Institutional Repository of Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences
|Place of Conferral||中国科学院海洋研究所|
|Keyword||沙海蜇毒素 蜇伤机理 协同效应 毒素差异 金属蛋白酶|
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.
|MOST Discipline Catalogue||海洋科学|
|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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