槲皮素对感染性性骨疾病骨生长重建的调控机制研究

Osteomyelitis and bone and joint infections promote osteoclast differentiation and activitation, which cause an imbalance of bone resorption by osteoclasts and bone formation by osteoblasts, an important cause leading to infective bone lysis and bone nonunion. In previous study we found that quercetin, a flavonoid, significantly inhibited LPS-induced osteoclast differentiation and bone resorption, and also reversed the down-regulated mRNA expression levels of osteoblast-related genes including osteocalcin (OCN), bone sialoprotein (BSP) and osterix. In future project when expose to LPS/IL-1 for an indicated periods, either osteoclasts or osteoblasts were treated with quercetin and cell functions were observed. The mRNA expression levels and protein levels of osteoclasts or osteoblasts were measured using real-time QPCR and western blot analysis. Pretreatment with MAPK or NF-кB inhibitors, the MAPK signaling pathway and NF-кB signaling pathway were determined by western blot analysis. Co-culture with osteoclasts and osteoblasts, cells were treated with LPS/IL-1 and quercetin. The effects of quercetin on cells interactions between osteoclasts and osteoblasts were assessed. Administration of quercetin treated into rats subjected to osteomyelitis; we evaluated the formation of osteoclasts or osteoblasts in staining skull of rats. Our project will clarify not only the effects of flavonoids on bone resorption by osteoclasts or bone formation by osteoblasts in bone inflammatory diseases, but also on bone regeneration and remodeling during inflammatory processes. Based on these experiments, quercetin would be a potential effective drug to treat human bone loss in chronic inflammatory diseases.

骨髓炎激活破骨细胞过度分化和活化，导致破骨细胞骨吸收与成骨细胞骨形成失平衡，是形成骨溶解、骨不愈合的重要因素。我们前期研究发现天然黄酮类药物槲皮素显著抑制脂多糖（LPS）诱导源性破骨细胞的分化及骨吸收，并逆转LPS对成骨细胞骨钙素（OCN）、骨涎蛋白（BSP）和 osterix mRNA表达的抑制作用。本项目在促炎介质LPS/白介素-1（IL-1）介导破骨细胞/成骨细胞过程中，采用槲皮素处理，观察破骨/成骨细胞的活性及其相关mRNA、蛋白表达变化；分别阻滞MAPK及NF-кB信号通路，检测p-MAPK和NF-кB活性水平变化。在LPS/IL-1介导破骨细胞-成骨细胞共培养体系和大鼠骨髓炎模型中采用槲皮素处理，观察槲皮素对细胞共培养耦联信号和鼠骨破骨细胞、成骨细胞形成的影响。从炎症介导下破骨、成骨细胞活性及细胞耦联的角度探讨槲皮素对骨生长重建的调控机制，为炎症性骨疾病的治疗提供新思路。

骨髓炎易造成炎症性骨溶解、骨不愈合。然而炎症反应导致破骨细胞骨吸收与成骨细胞骨形成失平衡的确切机制并不清楚。在前期研究发现槲皮素（Quercetin）显著抑制脂多糖（ LPS）诱导源性破骨细胞的分化及骨吸收，并逆转 LPS对成骨细胞骨钙素（ OCN）、骨涎蛋白（ BSP）和 osterix mRNA 表达的抑制作用，但其确切调控机制仍不清楚。项目在LPS/白介素-1（ IL-1）介导破骨/成骨细胞分化过程中，并采用槲皮素治疗，探讨炎症介质导致骨形成失平衡的确切机制及槲皮素治疗的分子调控机制。主要的研究发现：.[1] LPS可单独诱导破骨前体细胞RAW264.7细胞形成具有骨吸收功能的破骨细胞；单独应用IL-1α不能诱导RAW264.7细胞形成具有多核的破骨细胞；对已分化的破骨细胞LPS和IL-1α可上调其破骨相关基因表达。LPS和IL-1α通过上调MAPK ( p38、JNK和ERK)、NF-kB(IKK/IKBα)及β-catenin信号通路对促进破骨细胞分化和活化；.[2] LPS和IL-1不仅能抑制成骨细胞的分化和成骨活性，并促成骨细胞凋亡。LPS和IL-1 通过上调MAPK ( p38、JNK)信号通路实现对成骨细胞分化和成骨活性的抑制及促成骨细胞凋亡；.[3] 槲皮素治疗可恢复LPS和IL-1α抑制下成骨细胞分化和成骨活性；预防用槲皮素治疗不仅恢复LPS抑制下成骨细胞分化和成骨活性，也能减轻LPS促成骨细胞凋亡。槲皮素通过下调p38、JNK、上调ERK和Wnt/β-catenin信号蛋白实现对成骨细胞分化的调控。.实验结果证实：槲皮素能够多靶点调控炎症性骨形成失平衡，起着改善炎症性骨生长重建的重要作用。