自噬及其相关蛋白与IL-17相互级联作用持续活化牙槽骨破坏效应的信号传导机制研究

It is known that IL-17 regulates the inflammatory alveolar bone destruction, while the key factors and underlying molecular mechanisms of its continuous activation still remain unclarified. Our preliminary study unexpectedly found that IL-17 promoted alveolar bone resorption and osteoclast differentiation, which was accompanied by the increase of autophagy-related proteins Beclin1 and LC3 expression. Similar results have not been reported yet. Recent study found that dissociated Beclin1 appeared to activate downstream molecules of the RANKL-RANK signaling pathway. Moreover, LC3 co-localization with CatK to the actin ring and ruffled border in mature osteoclasts was Atg5 dependent on regulation of bone-resorbing activity. However, the cascade mechanisms among these key components are still unknown. Based on the above findings, this project will focus on the molecular mechanisms of how IL-17 mediates Beclin1 dissociation in pre-osteoclasts using co-immunoprecipitation, protein mass spectrometry, pull-down, immunofluorescence co-localization etc. Meanwhile, it will try to identify the biological functions of the key molecules in RANKL-RANK signaling pathway continuously activated by Beclin1. Last, it will testify how the cascade reactions of autophagy-related proteins are involved in mature osteoclasts function-related activity. The expected data may provide a new theoretical explanation for clarifying the mechanism by which the host immune response induces alveolar bone destruction and may lay a foundation for possible therapeutic targets of clinical periodontal diseases.

已知IL-17调控炎症性牙槽骨破坏，其持续活化牙槽骨破坏效应的关键因子及分子机制不明。项目组前期研究惊喜发现IL-17促牙槽骨破坏加剧/破骨细胞分化增强与自噬相关蛋白Beclin1、LC3表达增高呈一致性，类似结果未见报道。新近发现游离状态Beclin1能活化RANKL-RANK通路下游信号分子；成熟破骨细胞内LC3和CatK在肌动蛋白环、皱褶缘处共定位受Atg5调控，促骨吸收效应产生。而其关键信号分子相互级联作用机制不详。本项目拟用免疫共沉淀、蛋白质质谱分析、pull-down、免疫荧光共定位等技术重点关注IL-17影响破骨前体细胞内Beclin1呈游离状态的分子机制；鉴定Beclin1促RANKL-RANK信号通路持续活化中关键分子的生物学功能；明确自噬相关蛋白间级联作用对成熟破骨细胞骨吸收效应的影响。以期为宿主免疫反应介导牙槽骨破坏提供新理论解释，有望为牙周病治疗提供可能的干预靶点。

已知IL-17调控炎症性牙槽骨破坏，其持续活化牙槽骨破坏效应的关键因子及分子机制不明。本研究明确了自噬相关蛋白、IL-17 间的生物活动与慢性牙周炎牙槽骨破坏吸收密切相关。本项目采用慢病毒转染基因敲除自噬相关基因、体外构建成骨-破骨细胞共培养模型、RT-PCR、扫描电镜和各类细胞染色检测成骨、破骨细胞分化及骨吸收功能等从多方面多角度探索自噬相关蛋白对成骨-破骨细胞间相互作用、成骨细胞分化和钙化以及破骨细胞分化和骨吸收功能等的影响。此外，体内动物实验以IL-17、3-MA及联合干预建立实验性大鼠牙周炎模型，HE染色观察牙周炎性细胞浸润程度，Micro CT扫描重建牙槽骨结构并分析骨组织参数，免疫组织化学方法检测局部牙周组织内自噬相关蛋白的表达情况。上述研究发现：自噬及其相关蛋白参与IL-17介导的破骨细胞分化、吸收功能，而敲除破骨细胞自噬基因BECN1或加入自噬抑制剂3-MA后可明显抑制该现象；自噬相关蛋白可通过激活TRAF6/ERK/P38通路调控IL-17介导的破骨细胞分化过程；IL-17以Beclin-1/TAB3/ERK通路依赖方式促进破骨细胞分化；不同浓度IL-17在体外对原代成骨细胞的骨代谢作用不同，高浓度IL-17可通过抑制自噬并上调JAK2/STAT3通路来增强原代成骨细胞的RANKL表达。动物实验结果发现，自噬相关蛋白的表达与炎性牙槽骨破坏之间存在正相关，IL-17可明显促进牙周炎症及牙槽骨吸收作用，而抑制自噬可减弱此促进作用。为宿主免疫反应介导牙槽骨破坏提供依据。