实验海洋生物学重点实验室知识库

水母在世界范围内的频繁暴发，导致水母蜇伤成为最常见的海洋生物伤。美国国立卫生院的数据显示，每年约有1.5亿人被水母蜇伤，对公众健康造成了巨大威胁。水母蜇伤的临床表现从轻微的皮肤损伤、肌肉肿胀，到严重的休克，甚至死亡。水母皮炎是水母蜇伤造成的最直接和最广泛的伤害，通常表现为皮肤红肿，水泡以及瘙痒等局部皮肤症状。其中，疼痛和红肿等症状通常会在蜇伤后的两周内消退。然而，瘙痒持续时间长且难以忍受，几乎伴随整个蜇伤部位的恢复过程，甚至发展成迁延不愈的慢性瘙痒，困扰蜇伤者数十年。由于对水母毒素致痒的潜在机制和神经回路尚不清楚，到目前为止，没有特异性药物能够有效缓解或者治疗水母蜇伤导致的瘙痒症状。Mas相关G蛋白偶联受体（Mrgprs）是一种新型的G蛋白偶联受体，在皮肤瘙痒的发生中起着重要作用。因此，本论文以中国常见的大型有毒钵水母，沙海蜇（Nemopilema nomurai）为研究对象，研究毒素的致痒机制以及探究沙海蜇毒素的炎症刺激作用对痒行为的影响，主要研究结果如下：

1. 沙海蜇毒素为非组胺依赖性致痒：基于动物行为学实验的研究表明，水母毒素有显著的致痒活性，并且在一定浓度范围内，毒素诱导的小鼠抓挠行为呈浓度依赖性增加，50 μg的沙海蜇毒素在30 min内能诱导小鼠产生约100次的抓挠行为。并且，组胺受体1（H1R）和组胺受体4（H4R）拮抗剂对沙海蜇毒素的致痒效应没有显著性抑制作用，表明沙海蜇毒素通过非组胺依赖性通路致痒。通过活细胞钙离子成像实验，观察到沙海蜇毒素能够通过诱导胞外钙离子内流从而激活小鼠背根神经节（DRG）细胞，对DRG细胞的半数激活浓度为0.40 μg/mL，表明沙海蜇毒素对DRG细胞有较强的体外激活作用。

2. 沙海蜇毒素主要的痒效应受体是Mrgprc11/MRGPRX1：沙海蜇毒素对Mrgprc11和MRGPRX1受体的EC₅₀均为0.38 μg/mL。并且，与野生型小鼠相比，沙海蜇毒素对Mrgprc11^-/-小鼠痒行为的诱导作用显著降低，30 min内引起的抓挠次数从122次降低至67次，表明Mrgprc11/MRGPRX1是沙海蜇毒素的主要痒效应受体。

3. 沙海蜇毒素通过作用于Mrgprb2受体激活肥大细胞诱导炎症反应：沙海蜇毒素对特异性表达在肥大细胞膜上的Mrgprb2受体有显著的激活作用，并且能在体外激活肥大细胞，使钙信号显著增加。此外，沙海蜇毒素作用后，能诱导小鼠肥大细胞脱颗粒释放类胰蛋白酶β2以及少量的组胺和5-羟色胺。然而，肥大细胞脱颗粒抑制剂色甘酸钠并没有降低沙海蜇毒素的致痒作用，表明沙海蜇毒素的致痒效应不依赖于肥大细胞的激活。沙海蜇毒素能够引起急性炎症反应，使用肥大细胞稳定剂色甘酸钠作用后，毒素对小鼠的血管通透性和足趾肿胀作用均被显著性抑制，说明沙海蜇毒素能通过作用于Mrgprb2受体进一步激活肥大细胞产生炎症反应。

4. 沙海蜇毒素诱导的炎症反应可能会促进毒素的致痒效应：特异性磷脂酶A₂抑制剂伐瑞拉迪能显著降低沙海蜇毒素诱导的小鼠抓挠行为，毒素作用30 min内，小鼠的抓挠次数从100次左右降低至11次，而金属蛋白酶抑制剂巴马司他在30 min内对沙海蜇毒素的致痒效应没有抑制作用，表明沙海蜇毒素磷脂酶A₂是毒素中的痒效应分子之一。海洋天然多糖MNP不仅能通过显著降低沙海蜇毒素的磷脂酶A₂活性，还能通过降低皮肤细胞和机体产生的炎症因子白细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）以及单核细胞趋化蛋白-1（MCP-1）的表达水平，抑制氧化应激和NF-κB、MAPK通路激活降低炎症反应，从而抑制沙海蜇毒素的致痒效应。MNP是水母皮炎治疗的候选药物，沙海蜇毒素磷脂酶A₂可能是治疗水母皮炎的新靶点。

