基于Sema3A/Nrp1/PlexinA1信号通路探讨女贞子抗骨质疏松的分子机制和物质基础

Emerging evidence suggests that Semaphorin 3A (Sema3A) is one of key regulatory molecules in the development of osteoporosis through the regulation of osteoblasts-mediated bone formation and osteoclasts-dominated bone resorption. The results from our previous experiments demonstrated that Fructus Ligustri Lucidi (FLL) has the ability of increasing bone quantity and quality through inhibition of β-catenin phosphorylation (bone formative protein) and decreasing cathepsin K expression (bone resorptive key enzyme) in OVX rats. However, whether the anti-osteoporotic effect of FLL is associated with Sema3A remains unclear. Therefore, we proposed that FLL prevent the development of osteoporosis through regulation of Semaphorin 3A/Neuropilin 1(Nrp1)/PlexinA1 signaling pathway. In the current study, the mechanism of FLL on Sema3A in the management of osteoporosis was investigated from in vivo and in vitro. Firstly, the alterations of the molecules associated with Sema3A/Nrp1/PlexinA1signaling pathway were detected in the hypothalamus, femurs and tibias in OVX rats. Then, primary hypothalamus cells, hypothalamus dysfunctional rats and Sema3A neutralization antibody treated ovariectomized (OVX) rats were used to confirm the selective effect of FLL on Sema3A. Subsequently, the effect of the medicated serum and main active components on Sema3A signaling in the osteoblasts and osteoclasts, and on the activities of osteoblasts and osteoclasts were determined. All the experiments will contribute to reveal the molecular mechanism of FLL in improving bone quality through regulation of Sema3A/Nrp1/PlexinA1 signaling pathway in OVX rats. The results will provide experimental basis for clinical usage of this herb to management of the osteoporotic patients.

脑信号蛋白3A（Sema3A）不仅影响成骨细胞骨形成，而且调节破骨细胞骨吸收，是骨质疏松发生发展的关键调控分子。前期研究表明，女贞子能抑制去卵巢大鼠骨形成蛋白β-catenin磷酸化，降低骨吸收关键酶Cathepsin K活性，从而提高骨密度，但这种作用是否与其上游信号Sema3A相关尚未知。为此，本项目提出“女贞子通过调节Sema3A/Nrp1/PlexinA1通路抗骨质疏松”假说。我们拟从在体及离体层面，首先观察女贞子对下丘脑、股骨和胫骨Sema3A/Nrp1/PlexinA1通路的作用，然后用下丘脑细胞、下丘脑损伤大鼠和去卵巢大鼠分析女贞子对下丘脑和骨组织Sema3A作用的选择性，接着研究含药血清及其主要成分对Sema3A信号通路介导成骨和破骨细胞活性影响，揭示女贞子通过调节Sema3A/Nrp1/PlexinA1通路提高骨量和骨质量的分子机制和物质基础，为其临床应用提供实验依据。

脑信号蛋白3A（Sema3A）不仅影响成骨细胞骨形成，而且调节破骨细胞骨吸收，在骨质疏松发生和发展发挥着重要作用，但女贞子是否对其有调节作用尚不清楚。本项目利用去卵巢大、小鼠和斑马鱼模型以及细胞模型，并利用分子对接、UPLC/MS和组学分析等技术，发现女贞子能：（1）调节下丘脑和骨组织Sema3A的表达，进而促进β-catenin的核转移作用；（B）调节GH/IGF-1信号通路；（C）靶向下调miR-92a-2-5p，提高Sema3A，从而激活Wnt/β-catenin通路；（D）通过 PI3K/Akt信号通路，进而提高Sema 3A和 β-catenin的表达：（E）调节Sirt6/NF-κB/Cathepsin K信号通路有关。红景天苷和芹菜素可能是其发挥抗骨质疏松作用的物质基础。理论研究发现，女贞子可通过归经靶向于肝、肾，影响液、精、髓、血的物质转化，进而干预“肝、肾-脑-骨”调节体系，通过养血、化精、生髓发挥抗骨质疏松的作用。结合“肝、肾-脑-骨”调节体系与现代医学中“神经调节”概念有相通之处，提示女贞子也可能通过神经调节改善骨代谢，从而发挥抗骨质疏松作用。本项目研究揭示了女贞子通过调节Sema3A，激活Wnt/β-catenin通路，进而提高骨质疏松动物骨质量的分子机制和物质基础，这为进一步探索女贞子抗骨质疏松作用机理提供了中医理论指导。该项目研究为发展具有治疗骨骼类疾病的新药奠定了基础，同时也进一步丰富和发展中医药防治骨质疏松理论的科学内涵。