甘草查尔酮A调节中性粒细胞胞外陷阱清除金黄色葡萄球菌的作用机制研究

Ideally anti-infectious strategies should disarm weapon of bacteria invasion directly and render them more susceptible to host innate immune defenses. Neutrophil extracellular traps (NETs), as a mechanism of innate immune defense,are meshwork of extracellular DNA fibers mixed with their granular components in the extracellular space, which capture and kill exogenous microbes. It has been shown that the expression and secretion of staphylococcal nucleases by bacterial species allows bacteria to evade killing by the innate immune system through degradation of NETs. Our study found that Licochalcone A (LAA) as the main composition of licorice strengthens NETs production induced by Staphylococcus aureus (S. aureus) and extracellular S.aureus killing by NETs, and inhibits the nucleases expression of standard strain. So, whether the role of LAA in innate immune defense against infection associated with nuclease inhibited by LAA, need to be researched. This study will evaluate the expression of staphylococcal nucleases inhibited by LAA from the transcription and protein levels of nucleases. Next, we will establish the NETs model induced by S. aureus wild strain or S. aureus nuc1-mutant strain in vitro, using scanning electron microscope, immunofluorescence assay to evaluate the NETs, component of NETs (granular components and antibacterial peptides) in the extracellular space and killing of S. aureus mediated by NETs with LAA treatment. Further, we will establish the murine pneumonia model induced by S. aureus wild strain or S. aureus nuc1-mutant strain in vivo, to investigate the NETs, granular components, antibacterial peptides and bacterial load in lung tissue in lungs and bronchalveolar lavage fluid of infested mice with LAA treatment. The study will clarify the mechanism of LAA to regulate S.aureus clearance mediated by NETs, providing new strategy way in the treatment of bacterial infections as an immune enhancer.

理想的抗感染策略是抗菌药直接解除细菌的侵袭武器，并增强宿主天然免疫防卫对细菌的敏感性。中性粒细胞胞外陷阱（NETs）是由胞外DNA和颗粒蛋白组成的网状结构，能捕杀细菌。金黄色葡萄球菌（金葡菌）核酸酶能使金葡菌逃避NETs的捕杀。我们发现甘草查尔酮A（LAA）能增强NETs清除胞外金葡菌，且抑制金葡菌标准株核酸酶nuc1的表达。LAA在天然免疫抗金葡菌感染中的作用可能与抑制核酸酶有关。基于此，本课题从转录水平及蛋白活性方面考察LAA对核酸酶的影响；以金葡菌野生株及nuc1缺失株诱发NETs为模型，用扫描电镜、免疫荧光等技术检测LAA对NETs形成、组份（颗粒蛋白、抗菌肽）及其清除胞外菌能力的影响；构建小鼠肺炎模型，检测LAA对肺脏和肺泡灌洗液中NETs、颗粒蛋白、抗菌肽及肺脏载菌率的影响，最终阐明LAA调节NETs清除金葡菌的作用机制，为其作为免疫增强剂在抗细菌感染的治疗方面提供新思路。

耐甲氧西林金黄色葡萄球菌是侵袭性肺部感染的重要原因，极易造成肺损伤。金黄色葡萄球菌（SA）产生的多种毒力因子（如SPA、Sbi、脱氧核糖核酸酶等）有助于菌体侵袭宿主后在宿主中有效定植并逃避宿主的天然免疫防卫。在SA感染中，调节胞外陷阱(ET)形成和抑制NLRP3炎性小体激活已成为一种潜在的抗菌和抗感染策略。甘草的药理作用广且毒副作用小，主要成分为甘草查耳酮A（LAA），是极具开发潜力的小分子药物。本项目构建SA感染的细胞和动物模型，系统研究LAA调节METs清除SA和抗感染的作用机制。.结果显示：.1)LAA对临床分离株有很强的抑制作用，而这些菌株对常用抗生素却有不同程度的耐药，因此，LAA是非常有价值的抗SA感染的候选药物。.2)LAA能直接与SA核酸酶结合抑制核酸酶活性，增强METs的产生及胞外SA的杀伤活性，提示LAA可能通过抑制SA核酸酶的活性增强METs对SA的捕杀。.3)LAA降低SA肺炎小鼠的病理学损伤及肺组织的载菌数和肺泡灌洗液中总细胞数和中性粒细胞数，及小鼠血清和肺泡灌洗液中炎症因子IL-1β、IL-6和TNF-α的水平，起到保护SA肺炎的作用，这与LAA抑制NLRP3炎性小体、MAPK及NF-κB信号通路有关。.4)SPA和Sbi可以诱导巨噬细胞凋亡，激活巨噬细胞的NF-κB和NLRP3炎性小体信号通路，并导致NLRP3依赖的细胞焦亡，SPA激活的NLRP3炎性小体的活化还需要K+外流。SPA可以激活巨噬细胞的JNK和p38MAPK信号通路，诱导自噬发生，并且自噬调控NLRP3炎性小体依赖的细胞焦亡。.5)LAA抑制SPA和Sbi介导的NLRP3炎性小体的活化，及Sbi介导的NF-KB信号通路活化和细胞凋亡。.本项目从SA毒力因子出发，研究以核酸酶、SPA和Sbi为靶标的小分子药物的作用机制，为LAA作为免疫增强剂抗SA感染的治疗方向提供新思路。