总之，本论文探究了沙海蜇毒素的致痒机制，明确了沙海蜇毒素致痒的神经元受体，发现沙海蜇毒素诱导的炎症反应会进一步刺激瘙痒。本论文为水母毒素致痒的神经机制研究奠定基础，也为治疗水母蜇伤尤其是瘙痒特异性药物的研发提供了新思路。

Jellyfish outbreaks worldwide have resulted in jellyfish stings becoming the most prevalent form of marine life injury. According to the National Institutes of Health, it is estimated that approximately 150 million people are stung by jellyfish each year, posing a significant threat to public health. The clinical manifestations of jellyfish stings can vary from mild skin damage and muscle swelling to severe shock and even fatality. Among these manifestations, jellyfish dermatitis represents the most immediate and widespread injury resulting from jellyfish stings. Affected individuals typically exhibit localized skin symptoms such as skin redness, swelling, blister formation, and itching. While the pain and redness tend to subside within two weeks following the sting, the accompanying itching persists and becomes intolerable, often persisting throughout the entire recovery process at the sting site, even developing into a chronic itch that persists and plagues the stinger for decades. Due to limited understanding of the underlying mechanisms and neural circuits responsible for jellyfish toxin-induced itching, there are currently no targeted therapeutic interventions available to effectively alleviate or treat itching symptoms resulting from jellyfish stings. Mas-related G-protein-coupled receptors (Mrgprs) are a novel type of G-protein-coupled receptors that play an important role in the development of itch. Therefore, in this study, we have selected the large toxic scyphozoan jellyfish species Nemopilema nomurai as the research subject to systematically investigate the itch mechanism of Nemopilema nomurai venom (NnV) and explore the effect of inflammatory stimulation of NnV on itch behaviour. The main results are as follows:

1. The itching effect of NnV is histamine independent: Based on animal behavioral experiments, significant itching activity was demonstrated for NnV. The scratching behavior induced by the toxin in mice exhibited a concentration-dependent increase within a specific concentration range. Notably, administration of 50 μg of NnV resulted in approximately 100 scratching behaviors within a 30-min period. Moreover, the itching effect of the NnV was not significantly inhibited by antagonists targeting histamine receptor 1 and histamine receptor 4, suggesting that NnV induces itch through a non-histamine-dependent pathway. In a live cell calcium imaging assay, NnV was observed to elicit extracellular calcium inward flow, thereby activating mouse dorsal root ganglion (DRG) cells. The half-activation concentration of DRG cells was 0.40 μg/mL, indicating a potent in vitro activation effect of NnV on DRG cells.

2. The primary itch receptor for NnV is identified as Mrgprc11/MRGPRX1. The EC₅₀ for NnV were determined to be 0.38 μg/mL for Mrgprc11 and MRGPRX1. Notably, the induction of itch-related behaviors by NnV was significantly diminished in Mrgprc11^-/- mice compared to wild-type mice. The attenuation effect was substantial, with the number of scratches induced within a 30-minute period reduced from 122 to 67. These findings strongly suggest that Mrgprc11/MRGPRX1 serves as the primary itch receptor for NnV.

3. NnV activates mast cells to produce inflammatory response by acting on the Mrgprb2 receptor. NnV demonstrates a notable activating effect on the Mrgprb2 receptor, which is specifically expressed on mast cell membranes. In vitro experiments reveal that NnV significantly triggers an elevation in mast cell calcium signaling. Furthermore, NnV induces degranulation of mouse mast cells, leading to the release of tryptase β2, as well as small quantities of histamine and serotonin. However, the itching effect of NnV remains unaffected by sodium cromoglycate, an inhibitor of mast cell degranulation. This observation strongly suggests that the itching effect of NnV does not rely on mast cell activation. Additionally, NnV exhibits the ability to induce an acute inflammatory response. Remarkably, the toxin effectively inhibits vascular permeability and toe swelling in mice even after treatment with sodium cromoglycate, a mast cell stabilizer. These findings suggest that NnV may further stimulate mast cells to generate an inflammatory response through its interaction with the Mrgprb2 receptor.

