缩血管反应在水母毒素致延迟毒性综合征中的作用研究

Jellyfish sting is one of the most common marine life injuries around the world, and some of its victims would develop a delayed jellyfish envenomation syndrome (DJES) with serious multiple organ dysfunction or systemic damages. Our previous results demonstrated that the jellyfish venom can cause hypoxic-ischemic damage through contracting blood vessels that leads to hypoperfusion of vital organs such as heart and kidney, which can further develop into DJES. We also discovered that the elevation of Ca2+ concentration in the vascular smooth muscle cells and the endothelial cells plays an important role in the vasoconstriction effect of jellyfish venom. Starting from the isolation and purification of vasoconstriction component from the jellyfish Cyanea capillata nematocyst venom, we plan to explore the molecular mechanism underlying the Ca2+ overload in smooth muscle cells and endothelial cells by investigating the extracellular Ca2+ influx and intracellular Ca2+ release, to investigate the self-protective function of endothelial cells in terms of its nitric oxide release, and further to clarify the mechanism of the vasoconstriction effect in cellular and molecular level from the perspective of the intervention effects of the inhibitors. The contractile mechanism and the damage effects of the venom on the aorta, renal artery and coronary artery will be further testified in the isolated vascular rings and in vivo, and the intervention effects of the inhibitors will be also observed. Our research can shed light on the mechanism of vasoconstriction caused by jellyfish venom and its in vivo damage effects, providing new idea and drug candidates for the protection against the heart or renal failure caused by jellyfish envenomation.

水母蜇伤是最常见的海洋生物伤，部分患者出现全身多器官损伤，即延迟毒性综合征。我们的前期研究表明，水母毒素通过收缩血管使心、肾等重要脏器灌注不足而引起缺血缺氧性损伤，进一步发展为延迟毒性综合征；同时发现血管平滑肌和内皮细胞Ca2+升高在水母毒素缩血管效应中起重要作用，且内皮在血管收缩过程中还发挥了一定的功能性舒张作用。本课题拟从发形霞水母刺丝囊毒液中分离纯化缩血管组分，从细胞外Ca2+内流及内质网Ca2+释放与回摄途径分别探讨血管平滑肌和内皮细胞Ca2+升高的原因，从内皮细胞NO释放角度分析其自我保护作用，并结合抑制剂的拮抗作用阐述缩血管效应的细胞与分子机制；在离体血管及整体动物水平进一步验证毒素对主动脉、肾动脉和冠状动脉的收缩反应及其损伤效应。本项目将阐明水母毒素所致血管收缩的作用机理，也有助于明确血管收缩对心、肾功能的影响，为水母蜇伤引起的心、肾功能衰竭防治提供新的研究思路和候选药物。

水母蜇伤能引起局部或全身中毒症状，部分患者出现延迟性多器官损伤，水母毒素引起的血管收缩可能是水母蜇伤导致多器官衰竭的重要机制之一。本课题分别从血管效应细胞、离体血管及整体动物层面探讨了发形霞水母触手提取物（tentacle extract, TE）致血管收缩的作用机理，为水母蜇伤所致延迟毒性综合征提供理论依据。首先，在大鼠离体主动脉环上，我们发现TE具有直接的血管收缩效应，并且血管内皮在TE诱导的缩血管反应中发挥一定的保护作用。药物干预实验提示，TE引起的功能性舒血管效应可以受NO的调节，这可能与细胞外钙内流和内质网钙释放有关。其次，在人脐静脉内皮细胞上进一步分析了TE引起的舒血管效应分子机制。发现TE可以引起血管内皮细胞eNOS活力和NO的产生呈剂量和时间依赖性升高，并且激活了Akt和eNOS的磷酸化，说明其保护作用主要是通过PI3K/Akt/eNOS信号通路介导NO产生而发挥的。第三，共聚焦扫描检测血管内皮细胞和平滑肌细胞内[Ca2+]i，在细胞水平分析TE致血管收缩的作用机制。发现TE可以引起胞浆Ca2+水平显著上升，提示Ca2+信号异常可能是TE所致血管收缩及NO释放的主要原因。使用Ca2+信号通路抑制剂进一步分析Ca2+升高的来源，发现TE引起的钙升高既可以通过质膜上的穿孔通道由胞外内流，也可以来自细胞内的钙释放。第四，采用SR-μA和SR-μCT技术，我们直接清晰的观察到了TE作用后的实验动物的微血管结构。发现TE处理后肝、肾末梢小血管和对照组相比明显变细，甚至缺失，而金属蛋白酶抑制剂（BB-94）可显著改善TE诱导的肝、肾微血管损伤。总之，本课题探讨了TE引起血管收缩的作用机制，发现其主要与血管效应细胞钙超载有关，BB-94可以保护毒素引起的微血管形态变化，在水母蜇伤治疗方面具有良好的应用前景。