Inflammatory reactions activated by lipopolysaccharide (LPS) of gram-negative bacterium can lead to severe septic shocks. With recent emergence of multidrug-resistant gram-negative bacteria as well as lack of efficient ways to treat resulting infections, there is a need to develop novel anti-endotoxin agents. Antimicrobial peptides have been noticed as potential therapeutic molecules for bacterial infection and as candidates for new antibiotic drugs. We had previously designed the 9-meric antimicrobial peptide Pro9-3 and it showed high antimicrobial activity against gram-negative bacteria. To further examine its potency as an anti-endotoxin agent, in this study, we examined its anti-endotoxin activities and elucidated its mechanism of action. We performed a dye-leakage experiment, BODIPY-TR cadaverine assay, and limulus amebocyte lysate assay for Pro9-3, and its lysine-substituted analogue and their enantiomers. The results confirmed that Pro9-3 targets the bacterial membrane and the arginine residues play key roles in its antimicrobial activity. Pro9-3 showed excellent LPS-neutralizing activity and LPS-binding properties, which were superior to those of other peptides. Saturation transfer difference-nuclear magnetic resonance experiments to explore the interaction between LPS and Pro9-3 revealed that Trp² and Trp⁸ in Pro9-3 are critical for attracting Pro9-3 to the LPS in the gram-negative bacterial membrane. Moreover, anti-septic effect of Pro9-3 in vivo was investigated using an LPS-induced endotoxemia mouse model, demonstrating its dual activities: antibacterial activity against gram-negative bacteria and immunosuppressive effect to prevent LPS-induced endotoxemia. Collectively, these results confirmed the therapeutic potential of Pro9-3 against infection of gram-negative bacteria.

中文翻译：

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具有治疗内毒素血症治疗潜力的抗内毒素 9 聚体肽。

革兰氏阴性菌脂多糖 (LPS) 激活的炎症反应可导致严重的感染性休克。随着最近出现的多重耐药革兰氏阴性菌以及缺乏治疗由此产生的感染的有效方法，需要开发新型抗内毒素药物。抗菌肽已被注意到作为细菌感染的潜在治疗分子和新抗生素药物的候选者。我们之前设计了 9 聚体抗菌肽 Pro9-3，它对革兰氏阴性菌表现出高抗菌活性。为了进一步检查其作为抗内毒素剂的效力，在本研究中，我们检查了其抗内毒素活性并阐明了其作用机制。我们进行了染料渗漏实验，BODIPY-TR 尸胺测定，Pro9-3 及其赖氨酸取代类似物及其对映异构体的鲎变形细胞裂解物测定。结果证实 Pro9-3 靶向细菌膜，精氨酸残基在其抗菌活性中起关键作用。Pro9-3 显示出优异的 LPS 中和活性和 LPS 结合特性，优于其他肽。探索 LPS 与 Pro9-3 之间相互作用的饱和转移差异-核磁共振实验表明 Trp^{Pro9-3 中的2}和 Trp ⁸对于将 Pro9-3 吸引到革兰氏阴性细菌膜中的 LPS 至关重要。此外，使用 LPS 诱导的内毒素血症小鼠模型研究了 Pro9-3在体内的抗脓毒症作用，证明了其双重活性：对革兰氏阴性菌的抗菌活性和防止 LPS 诱导的内毒素血症的免疫抑制作用。总的来说，这些结果证实了 Pro9-3 对革兰氏阴性菌感染的治疗潜力。