4. The inflammatory reaction caused by jellyfish Nemopilema nomurai sting may further promote the itching effect of the venom: varespladib, a specific inhibitor of phospholipase A₂, exhibited a significant reduction in the scratching behavior induced by NnV in mice. Within a 30-minute timeframe, the number of scratches decreased from approximately 100 to 11. However, the metalloproteinase inhibitor barmastat had no discernible inhibitory effect on the itch-inducing properties of NnV within the same timeframe. These findings suggest that phospholipase A₂ is one of the effector molecules responsible for the itch response elicited by NnV. The Marine natural active polysaccharides MNP not only significantly reduces the phospholipase A₂ activity of NnV but also alleviated the toxin-induced inflammatory response by inhibiting the expression levels of inflammatory factors, including IL-6, TNF-α, and MCP-1, produced by skin cells and the organism, as well as reducing oxidative stress and the activation of the NF-κB and MAPK pathways, and thereby attenuating the pruritic effect of NnV. MNP emerges as a promising candidate for the treatment of jellyfish dermatitis, and the phospholipase A₂ in NnV represents a potential new target for therapeutic intervention of jellyfish dermatitis.

In conclusion, this thesis explores the itching mechanism of NnV, and provides insights into the neuronal receptors involved in the itch response to NnV. In addition, this study identifies the inflammatory response induced by NnV would further stimulate itching. This research lays the groundwork for future investigations into the neural mechanisms underlying jellyfish toxin-induced itch and offers new perspectives for the development of targeted therapies for jellyfish stings and related conditions.

第1章 绪论............................................................................... 1

1.1 水母蜇伤............................................................................................................ 1

1.1.1 水母蜇伤的症状......................................................................................... 1

1.1.2 水母毒素生物活性..................................................................................... 2

1.1.3 水母蜇伤的治疗......................................................................................... 4

1.2 痒觉的分子机制和治疗方法............................................................................ 4

1.2.1 组胺依赖性致痒......................................................................................... 6

1.2.2 非组胺依赖性致痒..................................................................................... 8

1.2.3 次要细胞和痒觉的关系........................................................................... 12

1.2.4 瘙痒的治疗............................................................................................... 15

1.3 炎症刺激作用和痒觉的关系.......................................................................... 16

1.3.1 炎性细胞因子对瘙痒的影响................................................................... 16

1.3.2 水母毒素致炎成分................................................................................... 18

1.4 选题意义与研究内容...................................................................................... 19

第2章 沙海蜇毒素致痒受体的研究........................................... 23

2.1 实验材料与仪器.............................................................................................. 23

2.1.1 样品采集................................................................................................... 23

2.1.2 实验试剂................................................................................................... 24

2.1.3 实验仪器................................................................................................... 24

2.2 实验步骤.......................................................................................................... 25

2.2.1 沙海蜇毒素的制备................................................................................... 25

2.2.2 毒素蛋白浓度的测定............................................................................... 25

2.2.3 痒行为学实验........................................................................................... 26

2.2.4 背根神经节的急性分离与体外培养....................................................... 27

2.2.5 真核表达载体的构建和质粒提取........................................................... 27

2.2.6 细胞培养................................................................................................... 28

2.2.7 细胞转染................................................................................................... 29

2.2.8 细胞活力测定........................................................................................... 30

2.2.9 活细胞钙离子成像实验........................................................................... 30

2.2.10 EC₅₀的计算.............................................................................................. 30

2.2.11 Mrgprc11^-/-小鼠的制备............................................................................ 30

2.2.12 统计分析................................................................................................. 31

2.3 实验结果.......................................................................................................... 31

2.3.1 沙海蜇毒素的致痒作用........................................................................... 31

2.3.2 组胺受体拮抗剂对沙海蜇毒素致痒效应的影响................................... 32

2.3.3 DRG神经节的提取................................................................................... 33

2.3.4 沙海蜇毒素对DRG神经元的体外激活效应......................................... 34

2.3.5 胞外钙离子对水母毒素刺激DRG神经节的作用................................. 35

2.3.6 沙海蜇毒素对HEK 293T细胞的毒性作用............................................ 36

2.3.7 沙海蜇毒素对小鼠Mrgprs受体的激活效应.......................................... 37

2.3.8 沙海蜇毒素对小鼠Mrgprc11受体的激活效应..................................... 39

2.3.9 沙海蜇毒素对Mrgprc11^-/-小鼠的致痒效应............................................ 40

2.3.10 沙海蜇毒素对Mrgprc11^-/-小鼠DRG神经元的激活效应................... 40

2.3.11 沙海蜇毒素对人MRGPRXs受体的激活效应..................................... 41

2.3.12 沙海蜇毒素对MRGPRX1受体的激活效应........................................ 42

2.4 本章小结.......................................................................................................... 44

