愈骨葆方调控RANKL/RANK/MITF通路介导的骨形成与骨吸收耦联的药效机理研究

New bone formation and bone resorption are both exist and maintain a homeostasis in the union of fracture, but how their effector cells: osteoblasts (Ob) and osteoclasts (Oc) to maintain such a balance is not clear? Recent studies have indicated that the nuclear transcription factor: MITF is an important transcription factor in the Oc activation process, it regulate Oc in the formation of proteinase K; Ob may affect Oc activity via the RANKL / RANK / MITF pathway in order to maintain the coupling balance of bone formation and bone resorption. Such an associated balance plays an important role in union of fracture. YuGuBao is a traditional Chinese medicine prescription which composed of 12 herbs, its excellent efficacy in fracture treatment has been verifying after long-term clinic practice. Our preliminary work has confirmed that YuGuBao significantly increase the differentiation and mineralization of osteoblasts, and can significantly up-regulated the expression of osteogenesis genes (OPN and COL-I). Although YuGuBao has an advantage in promoting osteanagenesis during union of fracture, but the key molecular mechanism remains unclear. Base on the preliminary studies, this project intends to investigate the effect of YuGuBao on bone formation and bone resorption by using the bone lineage cells and fracture animal models, and whether the bone formation and bone resorption coupled relationship are regulated via the RANKL / RANK / MITF pathway. This study will reveal the mechanism of YuGuBao in promote fracture healing, and to provide the scientific basis for the development of a new, efficient and safe Chinese medicine.

骨折愈合期间除新骨生成外，骨吸收同时存在且两者保持动态平衡，其效应细胞：成骨细胞（Ob）与破骨细胞(Oc)如何维持平衡？近期研究指出，核转录因子MITF是Oc活化过程中重要的转录因子；而Ob可能通过RANKL/RANK/MITF通路调控Oc活性以维持骨形成与骨吸收的耦联平衡。愈骨葆方是经民间长期实践验证具有确切接骨作用的中药验方。前期工作证实此方能显著增加Ob分化活性及矿化功能，且能明显上调成骨功能相关基因（OPN与COL-Ⅰ）的表达。虽然愈骨葆方在促进骨再生方面具有优势疗效，但其关键分子机理仍未明。本课题拟在前期研究基础上，利用骨系细胞和骨折修复动物模型，从组织、细胞和分子水平研究愈骨葆方在治疗过程中对骨生成和骨吸收的影响，及其是否通过调控RANKL/RANK/MITF通路而介导两者的耦联关系。本研究有助于揭示愈骨葆方促骨折愈合的疗效机制，为研发高效安全的接骨类新型中成药提供科学基础。

愈骨葆方是经民间长期实践验证具有确切接骨作用的中药验方，接骨类中药作为中药的优势药种，具有西医西药难以比拟的优势。虽然愈骨葆方在促进骨再生方面具有优势疗效，但其关键分子机理仍未明。骨折愈合期间除新骨生成外，骨吸收同时存在且两者保持动态平衡，其效应细胞：成骨细胞与破骨细胞通过RANKL/RANK/Mitf通路调控Oc活性以维持骨形成与骨吸收的耦联平衡。本课题在前期研究基础上，利用骨系细胞和骨折修复动物模型，从组织、细胞和分子水平研究愈骨葆方及其君药杜仲的生物活性成分：黄芩素在治疗过程中对骨生成和骨吸收的影响，及其是否通过调控RANKL/RANK/MITF通路而介导两者的耦联关系。研究结果归纳如下：.1..通过正交实验确定了愈骨葆方的最佳提取工艺，利用大鼠骨折模型证实愈骨葆方能显著地促进大鼠骨折愈合；并明确了愈骨葆方含药血清能显著地促进成熟破骨细胞的形成、粘附及其骨吸收功能；并探讨了RANKL/RANK/Mitf信号转导通路在其中的作用，证实愈骨葆方含药血清能影响上调Mitf蛋白在细胞核中的表达。此部分内容已申请专利。.2..明确复方中药中的有效功能成分是实现中药现代化的重要一步。经实验筛选，我们发现续断（愈骨葆方中的君药之一）中的提取物——黄芩素具有促进成熟破骨细胞的药效作用，包括显著地促进成熟破骨细胞的形成，上调破骨相关基因的表达，增强actin环的形成，及提高破骨细胞骨吸收活性。分子机制研究提示，黄芩素对破骨细胞的促进作用是通过RANKL/RANK/Mitf通路来实现的。此部分内容已整理成文，正在投稿。.本研究有助于揭示愈骨葆方及其促骨折愈合的疗效机制，为研发高效安全的接骨类新型中成药提供科学基础。