基于“肾主骨”理论探讨补肾中药通过TGF-β/Smad3信号转导通路调控骨发育不良的作用机制

Based on the theory of "kidney dominating bones", a large number of studies have proved that Chinese kidney-tonifying drugs can effectively regulate bone metabolism diseases and directional differentiation of mesenchymal stem cells to osteocyte lineage, but there is less research on the regulation of bone development process. Our previous study found that: (1) overexpression of Zeb1 in mouse mesenchymal stem cells inhibited the proliferation of chondrocytes in growth plate and increased its apoptosis, leading to delayed endochondral ossification and limb malformation; (2) Zeb1 downregulated TGF-β/Smad3 signaling pathway activity and inhibited the expression of TGFβ1 in embryonic bone tissue resulting in inhibition of chondrogenesis; (3) the medicated serum of Rhizoma Drynariae, Epimedium could upregulate TGF-β/Smad3 signaling pathway and promote cartilage formation. Therefore, we put forward the hypothesis of "Mechanism of Chinese Kidney-Tonifying Drugs in Regulating Bone Development Abnormalities through TGF-β/Smad3 signaling Based on TCM "Kidney Dominating Bone" Theory". As a model of bone development disorder, overexpression of Zeb1 in mesenchymal stem cells, we research the mechanism of Chinese kidney-tonifying drugs(Epimedium, Drynaria) and its effective components (icariin and naringin) on regulation bone developmen disorder model through in vivo and in vitro experiments. We observe the regulation of Chinese kidney-tonifying drugs on bone developmen and further enriched the theory of "kidney dominating bones".

大量研究从“肾主骨”理论出发证实补肾中药可有效调控骨代谢疾病或间充质干细胞向骨细胞谱系的定向分化，而对骨发育过程的调控研究较少。我们前期研究工作发现：（1）小鼠间充质干细胞过表达Zeb1会抑制生长板软骨细胞增殖，增加其凋亡，导致软骨内成骨障碍，出现四肢短缩畸形；（2）Zeb1下调TGF-β/Smad3信号通路活性及骨组织TGFβ1的表达，抑制软骨生成；（3）补肾中药（骨碎补、淫羊藿）含药血清可激活TGF-β/Smad3信号通路活性，促进软骨生成。因此，我们提出“基于“肾主骨”理论探讨补肾中药通过TGF-β/Smad3信号转导通路调控骨发育不良的作用机制”的假说。以间充质干细胞过表达Zeb1小鼠作为骨发育不良模型，通过体内外实验系统研究补肾中药（淫羊藿、骨碎补）及其有效组分（淫羊藿苷、柚皮苷）对骨发育不良的调控机制，从骨发育的角度阐述补肾中药的作用机理，进一步丰富“肾主骨”的理论内涵。

基于中医“肾主骨”理论探索及揭示补肾中药治疗骨骼疾病的作用机制具有重要的临床意义。1）本研究首先在体外分离出肢芽干细胞进行微团培养（模拟骨发育早期间充质干细胞凝集向软骨细胞分化过程），给予淫羊藿、骨碎补单味中药煎剂含药血清干预，发现淫羊藿、骨碎补可以促进软骨生成；为进一步探讨是否通过TGFβ信号通路以发挥作用，给予TGFβ信号通路抑制剂，证实淫羊藿、骨碎补通过TGFβ信号通路促进肢芽干细胞的增殖与分化。2）利用肢芽干细胞微团培养方法，分别给予淫羊藿及骨碎补有效组分淫羊藿苷及柚皮苷，筛选出促进软骨生成的最佳浓度（10μM），进一步将继续深入探究两种化合物的作用机制；3）在前期研究基础上，利用间充质干细胞过表达Zeb1小鼠(Zeb1Prx1)作为骨发育不良模型，该模式动物表型类似骨软化症：成年期骨基质矿化不全及骨强度的下降。基于以上我们给予淫羊藿苷及柚皮苷干预，发现淫羊藿苷及柚皮苷均能减轻Zeb1Prx1小鼠骨组织中软骨残留，改善骨基质矿化阻滞，增加骨强度，主要与促进骨矿化基质表达相关。提示补肾中药对先天导致的骨发育不良疾病同样具有治疗作用，为临床治疗该类疾病提供新思路。