伪羟基茜草素对山羊骨骼矿化沉积及生物学性能的影响

Pseudopurpurin extracted from madder is the main component of the natural dyes. In vital staining, pseudopurpurin can be specifically dyed with calcium ion, so that "calcium store"-bones present red in the body. Our previous experiments confirmed that the red matter is pseudopurpurin in red-boned Guishan goats, and found that pseudopurpurin could improve the microstructure and mechanical properties in the bones of red-boned Guishan goats. Therefore, we hypothesized that the chemical reaction between pseudopurpurin and calcium salt in the bone leads to promoted mineral deposit and improved metabolic. With application of molecular biology, chemistry and physics, through feeding pseudopurpurin to goats, we evaluated the change pattern of the morphological microstructure and mechanical properties of the bone tissues in various stages of goat growth. We observed under atomic force microscope the arrangement of, dispersion of, and the mineralization relation between collagen fibers and hydroxylapatite grain. We analyzed in micro-areas the ratios between calcium, phosphorus, or collagen fibers, and the mineral content in hydroxide apatite bone crystals. Further, via X-ray diffraction, we measured the crystalline substance content phase composition quantitatively and the chemical structure qualitatively. We aimed to confirm that the combination between pseudopurpurin and calcium during growth can promote calcium salt deposition and enhance the geometric structure, biomechanics and metabolic performance of bones, and further provide scientific evidence for the prevention and treatment of metabolic disorders in mineral elements of bones in animals and humans.

伪羟基茜草素是天然染料茜草中的主要成分。活体染色中，伪羟基茜草素可特异性着染钙离子，使体内"钙库"-骨骼呈现红色。本项目前期证实红骨山羊骨中红色物质为伪羟基茜草素，并发现其具有改善骨显微结构和生物力学性能的作用，在此基础上以伪羟基茜草素与骨中钙盐发生化学反应，进而促进骨的矿物质沉积和代谢性能为假设。运用分子生物学、化学和物理学等技术，通过山羊伪羟基茜草素饲喂试验，评价山羊各个生长阶段骨组织形态显微结构和生物力学性能的变化规律；原子力显微镜观察骨中胶原纤维和羟磷灰石晶粒的排列、分散性及其两者之间的矿化关系；微区范围内分析骨中羟磷灰石晶体中钙和磷、胶原纤维和矿物质含量的百分比；X射线衍射检测晶态物质物相组成的定量和化学结构的定性分析。确证伪羟基茜草素能与生长阶段骨中钙盐结合，促进钙盐的沉积，进而增强骨的几何结构、生物力学和代谢性能，为动物及人骨骼矿物质元素代谢紊乱性疾病的防治提供科学依据。

前期研究结果表明饲喂伪羟基茜草素能促进大鼠骨骼显微结构和生物力学性能，但伪羟基茜草素是如何影响骨骼矿化规律还尚未明确。本项目拟在前期试验结果的基础之上，选择大鼠骨骼作为靶器官，通过建立伪羟基茜草素大鼠体内动物模型。采用形态剂量学、扫描电镜、X射线能谱仪、骨显微CT、血液中生化指标和激素水平等方法，评价伪羟基茜草素对骨骼各个生长阶段骨组织形态显微结构和生物力学性能的变化规律；观察骨胶原显微和羟磷灰石晶粒的排列、分散性及其两者之间的矿化关系；以及骨骼晶态物质物相组成的定量和化学结构的定性分析。从分子和化学水平明确伪羟基茜草素引起骨骼生物性能优越的作用机制，揭示了伪羟基茜草素能与生长阶段骨中钙盐结合，促进钙盐的沉积，进而增强骨的几何结构、生物力学和代谢性能，为动物及人骨骼矿物质元素代谢紊乱性疾病的防治提供科学依据。.试验结果得出：实验组大鼠股骨远端被红色荧光标记，对照组大鼠显示出两条荧光条带；并且实验组大鼠骨的矿化和转化率要显著的优于对照组；扫描电镜观察得出实验组相对于对照组大鼠骨纤维排列整齐成，均匀并密集；应用纳米力学检测仪分析实验组大鼠的股骨骨干硬度和弹性模量要显著优于对照组大鼠骨骼；同时，实验组大鼠股骨远端和骨干显微结构显著优于对照组大鼠；并且实验组大鼠骨骼中钙磷比例优于对照组大鼠；实验组大鼠血液中1,25-二羟基维生素D3、降钙素以及羟基脯氨酸的含量比对照组显著增加。.通过以上结果得出伪羟基茜草素是一种粘合剂能与血液中金属阳离子结合，尤其是钙离子离子结合，并形成一种吸附力很强的钙盐，与骨骼中I型胶原纤维结合，促进钙在胶原纤维上沉积，进一步促进骨骼矿化。