基于巨噬细胞极化探索嗅结构域抑制剂在牙周炎中的作用研究

Periodontal disease, which is the primary reason leading to tooth loss in the older adults, also has adverse impacts on several systemic diseases. Macrophages are important immune effector cells with high plasticity. They can polarize into different phenotype (M1/M2), which play prominent roles in periodontal destruction and regeneration, respectively. Modulating macrophage polarization has been regarded as a potential therapeutic target in periodontitis. Bromodomain and extraterminal domain inhibitor (BETi) can epigenetically modulate histone acetylation with anti-inflammatory properties. However, whether BETi is involved in macrophage polarization has not yet reported. The preliminary work of this study revealed the role of BETi of downregulating the expression of M1marker and increasing M2 marker in macrophages under P. gingivalis LPS stimulation. Thus the present study aims to evaluate the precise role of BETi on macrophage polarization in periodontal inflammation. First, we want to determine the impacts of BETi macrophage phenotype and possible molecular mechanisms in gene level and protein level. Second, the effects of BETi-mediated macrophage polarization on biological properties of periodontal ligament cells in the same immune microenvironment are evaluated, including migration, apoptosis, osteogenesis. Third, in the mouse model of experimental periodontitis, the periodontal destruction level is determined by Micro-CT and histopathological analysis. In addition, the macrophage polarization switch pattern in the process will be evaluated. This study will provide data support for the understanding of the relationship between macrophage polarization and periodontal disease.

牙周病是中老年人牙齿缺失的重要原因，并可促进一些全身性疾病的发展。具有高度可塑性的巨噬细胞在局部微环境作用下可极化为不同的表型（M1/M2），分别在牙周炎症进展及组织修复中发挥了重要作用。调控巨噬细胞极性已成为牙周炎新的治疗靶点。嗅结构域抑制剂（BETi）可表观调控组蛋白乙酰化，具有抑制炎症的作用，而目前BETi对巨噬细胞极性的影响尚未报道。本课题前期发现，BETi可抑制P. gingivalis LPS刺激下巨噬细胞M1标志物升高和M2标志物降低。本课题拟深入研究BETi调控巨噬细胞极性在牙周炎中的作用，通过①从基因和蛋白层面分析BETi对巨噬细胞极性的影响及可能的机制；②观察BETi干预下极化的巨噬细胞对同一免疫微环境中牙周膜细胞迁移、凋亡、成骨等生物学行为的影响；③构建小鼠牙周炎模型，分析BETi在此过程中对牙周组织破坏及巨噬细胞极性时序性转换的影响，以期为牙周炎的治疗提供新的策略。

慢性牙周炎是中老年人牙齿缺失的重要原因。巨噬细胞因其具有高度的可塑性，可以极化为不同的表型（M1/M2），分别在牙周炎症进展和组织修复中发挥着关键作用。调控巨噬细胞极性已成为牙周炎新的治疗靶点。而目前嗅结构域抑制剂是否可调控巨噬细胞极性尚未见报道。本课题研究结果显示，（一）嗅结构域抑制剂apabetalone（Apa）能够降低牙龈卟啉单胞菌脂多糖（Porphyromonas gingivalis lipopolysaccharide, Pg LPS）诱导下巨噬细胞产生IL-1β和IL-6的水平。此外，Apa显著降低了Pg LPS诱导下巨噬细胞M1极性标志物CD86和iNOS的水平，并且这种作用是具有浓度依赖性的。与此相对应的是Apa促进了巨噬细胞M2表型标志物CD206和Arg-1的表达水平。通过多个与牙周炎相关的miRNA筛选，结果发现，Pg LPS能显著上调miR-130a水平，Apa则能抑制这种变化。miR-130a过表达能促进巨噬细胞M1极化。双荧光素酶报告基因显示，信号转导及转录激活子3（STAT3）是miR-130a的直接靶向基因。进一步的免疫印迹实验也印证了这一点。（二）将Apa干预下不同极化状态的巨噬细胞条件培养基与人牙周膜细胞（human periodontal ligament cell, hPDLC）共培养，结果显示，M1极化的巨噬细胞条件培养基显著抑制hPDLC的增殖，促进其凋亡。此外，Q-PCR结果显示，将与M1极化的巨噬细胞条件培养基共培养的hPDLC进行成骨诱导7d后，hPDLC表达RUNX2、OCN、OPN、BMP2等成骨相关因子的水平显著下降，碱性磷酸酶染色也得到了相同的结果。成骨诱导21d将hPDLC进行茜素红染色结果，结果显示M1极化的巨噬细胞条件培养基显著抑制hPDLC矿化结节的形成。而Apa干预后的巨噬细胞条件培养基可以逆转M1极化的巨噬细胞条件培养基对hPDLC增殖、迁移、成骨的抑制作用。（三）构建小鼠实验性牙周炎模型，结果显示，Apa可以有效降低结扎造成的牙槽骨吸收，抑制炎症细胞浸润、破骨细胞形成，降低组织中M1/M2比例。总的来说，这个新的嗅结构域蛋白抑制剂Apa对Pg LPS刺激下巨噬细胞炎症和极化具有非常重要的作用，能够抑制小鼠牙周炎的进展，并且这种调控作用与miR-130a-3p/STAT3信号轴有着密切的关系。