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沙海蜇毒素的致炎机理及酶成分的协同效应研究
于春林
Subtype博士
Thesis Advisor于华华
2022-05-10
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Degree Name理学博士
Degree Discipline海洋生物学
Keyword沙海蜇毒素 蜇伤机理 协同效应 毒素差异 金属蛋白酶
Abstract

水母蜇伤是全球范围沿海地区的公共健康问题。目前还没有水母蜇伤的专用药物大多采用验方、土法或参考其它皮炎的治疗方法,效果不理想,甚至加重病情。水母毒素是蜇伤的物质基础,毒素成分的蜇伤机理、协同效应以及毒素活性差异等制约着水母蜇伤专用药物的研制。毒素的致病机理研究能够揭示毒素成分、活性和疾病之间的联系,是指导治疗研究的重要前期基础;毒素成分的协同效应使毒素具有少量、高效的特点,并且影响毒素的毒性;水母毒素活性差异是导致蜇伤症状和治疗效果差异的重要原因。本论文以沙海蜇为研究对象,研究毒素的致炎机理、协同效应及不同海域沙海蜇毒素活性的差异,为水母蜇伤药物的研制提供理论依据。

沙海蜇是、韩中国海域常见的大型暴发水母,也是造成蜇伤事件的主要水母。金属蛋白酶和磷脂酶A2沙海蜇毒素的主要成分,研究表明它们是重要的蜇伤因子,但是尚未明确其蜇伤机理。本论文以沙海蜇毒素为研究对象,通过蛋白质组学和代谢组学分析了毒素的致炎机理;通过硫酸铵沉淀-离子柱层析-分子筛层析对毒素中的金属蛋白酶成分进行分离,分析了金属蛋白酶活性的协同效应;通过对比不同毒素的成分和酶活性分析了不同海域毒素活性的差异。本论文取得的主要研究成果如下:

通过皮下注射建立皮炎小鼠模型,利用皮炎小鼠与正常小鼠对照分析毒素导致皮炎的分子机制。皮肤组织蛋白质组分析发现毒素使组织中多种蛋白含量下调,包括黏附分子、通道蛋白、磷脂酰基转移酶等。关键差异蛋白的调控趋势在抑制毒素金属蛋白酶活性后减弱。结合关键差异蛋白的生物学功能发现毒素通过破坏细胞黏附、破坏渗透平衡、影响正常的磷脂合成造成了局部组织的急性炎症,并且阻碍机体的免疫应答和组织修复。血清的代谢组分析发现毒素处理使机体代谢水平下降,血液环境中多种炎性脂类代谢物含量下调,包括花生四烯酸和多种前体代谢物。这表明血液环境处于抑制花生四烯酸合成的抗炎状态。ELISA分析发现PI3K/Akt信号通路参与局部组织的促炎调控,NF-κB p65信号通路参与血液环境的抗炎调控。该部分研究结果表明金属蛋白酶成分在毒素引起皮炎中发挥重要作用。

通过层析技术分离沙海蜇毒素的金属蛋白酶成分,分析了金属蛋白酶活性的协同效应。金属蛋白酶活性在最后一步层析后失活,部分无金属蛋白酶活性的组分可通过适当混合恢复酶活性。LC-MS/MS分析发现协同组分中含有蛇毒P-1型金属蛋白酶和P-III型金属蛋白酶的同源序列。解离抑制因子和结合调节亚基两种蛇毒金属蛋白酶调控机制也可能存在于沙海蜇毒素的金属蛋白酶中。该部分研究结果表明沙海蜇毒素含有多种金属蛋白酶,且其中存在协同调控作用。

利用黄海海域采集的31份沙海蜇样本提取毒素和检测酶活性,明确了不同毒素样本的金属蛋白酶活性和磷脂酶A2活性的显著性差异以及变化趋势。不同毒素的SDS-PAGE条带及酶谱条带主要呈现强度差异,表明毒素的酶活性差异可能与酶成分的含量差异有关。毒素对草鱼和卤虫的致死性与金属蛋白酶活性相关。酶活性的等值线图显示金属蛋白酶活性表现为近岸水域活性偏低,磷脂酶A2表现为黄海中部活性偏低。结合沙海蜇相关研究文献,推测种群丰度高时毒素具有更高的磷脂酶A2活性,食物丰富高时毒素的金属蛋白酶活性偏低。

本论文以沙海蜇毒素为研究对象,揭示了毒素对皮肤的损伤模式和血液环境的代谢调控;发现了毒素金属蛋白酶活性的协同调控现象;明确了黄海海域中不同毒素的酶活性差异和变化趋势。本论文为沙海蜇蜇伤及治疗方法的研究奠定了基础。

Other Abstract

Jellyfish sting is a public health problem in coastal areas worldwide. At present, there is no special drug for jellyfish stings. Most of therapies use local methods or reference to other dermatitis treatment methods. The effect is not ideal, or even aggravate the condition. Toxin is the material basis of jellyfish stings. Problem that restricted the therapy research of jellyfish stings included in the pathogenesis, synergistic effect, and activity differences of jellyfish toxin. Researches on pathogenesis are important basis that could guide the therapy studies by revealing the relationship between toxin compositions, activities and symptoms. Synergistic effect of toxin components makes toxin have high efficiency with small amount and affects the toxicity. Toxin difference is an important reason for the symptom and therapeutic effect difference in sting cases. In this thesis, pathogenesis, synergistic effect and toxin differences in different waters of jellyfish Nemopilema nomurai toxin were studied, provided theoretical basis for the development of jellyfish sting drugs.

