathways in the regulation of bone reconstruction among the single use of Herba Epimedii or Fructus Ligustri Lucidi and the compatibility of Herba Epimedii and Fructus Ligustri Lucidi, analyze the compatibility mechanism of nourishing yin-Yang method in prevention and treatment of POP, and provide a scientific basis for the development of new drugs on the prevention and treatment of POP.

骨重建稳态失衡是原发性骨质疏松症（POP）发生的基本病机，成骨细胞（OB）及其主导的骨形成（属“阴”）和破骨细胞（OC）及其主导的骨吸收（属“阳”）通过彼此分泌的调节因子形成复杂作用网络（阴阳之间的生克制化）维系骨重建平衡。基于前期临床与实验基础，课题采用老年性及绝经后POP模型，结合细胞水平，以Micro-CT、骨病理、骨密度及生物力学评价药物作用；酶免法测血中骨转换标志物，实时荧光定量PCR、免疫组化、WB、共聚焦显微镜等测骨中由OC、OB分泌的，对应调节OB、OC功能的因子（RANKL、M-CSF、Wnt5a、OPG；TGF-β、IGF、BMP、ATP6v0d2）的表达；应用神经网络建模，动态评价药物干预OP进程的作用，并构建调节因子作用网络，比较淫羊藿、女贞子配伍与单用调节骨重建的靶点或途径的差异。分析阴阳双补法防治POP的配伍机理，为中药防治POP的临床及新药开发提供科学依据。

原发性骨质疏松症（POP）已成为老龄化社会卫生保健的一个突出问题，筛选及开发抗POP药物具有重要意义。骨重建稳态失衡是POP发生的基本病机，成骨细胞（OB）及其主导的骨形成（属“阴”）和破骨细胞（OC）及其主导的骨吸收（属“阳”）通过彼此分泌的调节因子形成复杂作用网络（阴阳之间的生克制化）维系骨重建平衡。. 动物研究分别采用去卵巢术后雌性大鼠模型及自然衰老雄性大鼠模拟绝经后和老年性骨质疏松（PMOP和SOP），比较淫羊藿、女贞子单用及配伍防治作用，并构建多层感知-人工神经网络模型（MLP-ANN）综合评价药物作用。结果显示，淫羊藿女贞子配伍可逆转骨微观结构的退化，提高骨量及骨质量；调节血清OPG、BGP、CTX-I、PICP、PINP、TGF-β1等水平，降低血清和骨组织中TRACP/ALP的比值维持骨转化稳态；对骨组织中关键的骨调节因子——RANKL、M-CSF、Wnt5a、Atp6v0d2、TGF-β1、OPG、IGF-1、BMP-2的蛋白和mRNA表达均有显著影响。MLP模型评价药物的综合作用显示，各给药组均取得显著疗效，配伍组显著优于单用组；淫羊藿、女贞子单用对PMOP大鼠的综合作用无显著差异；对SOP大鼠，淫羊藿优于女贞子。. 细胞研究分别采用正常培养、功能亢进及功能抑制的OB和OC，比较淫羊藿、女贞子单用及配伍的含药血清的作用。结果显示，淫羊藿女贞子配伍可抑制OC活性，促进OB增值及分化；通过下调OC中Atp6v0d2的表达，上调TGF-β1、IGF-1和BMP-2的表达，进而提高OB活性及其介导的骨生成能力；下调RANKL、M-CSF表达，上调OPG、TGF-β1的表达，调节OPG/RANKL比值进而抑制OC活性及其介导的骨吸收能力。. 该研究表明，淫羊藿女贞子配伍具有良好的防治POP的作用，其作用机制核心在于调节骨重建稳态，抑制骨吸收并促进骨形成，既促使OC分泌更多的有利于OB分化和增殖的细胞因子，又能促使OB分泌调节OC的细胞因子以抑制OC的分化和增殖。本研究阐明了平补阴阳，补肾壮骨立法的中药防治POP的综合作用及部分机制，为中药防治POP的临床及新药开发提供科学依据。构建的MLP模型用于后续的药物评价，为慢性衰老相关性疾病的药物评价提供了新的思路和方向。