β防御素在牙龈卟啉单胞菌脂多糖促动脉粥样硬化形成中的保护作用

Our previous studies observed that Pg-LPS stimulation contributes to an inflammatory response and injury of aortic endothelial cells. It enhances foam cells formation by facilitating lipid ingestion into macrophages/monocytes;it promotes atherosclerosis(AS) related gene expression.β defensin(BD) has anti-bacterial activity and can modify the inflammatory and immunological response as well. By nature, AS is an inflammatory disease, thus we hypothesize that: BD can inhibit Toll like receptors(TLRs)-nuclear factor-κB(NF-κB)-proinflammatory cytokine signal pathway. Therefore, it may inhibit the acceleration activity of Pg-LPS in AS development.In this project, Pg-LPS endotoxemia is to be established in ApoE-/- mice in vivo, and then BD is to be utilized to inhibit the inflammatory process. Lipid metabolism, atherosclerotic plaque formation, inflammatory reaction in arterial wall, TLRs expression and NF-κB pathway activation are to be analyzed, in order to examine the role of BD and Pg-LPS endotoxemia in AS. Furthermore, macrophages are to be cultured in vitro, and stimulated by Pg-LPS and BD. Molecular biological techniques, such as real time PCR, western blot and EMSA, are to be used to explore the influence and mechanism of Pg-LPS and BD in inflammatory response in macrophages.Overall,this study aims to provide theoretical and experimental evidence to unravel the correlation between periodontal disease and AS, their molecular mechanism, and to select optimal treatment and prevention method as well.

前期研究发现：Pg-LPS可损伤主动脉内皮细胞，致其发生炎症反应，促进单核/巨噬细胞吞噬脂质形成泡沫细胞，促进动脉粥样硬化（AS）相关基因的表达。β防御素（BD）具有抗菌活性，能调节机体的炎症免疫反应。AS的本质是炎性反应，因此我们推测：BD能抑制TLR- NF-κB- 促炎细胞因子信号通路，阻断Pg-LPS促AS的形成。本项目通过ApoE-/-小鼠建立Pg内毒素血症模型，并应用BD，从脂质代谢、斑块形成、血管壁炎症反应、Toll样受体表达、NF-κB信号通路激活等多个角度，分析Pg内毒素血症促AS作用和BD的阻断作用及其机制；通过体外细胞实验，对巨噬细胞给予Pg-LPS刺激和BD干预，应用实时定量PCR、免疫印迹、EMSA等技术，深入分析Pg-LPS和BD对巨噬细胞炎症反应的影响和分子学机制。以期深入了解牙周病与AS的相互关系和分子学机制，为选择适宜的治疗和预防手段提供理论和实验依据。

前期研究发现，牙龈卟啉单胞菌内毒素（Porphyromonas gingivalis lipopolysaccharide, Pg-LPS）是一种致炎性物质，能促进单核/巨噬细胞吞噬脂质形成泡沫细胞，促进动脉粥样硬化（atherosclerosis, AS）相关基因的表达。人β防御素3（human β-defensin 3, hBD3）是一种天然存在的抗菌多肽，被证明具有抗菌、抗病毒、免疫调节等多种生物学活性.因此，本研究的目的是探究hBD3在Pg-LPS内毒素血症产生的炎症反应和促AS病变中的作用；研究hBD3在Pg-LPS促巨噬细胞炎症反应中的作用。本课题首先构建了ApoE-/-小鼠Pg-LPS内毒素血症模型，并使用hBD3进行干预。此外，本课题还探究了hBD3在Pg-LPS刺激的巨噬细胞炎症反应中的作用，从脂质代谢、斑块形成、血管壁炎症作用、MAPK信号通路等多个角度验证hBD3的生物学功能。实验结果表明：hBD3体内应用能显著降低Pg-LPS刺激下ApoE-/-小鼠炎症水平；此外，hBD3显著改善了Pg-LPS内毒素血症促动脉粥样硬化病变的形成，表现为内膜下巨噬细胞浸润减少，血管壁内-中膜厚度降低，以及主动脉根部和主动脉树斑块形成减少；体外细胞实验中，hBD3能显著减少Pg-LPS刺激下小鼠巨噬细胞RAW264.7和人单核细胞THP-1分泌炎症因子；hBD3显著抑制Pg-LPS刺激下RAW 264.7细胞一些炎症基因的表达上调；hBD3能显著抑制炎症状态下血管内皮细胞的活化；在一个实验性牙周炎模型中，hBD3牙周袋内应用能显著抑制牙槽骨的吸收和牙周组织中炎症因子的表达。