基于H型血管与成骨偶联探讨健骨颗粒防治绝经后骨质疏松症的作用机制

Jiangu granule is a theoretical prescription of tonifying kidney and spleen, activating blood and invigorating qi, which has a significant curative effect on the prevention and treatment of postmenopausal osteoporosis (PMOP).Previous studies based on the kidney-tonifying and bone-strengthening theory showed that jiangu granule could improve osteoporosis by promoting the proliferation and differentiation of osteoblasts and inhibiting osteoclast bone resorption, but the mechanism of the relationship between its blood-activating effect and bone-strengthening was not clear.Many studies have shown that the blood-activating effect of traditional Chinese medicine is closely related to the regulation of angiogenesis.H-type blood vessels in bone tissue can promote bone formation, while osteoblasts conversely affect angiogenesis through the pro-angiogenic factor SLIT3 and its regulatory protein SHN3.The coupling of bone formation and angiogenesis is expected to be a new target in the treatment of osteoporosis.This study intends to establish the osteoporosis rats model by ovariectomy and shn3 knockout osteoblast. After the intervention with JianGu particles, We'll detecte Bone morphology, bone metabolism level, H-type blood vessels, expression of osteoblastkc SLIT and its angiogenesis promoting effect, by using of μCT, ELISA, laser confocal microscope, flow cytometry, realtime quantitative PCR and Western Blot techniques. The purpose of this study is to explore the mechanisms of the effect of JianGu granule on PMOP, from the aspect of bone formation and angiogenesis coupling, to provide preliminary experimental basis of improving effect and decreasing toxicity of traditional Chinese medicine by molecular compatibility.

健骨颗粒以补肾健脾和活血益气的理论组方，用于防治绝经后骨质疏松症（PMOP）疗效显著。前期基于补肾健骨理论研究表明，健骨颗粒可通过促进成骨细胞增殖分化和抑制破骨细胞骨吸收改善骨质疏松，但其活血功效与健骨的关系机理未明。研究表明中药的活血功效与调控血管生成密切相关。骨组织中的H型血管能促进骨形成，而成骨细胞反之通过促血管生成因子SLIT3及其调节蛋白SHN3影响该血管生成。这种骨形成与血管生成的偶联有望成为治疗骨质疏松的新靶标。本研究拟建立去卵巢骨质疏松大鼠模型和shn3基因敲除成骨细胞，以健骨颗粒干预后，通过μCT、激光共聚焦显微镜、流式细胞术、荧光定量PCR和Western Blot等技术检测骨形态、骨代谢、H型血管、成骨细胞SLIT表达及其促血管生成效应，观察健骨颗粒对骨形成和血管形成偶联的影响，探讨该中药方防治PMOP的相关作用机制，为中药通过“分子配伍”增效减毒提供前期实验基础。

健骨颗粒以补肾健脾和活血益气的理论组方，用于防治绝经后骨质疏松症疗效显著。前期基于补肾健骨理论研究表明，健骨颗粒可通过促进成骨细胞增殖分化和抑制破骨细胞骨吸收改善骨质疏松，但其活血功效与健骨的关系机理未明。研究表明中药的活血功效与调控血管生成密切相关。骨组织中的H型血管能促进骨形成，而成骨细胞反之通过促血管生成因子SLIT3及其调节蛋白SHN3影响该血管生成。这种骨形成与血管生成的偶联有望成为治疗骨质疏松的新靶标。本研究建立去卵巢骨质疏松大鼠模型，健骨颗粒干预后，通过μCT观察骨形态，骨生物力学检测骨强度和ELISA检测骨代谢指标。发现去卵巢后大鼠骨密度下降，骨微结构破坏，骨强度下降，补充雌激素可恢复至正常水平。健骨颗粒干预后，骨密度，骨微结构和骨代谢水平有所恢复，表明健骨颗粒干预去卵巢后骨质疏松有效。荧光染色激光共聚焦显微镜观察以及流式细胞检测发现，骨髓中CD31hiEMCNhi H型血管在去卵巢后表达下降，补充雌激素或健骨颗粒后表达升高，表明去卵巢后骨质疏松与H型血管的表达水平相关，健骨颗粒可影响其表达。通过敲除成骨细胞shn3基因，使slit3表达下降，以健骨颗粒含药血清干预后观察其对血管内皮细胞的影响，发现健骨颗粒含药血清干预后的成骨细胞上清液可促进正常血管内皮细胞迁移和成血管效应，而对shn3基因敲除后的内皮细胞影响较小。表明健骨颗粒可能通过SHN3-slit3通路促血管生成，从而改善去卵巢后骨质疏松。