基于成分-靶点相关网络的二至丸调控骨重建的作用机制

A biological network that mediates the coupling of bone formation and bone resorption, maintains the homestasis of bone remodeling.The imbalance of the biological network resulted in bone-related diseases,such as osteoporosis. Er-Zhi-Wan is a "kidney-tonifying" traditional Chinese formulation.Modern research shows that antiosteoporotic activity of Er-Zhi-Wan is carried out by regulation of bone resorption and bone formation, to develop an new class of dual-action therapeutic agent for clinic usage. However,current knowledge whether Er-Zhi-Wan and its components have multi-target effect should be evaluated.This project is to establish the bilogical network involved in regulating the couple of bone degradation and bone building,based on the anlysis of the signaling pathways of Wnt/beta-catenin,ER,and RANKL/RANK in the co-culture of bone mesenchymal stem cells, pre-osteoclasts and ovarian follicular granulosa cells. Further, the impact of Er-Zhi-Wan and its components on the biological network is investigated. In addition, by docking the crystal structure of targets for compounds,we detemine the binding affinity of targets for active constituents.We expect to demonstrate that the compounds target the sinaling system,therefore simultaneously reducing bone destruction and increasing bone synthesis.These studies are expected to show for the first time a role for Er-Zhi-Wan in regulation of biological network of bone remodeling. The outcomes of the project have signifcant implication for understanding the mechansim by which Er-Zhi-Wan regulates bone remodeling,and for developing the novel dual-action agents from the traditioanl Chinese formulation of Er-Zhi-Wan. Additionally, these established methods will contribute to validating the multi-target mechanism of other traditional Chinese medicine.

骨重建是由分子网络调控的骨形成与骨吸收介导的功能耦联过程。骨重建失衡是骨质疏松的关键致病因素。经典方剂-二至丸临床用于治疗骨质疏松，可调控骨重建多环节，但目前对其研究以单靶点评价为主，且调控骨重建的多靶成分认识不够充分和深入。本课题拟构建调控骨形成与骨吸收耦联的骨重建生物信号网络，结合前期建立的细胞共培养体系，反映骨形成的Wnt/beta-catenin、骨吸收的RANKL/RANK、ER等途径的动态变化规律，以此为二至丸及其成分调控骨重建的作用评价提供多靶标的整合环境。在不同靶标可同时分析基础上，结合分子模拟等手段，计算二至丸成分与靶点的结合能力，为从靶点及其调控网络角度研究揭示二至丸的多靶点、整合作用机理。本研究建立的生物网络建模-成分靶点对接-调控网络构建的二至丸调控骨重建的方法体系为中药多成分、多靶点、整合调节机制研究提供了新的研究模式，有望加快二至丸防治骨质疏松的创新中药研发。

骨重建是由分子网络调控的骨形成与骨吸收介导的功能耦联过程。骨重建失衡是骨质疏松的关键致病因素。经典方剂-二至丸临床用于治疗骨质疏松，可调控骨重建多环节，但目前对其研究以单靶点评价为主，且调控骨重建的多靶成分认识不够充分和深入。本项目首先构建了骨形成与骨吸收耦联的骨重建生物信号网络，发现了关键节点间的相互作用关系，并结合骨重建细胞共培养模型，揭示了 Wnt/β-catenin、RANKL/RANK及 ER 途径不同靶标间的相互影响及内在联系。通过分子对接等计算方法，计算了二至丸成分与靶点的结合能力，分析了女贞子中5种成分的作用靶点，包括CK2alpha, GSK-3Beta等靶点亲和力。通过女贞子成分高效制备获取，利用骨重建细胞共培养模型，采用各种干扰手段，证实女贞子多种成分通过Wnt途径、凋亡途径促进骨形成，抑制骨吸收，发挥抗骨质疏松作用的多靶点调节作用。同时，我们首次发现了墨旱莲中的活性成分蟛蜞菊内酯，其通过与雌激素受体及GSK-3Beta结合，促进骨形成同时抑制骨吸收，发挥双功能多靶点作用效果。墨旱莲成分蟛蜞菊内酯与女贞子成分配伍，发挥了协同增效，拮抗减毒的配伍效果。蟛蜞菊内酯与女贞苷、特女贞苷、油女贞苷等配伍，能够不同程度增加骨形成作用，拮抗高剂量蟛蜞菊内酯的细胞毒性，增加细胞存活率，增强ALP的表达。油女贞苷通过Wnt/Beta-catenin途径增强蟛蜞菊内酯对成骨细胞生成的作用；通过Wnt/CK2Alpha途径减少蟛蜞菊内酯细胞毒作用。总之，本研究建立的生物网络建模-成分靶点对接-调控网络构建的二至丸调控骨重建的方法体系，推动了二至丸防治骨质疏松临床应用及创新中药研发。本项目完成的二至丸作用靶点研究在医院推广应用，二至丸成分高效制备获取及系列标准品在企业应用，获得明显社会经济效益。本项目共发表文章13篇，其中8篇SCI文章，2篇中文核心期刊论文，2篇中国科技核心期刊论文。申请专利3件，授权专利1项，培养研究生5名，获得辽宁省中西医结合学会科技进步二等奖1项，辽宁省政府科技进步二等奖1项。