镁离子通过调控IκBα泛素化诱导巨噬细胞极化促进成骨机制研究

According to the concept of osteoimmunomodulation, the polarization of macrophage plays a crucial role in the osteogenic process of bone grafting materials in vivo. In our previous study, magnesium was found to affect the osteogenic process of calcium phosphate. It was reported that magnesium down-regulated the expression of inflammatory cytokines in the NF-κB pathway, by sustaining the intracellular concentration of IκBα, which may affect the polarization of macrophage and promote bone formation subsequently. However, the underlying mechanism of magnesium regulating the intracellular concentration of IκBα remains unknown. The proteolysis of IκBα depends on the modification of a Lys-48 ubiquitin poly-chain, whereas the Lys-48 of ubiquitin was found to be bonded by magnesium, leading to a decrease of free Lys-48 in a crystallographic study. This suggests that magnesium may inhibit the formation of Lys-48 ubiquitin poly-chains in vivo. Hence, we hypothesize that magnesium may inhibit the proteolysis of IκBα by bonding to Lys-48, sustain the inhibitory effect of IκBα on NF-κB, leading to a lower expression of inflammatory cytokines, the polarization of macrophage from M1 to M2 and new bone formation consequently. Thus, this study is firstly to reconfirm the effect of magnesium on the osteoimmunomodulatory function of macrophage by investigating the inflammatory cytokines and osteogenic genes in the presence of magnesium in the culture media; and secondly to elucidate the mechanism of magnesium regulating the ubiquitylation of IκBα by analyzing the change of Lys-48 in ubiquitin from macrophage. Additionally, magnesium-incorporated calcium phosphate is to be synthesized, and its osteogenic capability is to be evaluated in vitro and in vivo, so as to enrich the theoretical basis for the research and application of a new bone grafting material.

骨免疫调控中巨噬细胞的极化对骨移植材料的体内成骨影响显著。前期研究发现镁离子可影响磷酸钙成骨；有研究发现镁离子通过维持巨噬细胞IκBα浓度、下调NF-κB通路炎症因子表达，诱导巨噬细胞极化而促进成骨，但镁离子维持IκBα浓度的机制不明。IκBα经泛素Lys-48多链修饰后水解；而体外晶体研究发现镁离子可与泛素Lys-48结合，提示镁离子可能影响体内Lys-48多链的形成。据此提出科学假说：镁离子与Lys-48结合，抑制IκBα泛素化水解，下调NF-κB通路炎症因子表达，诱导巨噬细胞向M2型极化而促进成骨。为此，本研究通过调控微环境中镁离子浓度，检测巨噬细胞炎症因子和促成骨因子的表达，验证镁离子对巨噬细胞骨免疫调控功能的影响；检测分析泛素Lys-48的变化，深入阐明镁离子调控IκBα泛素化的具体机制；在此基础上，制备掺镁磷酸钙并评估其体内外成骨性能，为新型骨移植材料的研发和应用提供理论依据。

外伤、疾病、手术和先天性畸形而引起的骨缺损，是世界范围内广泛面临的健康问题。尽管生物源性磷酸钙类骨替代材料已经广泛应用于临床，但由于传统的材料设计理念倾向于制备惰性的异种骨替代材料，其成骨效果并不能满足临床需求。本项目应用基于镁离子的骨免疫调控策略，优化了猪骨源性骨替代材料（PHA）的成骨效能。通过简便、经济、高效的水热反应法，保留PHA固有的三维孔隙结构作为框架；通过镁离子进行表面改性（nMgHA），明显观察到新骨生成率和微血管生成率提升，这可能是由于nMgHA释放的镁离子抑制了巨噬细胞M1方向极化。体外研究发现巨噬细胞中NF-κB通路与炎性细胞因子表达水平降低，归因为镁离子通过影响IκBα泛素-蛋白酶体途径降解，从而维持IκBα蛋白稳定，持续抑制信号通路。综上，本项目从解决临床问题的角度出发，通过研究基于镁离子的骨免疫调控策略，从而调控生物源性磷酸钙类骨替代材料的生物学活性，研发出一种具有骨免疫调控性能的新型骨替代材料，为临床骨缺损修复提供新的思路。