Fan Lanlan, Luo Jun, Li Xiaoyong, Chen Ming, Shu Wei, Qu Xiaosheng
School of Pharmacy, Guangxi University of Chinese Medicine, 530001, Nanning, China.
National Engineering Laboratory of Southwest Endangered Medicinal Resources Development, Guangxi Botanical Garden of Medicinal Plants, 530023, Nanning, China.
Toxicon. 2017 Jul;133:162-168. doi: 10.1016/j.toxicon.2017.05.016. Epub 2017 May 17.
We previously reported unexpected apoptosis-like cell death induced by nematocyst venom (NV) from Chrysaora helvola Brandt (C. helvola) jellyfish. To assess whether the pore formation mechanism underlay the action of NV, the change in cell membrane permeability was studied in both chicken erythrocytes and human CNE-2 cells. Initially, paradoxical results were derived from osmoprotectant protection assays. Polyethylene glycol (PEG), which completely inhibited the NV induced hemolysis, failed to protect CNE-2 cells. Detailed experiments showed that PEG protection from hemolysis is concentration dependent and indicated caution when estimating the pore size formed by NV with the osmotic protection method. NV-treated CNE-2 cells remained impermeable to dyes with various molecular weights (MWs) (622.6-40,000 Da). Furthermore, membrane depolarization and selective permeability to Na other than K were induced in CNE-2 cells. No oxidative damage to the cell membrane was detected. Amiloride, an inhibitor of Na/H exchanger (NHE), substantially protected both CNE-2 cells and erythrocytes from NV. Combined with the previously reported increase in intracellular pH, we supposed that NV activated plasma membrane NHE without forming transmembrane pores. Interestingly, glutathione (GSH) showed significant protection to CNE-2 cells while potentiating the hemolytic power of NV. This finding may suggest a key role of reactive oxygen species (ROS) in the cytotoxicity of NV. To the best of our knowledge, this is the first report that a hemolytic jellyfish venom acts through NHE in a manner other than compromising membrane integrity. The current work provides new insight into the arsenal of toxic jellyfishes.
我们之前报道过，来自金色海蜇（Chrysaora helvola Brandt，简称C. helvola）水母的刺丝囊毒液（NV）可诱导意外的凋亡样细胞死亡。为了评估孔形成机制是否是NV作用的基础，我们研究了鸡红细胞和人CNE-2细胞中细胞膜通透性的变化。最初，渗透压保护剂保护试验得出了矛盾的结果。聚乙二醇（PEG）完全抑制了NV诱导的溶血，但未能保护CNE-2细胞。详细实验表明，PEG对溶血的保护作用具有浓度依赖性，这表明在用渗透压保护法估计NV形成的孔径时需谨慎。经NV处理的CNE-2细胞对各种分子量（MW）（622.6 - 40,000 Da）的染料仍具有不透性。此外，CNE-2细胞中诱导了膜去极化以及对Na而非K的选择性通透性。未检测到对细胞膜的氧化损伤。钠/氢交换体（NHE）抑制剂氨氯地平可显著保护CNE-2细胞和红细胞免受NV的损伤。结合之前报道的细胞内pH升高，我们推测NV激活了质膜NHE，但未形成跨膜孔。有趣的是，谷胱甘肽（GSH）对CNE-2细胞显示出显著的保护作用，同时增强了NV的溶血能力。这一发现可能表明活性氧（ROS）在NV的细胞毒性中起关键作用。据我们所知，这是第一份关于溶血性水母毒液通过NHE以不损害膜完整性的方式发挥作用的报告。目前的工作为有毒水母的毒性作用提供了新的见解。