第3章 肥大细胞在沙海蜇毒素致痒效应中的作用研究............... 45

3.1 实验材料与仪器.............................................................................................. 45

3.1.1 样品采集................................................................................................... 45

3.1.2 实验试剂................................................................................................... 45

3.1.3 实验仪器................................................................................................... 46

3.2 实验步骤.......................................................................................................... 47

3.2.1 沙海蜇毒素的制备................................................................................... 47

3.2.2 腹腔肥大细胞的分离与体外培养........................................................... 47

3.2.3 活细胞钙离子成像实验........................................................................... 48

3.2.4 肥大细胞嗜碱性颗粒蛋白表达量的测定............................................... 48

3.2.5 肥大细胞嗜碱性颗粒基因表达水平的测定........................................... 48

3.2.6 肥大细胞稳定剂对沙海蜇毒素致痒效应的影响................................... 50

3.2.7 伊文思蓝渗漏实验................................................................................... 50

3.2.8 小鼠足趾肿胀实验................................................................................... 51

3.2.9 统计分析................................................................................................... 51

3.3 实验结果.......................................................................................................... 51

3.3.1 沙海蜇毒素对小鼠Mrgprb2受体的体外激活效应............................... 51

3.3.2 沙海蜇毒素对小鼠腹腔肥大细胞的体外激活效应............................... 52

3.3.3 沙海蜇毒素对肥大细胞脱颗粒蛋白表达量的影响............................... 52

3.3.4 沙海蜇毒素对肥大细胞脱颗粒基因表达水平的影响........................... 55

3.3.5 沙海蜇毒素对肥大细胞的体内激活效应............................................... 55

3.3.6 肥大细胞在沙海蜇毒素诱导急性炎症反应中的作用........................... 56

3.4 本章小结.......................................................................................................... 58

第4章 沙海蜇毒素的炎症刺激作用对痒行为的影响.................. 59

4.1 实验材料与仪器.............................................................................................. 59

4.2 实验步骤.......................................................................................................... 61

4.2.1 沙海蜇毒素的制备................................................................................... 61

4.2.2 痒行为学实验........................................................................................... 61

4.2.3 金属蛋白酶活性测定............................................................................... 62

4.2.4 磷脂酶A₂活性测定................................................................................. 62

4.2.5 体外溶血活性........................................................................................... 63

4.2.6 细胞培养................................................................................................... 63

4.2.7 细胞活力测定........................................................................................... 63

4.2.8 细胞中炎症因子的基因表达水平测定................................................... 64

4.2.9 皮肤毒性实验........................................................................................... 64

4.2.10 ELISA....................................................................................................... 65

4.2.11 Western blot.............................................................................................. 65

4.2.12 肌肉毒性实验......................................................................................... 66

4.2.13 MNP对水母皮炎的治疗......................................................................... 67

4.2.14 免疫组织病理切片................................................................................. 67

4.2.15 统计分析................................................................................................. 69

4.3 实验结果.......................................................................................................... 69

4.3.1 沙海蜇毒素金属蛋白酶对毒素致痒的影响........................................... 69

4.3.2 沙海蜇毒素磷脂酶A₂对毒素致痒的影响............................................. 70

4.3.3 MNP对沙海蜇毒素金属蛋白酶和磷脂酶A₂的抑制作用..................... 70

4.3.4 MNP对沙海蜇毒素溶血活性的抑制作用............................................... 71

4.3.5 MNP对沙海蜇毒素细胞毒性的抑制作用............................................... 72

4.3.6 MNP对沙海蜇毒素皮肤毒性的影响....................................................... 75

4.3.7 MNP对沙海蜇毒素肌肉毒性的影响....................................................... 78

4.3.8 MNP对沙海蜇毒素致炎抑制作用机制................................................... 80

4.3.9 MNP对水母皮炎炎症症状的治疗作用................................................... 83

4.3.10 MNP对水母皮炎瘙痒症状的治疗作用................................................. 85

4.4 本章小结.......................................................................................................... 86

第5章 总结与创新................................................................... 89

5.1 总结.................................................................................................................. 89

5.2 创新点.............................................................................................................. 90

5.3 展望.................................................................................................................. 90

附录............................................................................................ 91

参考文献..................................................................................... 93

致谢........................................................................................... 107

作者简历及攻读学位期间发表的学术论文与学术成果................. 109