中国科学院昆明动物研究所机构知识库

研究表明有毒动物及昆虫导致的过敏反应病例中膜翅目昆虫蜜蜂、胡峰及马蜂占绝大多数，这些昆虫的广泛分布及其叮咬导致的严重临床症状使得其研究具有重要的临床意义。中国大虎头蜂（Vespa mandarinia）是世界上最大的胡峰，其毒素会导致溶血、凝血障碍以及横纹肌溶解等由此宏观表现为皮肤瘙痒、剧烈的疼痛以及严重的水肿等临床症状，然而胡峰毒素导致这些症状的具体分子机制还不是特别清楚，在临床治疗中缺乏胡峰叮咬中毒后的诊断标志物以及快速有效的治疗手段，治疗预后不容乐观。细胞膜是构成人类表面生物屏障的重要基础，胡峰毒液导致的严重水肿等症状正是基于对细胞膜的破坏。利用G50分子筛以及反向高效液相色谱我们从胡峰粗毒中纯化到一个细胞膜穿孔活性多肽，我们将其命名为VESCP-M2，利用MALDI-TOF质谱分析以及Edman 降解N端测序我们获得了该多肽分子的分子量及氨基酸序列信息。NCBI比对发现该多肽为首次被发现，其半胱氨酸构型同已报到的细胞因子趋化肽（Chemotactic peptides）一致。基于氨基酸的一级序列信息我们利用 Discovery Studio 3.1模拟了该多肽的结构，并使用膜片钳技术对该多肽的电生理特性进行了研究，内膜向外（Inside-out）膜片钳记录方式下，低浓度毒素的灌流能记录到单通道电流发放，提示该多肽能在膜上聚合为孔道，全细胞（Whole-cell）记录下,高浓度的多肽灌流能记录到巨大的漏电流，能直接观察到细胞被大量穿孔后裂解， 同时，外膜向外（Outside-out）和内膜向外（Inside-out）膜片钳记录方式下VESCP-M2的活性保持一致，提示VESCP-M2在细胞膜上的作用没有膜的方向选择性。接下来我们利用动物试验验证了VESCP-M2的促炎反应，试验结果表明VESCP-M2能引起动物持续且严重的水肿和组织坏死，在坏死组织及水肿液中我们检测到了大量的载脂蛋白A-1以及炎症因子，坏死组织的免疫组化结果观察到了大量的角蛋白产生以及炎症细胞侵润，这些结果提示VESCP-M2能引发强烈的组织损伤和炎症反应。本研究从胡峰毒素中鉴定到了一条膜穿孔多肽毒素VESCP-M2，对该多肽的研究揭示了胡峰毒素造成人严重临床症状如水肿、组织损伤等的分子机制，并为胡峰叮咬提供了临床干预治疗的分子基础。

Specific global statistics have indicated that bees, wasps and hornets, all members of the order Hymenoptera, account for majority of anaphylactic reactions resulting from venomous animals and insects. Hymenopterans are of fundamental clinical significance because of their ubiquitous distribution in nature as well as the vast array of histological and clinical manifestations resulting from their stings. The Asian giant hornet, Vespa mandarinia, is the world’s largest social wasp and stings inflicted by this hornet induce a wide range of local reactions such as transient pain, itching and swelling as well as generalized systemic reactions such as hemolysis, coagulopathy, rhabdomyolysis among others. However, the available knowledge on the mechanism involved in the development of tissue damage resulting from Vespa mandarinia envenomation is inadequate. Understanding the mechanism of Vespa mandarinia envenomation presents an invaluable window in the assessment of specific biomarkers that are vital in providing insight into the mechanisms of tissue damage, prognosis, as well as therapeutic interventions. The cell membrane builds a barrier that prevents all harmful substances from accessing the cell, therefore in this study, the membrane disrupting peptides in high concentration were explored from the hornet venom. By using gel filtration chromatography and reverse phase high-performance liquid chromatography (RP-HPLC), a new membrane disrupting peptide, named here as VESCP-M2 was isolated from crude venom and subsequent peptide characterization carried out using matrix- assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometry and automatic Edman degradation to determine the molecular mass and amino acid sequence respectively. A NCBI protein blast search of the tridecapeptide showed no similarity with other peptides earlier reported. The alignment of the peptide sequence of VESCP-M2 showed some homologues from other vespid venoms which are classified as chemotactic peptides. The structure model of the peptide was determined by the software Discovery Studio 3.1 displayed its positive and negative electrostatic potential. Membrane disrupting activity of the peptide on the cell membrane of different cells was tested using whole-cell electrophysiology whereby a huge leaking current was observed. Outside-out and inside-out recordings demonstrated that the action concentrations were indiscriminating, indicating that the interaction between VESCP-M2 and the cell membrane is not associated with the polarity of cell membrane. These assays demonstrated that VESCP-M2 exhibits a potent and non-selective ability to induce membrane permeation. Using animal model assays, the local inflammatory reactions caused by VESCP-M2 were examined. The response was characterized by intense inflammatory reaction with significant and persistent edema confirmed by the presence of apolipoprotein A-1 in the exudates as well as induction of pain. In addition, phenotypic intense tissue necrosis was observed which was characterized by increased level of keratin in the wound exudates and intense influx of inflammatory cells into the tissue. Increased levels of pro-inflammatory chemokines and cytokines were also observed in the paw tissue homogenates. The study presents the discovery of VESCP-M2 which provides a critical insight into the assessment of the mechanism of tissue damage associated with wasp envenomation. The elucidated underlying molecular mechanism and symptom-related function of VESCP-M2 forms a basis for prognosis as well as therapeutic interventions.