纳米银颗粒与Ebselen对牙周致病菌的协同杀菌作用及机制研究

Bacterial infection is a pivotal factor in the development of periodontal diseases. Bacteria can exist in the biofilms and even invade into gingival epithelial cells, where they can proliferate and directly spread intercellularly. Despite antibiotics are the most popular antibacterial drugs, their tendency to develop antibiotic resistance raises lots of attention. On the contrary, silver nanoparticles (AgNPs), a chemical antimicrobial agent, are widely explored due to their superior antibacterial properties and less tendency to form a resistance. Nevertheless, AgNPs have a limited therapeutic window due to their cytotoxicity related to their increase of reactive oxygen species. Ebselen is an antioxidant and anti-inflammatory drug. It can serve as a substrate of mammalian cellular thioredoxin reductase (TrxR), which is the key enzyme of the major redox pathway named thioredoxin (Trx) system while it can function as a competitive inhibitor of bacterial TrxR. Therefore, the combination of AgNPs with Ebselen may contribute to the enlargement of the therapeutic window of AgNPs and synergistically kill periodontal pathogens both extracellularly and intracellularly. Based on my previous studies about the antibacterial properties of AgNPs, the current project aimed to explore both in vitro and in vivo 1)whether the combination of AgNPs and Ebselen can have a synergistic bactericidal effect on periodontal pathogens, 2) whether the combination of AgNPs and Ebselen can enlarge the therapeutic window of AgNPs, 3) whether the combination of AgNPs and Ebselen can effectively kill the intracellular periodontal bacteria, and to elucidate the molecular mechanisms by analyzing the Trx system and glutathione (Grx) system. This project may provide a novel therapeutic strategy to improve the clinical outcome of patients.

细菌在牙周病发生发展中起重要作用，既可存在于菌斑微生物膜，又可侵入细胞在胞内增殖乃至细胞间直接传播。目前最主流的抗菌物质抗生素存在抗生素抵抗的风险。纳米银颗粒(AgNPs)以其优良抗菌性能与不易产生抵抗而获得广泛研究。然而，AgNPs易引起细胞过氧化而导致细胞毒性，使其抗菌浓度窗口有限。Ebselen是一种抗氧化药物，是细胞氧化还原系统关键酶TrxR的合成底物，却是细菌TrxR的竞争抑制剂。Ebselen与AgNPs联用有望通过拮抗AgNPs引起的过氧化而增加其治疗浓度窗口，同时又可协同对牙周致病菌直接抗菌乃至胞内抗菌。本课题基于前期对AgNPs抗菌性能的研究，进一步联合应用Ebselen，拟通过体外内实验探讨两者联用对牙周致病菌的协同杀菌作用、对细胞毒性的影响及对胞内细菌的杀菌效能，并通过氧化还原通路Trx系统和Grx系统来探索其作用机制，从而为临床改善牙周病的治疗效果提供新治疗策略。

牙龈卟啉单胞菌（Porphyromonas gingivalis，P. gingivalis）是牙周炎发生发展的关键致病菌，多以菌斑生物膜的形式存在，也可侵入细胞进行免疫逃逸致使许多抗生素无法发挥有效抗菌作用，开发对其具有高效抗菌效果的新策略对牙周炎的预防和治疗具有重要意义。本项目以此作为切入点，在构建新型牙周抗菌策略、促进牙槽骨修复再生方面做出贡献。在本项目的资助下，课题组在International Journal of Biological Macromolecules, Bioactive Materials, Journal of Leukocyte Biology，ACS Omega, ACS Applied Materials & Interfaces等杂志上发表论文12篇，均标明NSFC资助号81901009。在本项目的资助下，获得山东大学优秀博士后二等奖、山东医学科技奖一等奖等6项奖项，获批发明专利2项，培养了齐鲁卫生与健康杰出青年人才一名、泰山学者特聘专家1名，并且辅助培养多名研究生，项目组成员多次参加国内外生物材料和口腔相关学术会议汇报成果，获得同行专家一致认可与好评，主要内容如下：.1、纳米银颗粒（silver nanoparticles, AgNPs）与Ebselen联合应用可对浮游态、生物膜态及胞内感染的P. gingivalis发挥协同抗菌作用，该组合可以降低AgNPs的治疗阈值浓度，并使其能够对抗免疫逃逸细菌。.2、AgNPs与Ebselen联合应用无明显细胞毒性，Ebselen还可作为抗氧化剂明显改善AgNPs导致的氧化应激，从而减轻细胞氧化损伤。.3、AgNPs与Ebselen联合应用可有效抑制P. gingivalis诱导的细胞炎性因子表达，这可能是协同抗菌作用、抗氧化作用、自噬水平增加的综合结果。.4、AgNPs与Ebselen联合应用可有效改善P. gingivalis诱导的大鼠牙槽骨破坏，降低局部促炎因子的表达。.5、AgNPs与Ebselen联合应用有望成为一种有效应对P. gingivalis感染的抗菌治疗新策略，从而为牙周炎治疗提供新策略。