Potential role of an antimicrobial peptide, KLK in inhibiting lipopolysaccharide-induced macrophage inflammation

Antimicrobial peptides (AMPs) are attractive alternatives to antibiotics. Due to their immune modulatory properties, AMPs are at present emerging as promising agents for controlling inflammatory-mediated diseases. In this study, anti-inflammatory potential of an antimicrobial peptide, KLK (KLKLLLLLKLK) and its analogs was evaluated in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. The results herein demonstrated that KLK peptide as well as its analogs significantly inhibited the pro-inflammatory mediator nitric oxide (NO), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) production in LPS-stimulated RAW 264.7 macrophages in dose-dependent manners, and such inhibitory effects were not due to direct cytotoxicity. When considering inhibition potency, KLK among the test peptides exhibited the most effective activity. The inhibitory activity of KLK peptide also extended to include suppression of LPS-induced production of prostaglandin E2 (PGE2). KLK significantly decreased mRNA and protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) as well as mRNA expression of IL-1β and TNF-α. Moreover, KLK inhibited nuclear translocation of nuclear factor-κB (NF-κB) p65 and blocked degradation and phosphorylation of inhibitor of κB (IκB). Taken together, these results suggested that the KLK peptide inhibited inflammatory response through the down-regulation of NF-κB mediated activation in macrophages. Since peptide analogs with different amino acid sequences and arrangement were investigated for their anti-inflammatory activities, the residues/structures required for activity were also discussed. Our findings therefore proved anti-inflammatory potential of the KLK peptide and provide direct evidence for therapeutic application of KLK as a novel anti-inflammatory agent.抗菌肽（Antimicrobial peptides，AMPs）是极具潜力的抗生素替代物。因其具备免疫调节特性，当前抗菌肽正逐渐成为治疗炎症介导类疾病的候选治疗制剂。本研究针对抗菌肽KLK（KLKLLLLLKLK）及其类似物的抗炎活性，在脂多糖（Lipopolysaccharide，LPS）诱导的RAW 264.7巨噬细胞模型中开展了评估。实验结果显示，KLK肽及其类似物均可通过剂量依赖性方式，显著抑制脂多糖活化的RAW 264.7巨噬细胞中促炎介质一氧化氮（Nitric oxide，NO）、白细胞介素-1β（Interleukin-1β，IL-1β）与肿瘤坏死因子-α（Tumor necrosis factor-α，TNF-α）的生成，且该抑制作用并非由直接细胞毒性所介导。在抑制效能方面，受试肽段中KLK展现出最优的抗炎活性。KLK肽的抗炎活性还可覆盖脂多糖诱导的前列腺素E2（Prostaglandin E2，PGE2）生成的抑制作用。KLK可显著下调诱导型一氧化氮合酶（Inducible nitric oxide synthase，iNOS）与环氧合酶-2（Cyclooxygenase-2，COX-2）的mRNA及蛋白表达水平，同时也能降低IL-1β与TNF-α的mRNA表达量。此外，KLK可抑制核因子κB（Nuclear factor-κB，NF-κB）p65的核转位，并阻断κB抑制蛋白（Inhibitor of κB，IκB）的降解与磷酸化。综上，上述结果表明KLK肽可通过下调巨噬细胞中核因子κB介导的信号通路活化，从而抑制炎症反应。由于本研究针对不同氨基酸序列与排布的肽类似物的抗炎活性进行了探究，因此本研究还讨论了维持该活性所必需的残基与结构。本研究结果证实了KLK肽的抗炎潜力，并为KLK作为新型抗炎制剂的治疗应用提供了直接实验依据。