Giant jellyfish Nemopilema nomurai is the main species causing jellyfish bloom and stings in Japan, Korea and China Waters. The enzymes, including Metalloproteinase and phospholipase A2, are main components of Nemopilema nomura toxin. Studies have shown that they were important sting factors. But their sting mechanism has not been clarified. In this thesis, Nemopilema nomura toxin was studied. The inflammatory mechanism was analyzed by proteomics and metabolomics. The toxin metalloproteinase components were isolated by “ammonium sulfate precipitation - ion exchange chromatography - molecular sieve chromatography to analyse the synergistic effect. The toxin differences in different waters were analyzed by comparing enzyme activities of different toxin samples. The main research results of this thesis were as follows:

Dermatitis mouse model was established by subcutaneous injection of venom samples, and the inflammatory mechanism was analyzed by comparing dermatitis mouse with normal mouse. Proteomic analysis of skin tissue showed that the venom down-regulated several proteins, including adhesion molecules, channel proteins and lysophospholipid acyltransferase. The down-regulation trend of key differential proteins weakened after inhibiting the metalloproteinase activity of venom. Combined with the biological functions of key differential proteins, we found that the venom caused acute inflammation in local tissues by destroying the cell adhesion, disrupting the osmotic balance, affecting the synthesis of phospholipids, and obstructing the immune response and tissue repair. Metabolomics analysis of serum showed that venom treatment decreased metabolism level, and many inflammatory lipid metabolites in blood were down-regulated, including arachidonic acid and its precursor metabolites. It indicated that the blood environment of dermatitis mouse was in an anti-inflammation state that inhibited arachidonic acid synthesis. ELISA analysis showed that PI3K/Akt signaling pathway was involved in pro-inflammation regulation of stung tissues, and NF-κB p65 signaling pathway was involved in anti-inflammation regulation of blood environment. These results indicated that metalloproteinases play an important role in Nemopilema nomurai venom-induced dermatitis.

The Nemopilema nomurai venom metalloproteinase components were isolated by chromatography to analyse the synergistic effect. The metalloproteinase activity was inactivated after the last purification step, while it could be restored by mixing some of fractions. LC-MS/MS analysis found that the synergistic fractions contained homologous sequences of P-I and P-III snake venom metalloproteinase. The regulation mechanisms of snake venom metalloproteinase, dissociation inhibitor and binding subunit, may also exist in Nemopilema nomurai venom metalloproteinase. The results showed that Nemopilema nomurai venom contained multiple kinds of metalloproteinases, and there was synergistic regulation effect between them.

The significant differences and variation trend in the activities of metalloproteinase and phospholipase A2 were confirmed by extracted toxin and tested enzyme activities of 31 Nemopilema nomura samples which collected from different area in the Yellow Sea. The electrophoretic bands in SDS-PAGE and zymography of different venom samples were different in intensity, which indicated that the enzyme activity variation might be related to the difference of enzyme content. The lethal toxicity to artemia and grass carp was related to metalloproteinase activity. The counter map of enzyme activities showed that the metalloproteinase activity was lower in the coastal waters, and the phospholipase A2 activity was lower in the middle of the Yellow Sea. Combined with Nemopilema nomura related research literature, it was speculated that the venom had higher phospholipase A2 activity when the population abundance was higher, and the venom had lower metalloproteinase activity when the food supply was more abundant. These results provided a theoretical basis for the selection and preparation of venom samples for subsequent experiments.

Giant jellyfish Nemopilema nomurai venom was selected as the subject in this thesis. The mode of skin damage and blood metabolic regulation induced by venom were revealed. The synergistic regulation of metalloproteinase activity was identified . Enzyme activities difference and variation trend of different toxin samples in the Yellow Sea were clarified. This thesis lays the foundation for the Nemopilema nomurai sting and therapy studies.

Subject Area海洋生物学
MOST Discipline Catalogue海洋科学
Pages130
Language中文
Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/178285
Collection实验海洋生物学重点实验室
Recommended Citation
GB/T 7714
于春林. 沙海蜇毒素的致炎机理及酶成分的协同效应研究[D]. 中国科学院海洋研究所. 中国科学院大学,2022.
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