Laboratório de Fisiopatologia Experimental, Programa de Pós Graduação Em Ciências da Saúde, Universidade Do Extremo Sul CatarinenseCriciúma, Santa Catarina, Brazil.
Programa de Pós-Graduação Em Ciências da Saúde: Infectologia E Medicina Tropical, Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, 30130-100, Brazil.
Immunol Res. 2024 Aug;72(4):788-796. doi: 10.1007/s12026-024-09485-6. Epub 2024 May 2.
The pathological manifestation of the inflammatory process primarily stems from the heightened release of pro-inflammatory cytokines, with IL-1β standing out as a pivotal cytokine. The excessive presence of IL-1β disrupts immune signaling, thereby assuming a pathogenic and exacerbating role in the pathophysiology of numerous inflammatory diseases. Regulating IL-1β levels becomes crucial, and the IL-1Ra molecule serves this purpose by binding to the IL-1R1 receptor, thereby impeding the binding of IL-1β. Several pharmaceuticals have entered the market, aiming to neutralize IL-1β's biological function through diverse mechanisms. However, the existing IL-1β inhibitors are recombinant proteins, characterized by a high production cost and limited stability. Therefore, this study aimed to predict a peptide, named DAP1-2, based on the IL-1Ra molecule. DAP1-2 was designed to attenuate responses triggered by IL-1β by blocking the IL-1R1 receptor. The selection of amino acids from the IL-1Ra molecule (PDB: I1RA) that interact with the three domains of the IL-1R1 receptor was performed using Swiss PDB Viewer. After prediction, chemical synthesis was made using the Fmoc-Synthesis technique. The efficacy of DAP1-2 was assessed using RAW 264.7 cells, which were exposed to LPS (5 μg/mL) for 24 h to induce IL-1β expression and treated with the peptides in different concentrations. IL-1β levels were assessed using ELISA, and the gene expression of IL-1β was measured by RT-qPCR, additionally to the viability test. Results revealed a significant reduction in IL-1β levels and gene expression in cells stimulated by LPS and treated with DAP1-2 in different concentrations. Furthermore, the MTT assay confirmed the nontoxic nature of the peptides on the cell lineage. This alternative approach shows promise as an IL-1 inhibitor, due to the stability, ease of production, and cost-effectiveness provided by the use of synthetic peptides.
炎症过程的病理表现主要源于促炎细胞因子的过度释放，其中白细胞介素-1β（IL-1β）尤为突出，是一种关键的细胞因子。IL-1β 的过度存在破坏了免疫信号，因此在许多炎症性疾病的病理生理学中具有致病和加重作用。调节 IL-1β 水平至关重要，IL-1Ra 分子通过与 IL-1R1 受体结合来实现这一目的，从而阻止 IL-1β 的结合。已有几种药物进入市场，旨在通过多种机制中和 IL-1β 的生物学功能。然而，现有的 IL-1β 抑制剂是重组蛋白，生产成本高且稳定性有限。因此，本研究旨在基于 IL-1Ra 分子预测一种名为 DAP1-2 的肽。DAP1-2 设计用于通过阻断 IL-1R1 受体来减弱由 IL-1β 触发的反应。使用 Swiss PDB Viewer 从与 IL-1R1 受体的三个结构域相互作用的 IL-1Ra 分子（PDB：I1RA）中选择氨基酸。经过预测，使用 Fmoc 合成技术进行化学合成。使用 RAW 264.7 细胞评估 DAP1-2 的功效，将细胞用 LPS（5μg/mL）孵育 24 小时以诱导 IL-1β 表达，并在不同浓度下用肽处理。使用 ELISA 评估 IL-1β 水平，并通过 RT-qPCR 测量 IL-1β 的基因表达，此外还进行了细胞活力测试。结果表明，用不同浓度的 DAP1-2 处理 LPS 刺激的细胞后，IL-1β 水平和基因表达均显著降低。此外，MTT 测定证实了肽对细胞系的非毒性。由于使用合成肽提供了稳定性、易于生产和成本效益，因此这种替代方法作为 IL-1 抑制剂具有很大的前景。
