异甘草素基于IKKβ/NF-κB信号通路防治根尖周骨破坏的作用机制研究

The IKKβ/NF-κB signaling pathway was newly discovered to have dual roles in bone metabolism. It works to both aggravate osteoclastic bone resorption and impact osteoblastic bone formation, which arises as a promising molecular target of inflammatory lytic bone diseases. Until now structure-activity relationship evidence has indicated that isoliquiritigenin, a natural flavonoid derived from licorice, blocks IKKβ/NF-κB signaling. Our recent findings indicate that isoliquiritigenin efficiently suppresses RANKL-induced osteoclastogenesis and bone resorption. However, the action mechanism of isoliquiritigenin on bone metablism remains unclear. This project aims to comprehensively investigate the potential action of isoliquiritigenin targeting IKKβ/NF-κB to alleviate periapical bone destruction. Thus the thesis is devided into two parts: Part one is in vivo experiment: 1.IKKβ/NF-κB, osteoclast- and osteoblast-related factors are detected and measured in periapical tissues obtained as biopsy samples during periapical surgery and in experimental periapical lesions of wild-type and NF-κB-/- KO mice. 2.The bone destrution in the periapical regions is subjected to histomorphometric and microradiographic examinations, and the relationship between isoliquiritigenin administration, IKKβ/NF-κB, and osteoclast/osteoblast-related factors is analysed. Part two is in vitro experiment: 1.Cultured osteoclasts/ostoblasts are either treated with isoliquiritigenin or genetically manipulated for NF-κB overexpression, then its osteoclastogenic/mineralizing potential was analysed. 2.The three-dimensioned co-culture system of osteoblast and osteoclast is pretreated as mentioned above, and morphological and functional assessment of osteoclasts is carried out. Therefore, the present project may both deepen molecular understanding of periapical bone destruction and lay foundations for exploitation of isoliquiritigenin as a novel therapeutic and/or preventive strategy against lytic bone diseases.

新发现IKKβ/NF-κB信号通路在骨代谢中具有双重调节效应-促进破骨和抑制成骨，可能作为炎性溶骨性疾病的一个理想治疗靶点。异甘草素作为甘草中黄酮类活效单体，构效关系研究表明可阻断IKKβ/NF-κB信号通路。我们前期研究发现异甘草素可高效抑制破骨细胞分化及骨吸收，但其对骨代谢的具体作用机理尚不清楚。本课题旨在探讨异甘草素基于IKKβ/NF-κB信号通路防治根尖周骨破坏的作用及其机制：拟通过人病变组织和基因敲除小鼠模型，检测IKKβ/NF-κB、破骨/成骨相关因子在根尖周病变不同阶段的动态变化，并观察局部注射异甘草素作用后IKKβ/NF-κB、骨吸收/骨形成的改变情况；同时将异甘草素分别作用于破骨、成骨细胞分化体系及成骨-破骨共培养体系，探讨其防治骨破坏的机理。本系列研究将进一步阐明根尖周病骨破坏机制并为今后开发异甘草素作为防治溶骨性疾病的新药提供实验依据。

新发现IKKβ/NF-κB信号通路与自噬关联，但其在炎性溶骨性疾病中的作用联系尚不明确。本研究探讨了黄酮类活效单体-异甘草素基于IKKβ/NF-κB-自噬信号轴防治根尖周骨破坏的作用及其机制。本研究发现：①临床根尖病变标本包括根尖囊肿和根尖肉芽肿，存在NF-κB及自噬信号的异常表达活化；②小鼠根尖周炎病损进展过程中，破骨细胞表达在急性期上调、慢性期回落，而成骨相关分子Runx2/OCN则呈现逐渐上调趋势；③IKKβ/NF-κB-自噬作为信号联动轴，调控体外破骨细胞分化及骨吸收；④异甘草素强效抑制体外破骨细胞分化和骨吸收，机制学则在于其阻遏IKKβ/NF-κB-自噬信号轴；⑤异甘草素对小鼠根尖周炎骨破坏和LPS诱发的颅盖骨骨噬模型都具有显著保护效应，并高效抑制颅盖骨骨噬病灶区IKKβ/NF-κB和自噬信号；⑥新型“即用型”纳米生物陶瓷iRoot BP Plus具有优越的生物活性（bioactivity）和生物相容性（biocompatibility），显著提升体外成骨细胞存活、材料表面铺展和矿化，并促进牙髓干细胞迁移及体内牙髓损伤修复。上述研究结果提示：IKKβ/NF-κB-自噬信号轴调控破骨细胞分化及骨吸收；异甘草素基于IKKβ/NF-κB-自噬信号轴抑制破骨细胞分化和功能，从而有效防治骨破坏疾病。iRoot BP Plus的相关研究则为“即用型”纳米生物陶瓷推广应用于牙髓根尖周病的防治提供针对性实验依据。通过本项目的研究工作，我们建成了多项技术平台，申请国家发明专利2项，在牙髓根尖周病领域发表SCI论文10篇（含SCI一区2篇），其中2篇分别被选为牙科学顶级杂志《J Dental Res》封面文章以及材料学权威杂志《J Mater Chem B》Hot Paper。