A potent anti-inflammatory peptide from the salivary glands of horsefly.
A potent anti-inflammatory peptide from the salivary glands of horsefly.
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来自马蝇唾液腺的有效抗炎肽
DOI：
10.1186/s13071-015-1149-y
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发表时间：
2015-10-24
影响因子：
3.2
通讯作者：
Xu W
中科院分区：
文献类型：
--
作者：
Wei L;Huang C;Yang H;Li M;Yang J;Qiao X;Mu L;Xiong F;Wu J;Xu W
研究背景瑶虻（Tabanusyao）唾液腺中存在一组生理活性肽/蛋白质，它们促进了其对血餐的获取。然而，它们在局部炎症调节中的作用仍然知之甚少。在细菌饲喂后通过定量PCR（qPCR）分析yao。在脂多糖（LPS）刺激的小鼠腹腔巨噬细胞中选择一个显著上调的分子（天蚕素-TY 1）进行抗炎试验。通过qPCR定量诱导型NO合酶（iNOS）和促炎细胞因子的转录水平。用Griess试剂测定一氧化氮（NO）的生成量。通过酶联免疫吸附测定（ELISA）测定促炎细胞因子的产生。Western blotting分析炎症信号。用圆二色谱法测定了抗菌肽TY 1的二级结构。通过异硫氰酸荧光素（FITC）结合的LPS聚集体的解离以及定量显色终点鲎变形细胞溶解物（TAL）测定试剂盒确定的LPS中和作用来评估抗菌肽-TY 1与LPS的相互作用。通过同源模建结构分析和关键残基/结构的突变来了解其构效关系. ResultsCecropin-TY 1具有较高的抗炎活性和对小鼠巨噬细胞的低细胞毒性。在LPS刺激的小鼠腹腔巨噬细胞中，添加天蚕素-TY 1显著抑制一氧化氮（NO）和促炎细胞因子的产生。进一步的研究表明，天蚕素TY 1通过阻断丝裂原活化蛋白激酶（MAPK）和转录核因子-κB（NF-κB）信号的激活来抑制炎性细胞因子的产生。抗菌肽TY 1甚至与LPS相互作用并中和LPS。二级结构分析表明，抗菌肽TY 1在疏水性环境中呈无序结构，但在模拟膜环境中转变为α-螺旋结构。同源模建结构分析表明，天蚕抗菌肽TY 1采用两个α-螺旋（Leu 3-Thr 24，Ile 27-Leu 38）通过铰链（Leu 25-Pro26）连接，结构表面部分带正电荷。构效关系分析表明，α-螺旋、芳香残基Trp 2、带正电荷残基Lys和Arg、铰链残基Pro26和N-末端酰胺化是影响其抗炎活性的关键残基/结构。为更好地理解马蝇与宿主的外寄生虫-宿主相互作用奠定基础，并突出其在革兰氏阴性细菌感染引起的脓毒症和内毒素休克的抗炎治疗中的效力。
BackgroundA diverse group of physiologically active peptides/proteins are present in the salivary glands of horseflyTabanus yao(Diptera, Tabanidae) that facilitate acquisition of blood meal. However, their roles in the regulation of local inflammation remains poorly understood.MethodsInduction expression profiles of immune-related molecules in the salivary glands ofT. yaowas analyzed by quantitative PCR (qPCR) after bacterial feeding. A significantly up-regulated molecule (cecropin-TY1) was selected for anti-inflammatory assay in lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages. The transcription levels of inducible NO synthase (iNOS) and pro-inflammatory cytokines were quantified by qPCR. Nitric oxide (NO) production was determined by Griess reagent. Pro-inflammatory cytokine production was determined by an enzyme-linked immunosorbent assay (ELISA). The inflammatory signals were assayed by Western blotting analysis. The secondary structure of cecropin-TY1 was measured by Circular dichroism (CD) spectroscopy. Interaction of cecropin-TY1 with LPS was evaluated by the dissociation of fluorescein isothiocyanate (FITC)-conjugated LPS aggregates and neutralization of LPS determined by a quantitative Chromogenic End-point Tachypleus amebocyte lysate (TAL) assay kit. Homology modeled structure analysis and mutation of key residues/structures were performed to understand its structure-activity relationship.ResultsCecropin-TY1 was demonstrated to possess high anti-inflammatory activity and low cytotoxicity toward mouse macrophages. In LPS-stimulated mouse peritoneal macrophage, addition of cecropin-TY1 significantly inhibited the production of nitric oxide (NO) and pro-inflammatory cytokines. Further study revealed that cecropin-TY1 inhibited inflammatory cytokine production by blocking activation of mitogen-activated protein kinases (MAPKs) and transcriptional nuclear factor-κB (NF-κB) signals. Cecropin-TY1 even interacted with LPS and neutralized LPS. The secondary structure analysis revealed that cecropin-TY1 adopted unordered structures in hydrophobic environment but converted to α-helical confirmation in membrane mimetic environments. Homology modeled structure analysis demonstrated that cecropin-TY1 adopted two α-helices (Leu3-Thr24, Ile27-Leu38) linked by a hinge (Leu25-Pro26) and the structure surface was partly positively charged. Structure-activity relationship analysis indicated that several key residues/structures are crucial for its anti-inflammatory activity including α-helices, aromatic residue Trp2, positively charged residues Lys and Arg, hinge residue Pro26 and N-terminal amidation.ConclusionsWe found a novel anti-inflammatory function of horsefly-derived cecropin-TY1 peptide, laying groundwork for better understanding the ectoparasite-host interaction of horsefly with host and highlighting its potency in anti-inflammatory therapy for sepsis and endotoxin shock caused by Gram-negative bacterial infections.