基于人α-防御素5改造的新型纳米抗生素通过维持肠道微生态改善脓毒症预后的作用与机制研究

Intestinal microecology plays an important role in combating pathogen infections. While it is disrupted in septic patients who undergo long-term antibiotic treatment and as a result, it is related to secondary infection and increased morbidity. Human alpha-defensin 5 (HD5), a member of cationic antimicrobial peptides, tends to target the more negative-charged pathogens when compared with less negative-charged commensal bacteria. As a result, HD5 selectively kills pathogens. While it is easy to degradation with diverse enzymes and its killing activity is relatively weak. In view of the shortcomings of HD5, our preliminary studies have myristoylated HD5 into a micelle named HD5-myr based on nanotechnology and found that: ① the killing activities against frequently-isolated pathogens of sepsis such as E. coli, S. aureus were enhanced while the killing activities against commensal bacteria such as B. thetaiotaomicron were negligible; ② the therapeutic effect against septic mice was improved; ③ resistance to secondary infection was improved when compared with traditional antibiotics; ④ the stability was significantly improved. Therefore, we hypothesize that HD5-myr will protect intestinal microecology by selectively killing pathogens. As a result, it will protect body from secondary infection and then improve the prognosis of sepsis. The present project will, from systemic, bacteria and molecular levels, investigate the action and mechanism of how HD5-myr improve prognosis of sepsis via maintaining intestinal microecology. Ultimately, our project will provide a candidate in developing new drugs for treating sepsis targeting intestinal microecology.

肠道微生态在抵抗病原体感染中发挥重要作用。脓毒症患者长期应用抗生素破坏了肠道微生态，易继发二次感染，增加死亡率。阳离子抗菌肽人α-防御素5（HD5）更易结合带负电荷更多的致病菌，而对负电荷较少的共生菌结合能力弱，从而选择性杀灭致病菌。但易被酶降解、杀菌效果差。针对其缺点，我们前期利用纳米技术肉豆蔻酰化HD5成为胶束HD5-myr后发现①对脓毒症常见的金黄色葡萄球菌、大肠杆菌等致病菌的杀灭能力显著增强，对多形拟杆菌等肠道共生菌影响小 ②显著改善脓毒症小鼠预后 ③与传统抗生素对比，增强小鼠抵抗二次感染能力 ④稳定性显著提高。基于上述数据我们提出科学假说：HD5-myr通过选择性杀灭致病菌保护肠道微生态，抵抗病原菌再次感染，进而改善脓毒症预后。为此我们将从整体、细菌、分子水平分析HD5-myr通过维持肠道微生态改善脓毒症的作用及机制。最终为研发靶向肠道微生态改善脓毒症预后的药物提供参考。

随着全球抗生素耐药问题的日益严峻，革兰氏阴性菌感染导致的脓毒症已成为全球的主要医疗负担之一。但是新抗生素开发相对滞后。抗生素在杀灭革兰氏阴性菌的同时，可能促进其LPS释放，导致炎症反应加重。LL-37是唯一一种在人体中发现的Cathelicidin，具有多种生物活性，包括直接杀菌作用、调节炎症和中和LPS。KR-12是LL-37中最小的活性片段，具有杀灭细菌和中和LPS的作用。在本研究中，我们将KR-12进行纳米化修饰，研究其杀菌活性及中和LPS的特性，我们合成了KR-12的两种肉豆蔻酰化衍生物KR-121和KR-122，当与去离子水混合时，它们可以自组装形成纳米颗粒。我们发现肉豆蔻酰化KR-12衍生物通过在细菌细胞膜表面“打孔”的方式杀灭细菌。这种独特的杀菌方式使细菌不容产生耐药性，且对耐药细菌的依然保持较强的杀菌作用。在腹腔注射大肠杆菌的脓毒症小鼠模型上，KR-121能显著改善脓毒症小鼠的生存率，改善脓毒症小鼠肝脏、肾脏、脾脏、肺脏、血液及腹腔灌洗液的细菌负荷及脏器损伤，其治疗效果优于临床常用的强效抗生素美罗培南。我们还证明，肉豆蔻酰化的KR-12纳米抗生素可以在体外与LPS结合并抑制炎症反应。在LPS诱导的脓毒症小鼠模型中，KR-121可以显著改善该模型脓毒症小鼠的生存率，且其治疗效果优于临床常用的多粘菌素B。毒性实验表明，KR-121和KR-122纳米抗生素的溶血毒性低，且肝肾毒性低。因此，我们开发了一种具有杀菌和LPS中和双重作用的新型纳米抗生素，这可能为抗菌肽的临床转化和新抗生素的研发提供参考。