Study on the Mechanism of Erythrocyte and NRK-52E Cell Injury Induced by Tentacle Extract from the Jellyfish Cyanea capillata
|School||Second Military Medical University|
|Course||Preventive Medicine in Military|
|Keywords||jellyfish tentacle extract hemolysis lipid peroxidation pore-formation apoptosis ROS mitochondrial|
IntroductionWith the increasing number of jellyfish blooms and human sea-related activities,poisonous jellyfish stings have become the most common marine life injuries, which canproduce a variety of local and systemic symptoms. The jellyfish venoms are composed ofproteinaceous toxins with novel structure, and exhibit a variety of toxicities such ashemolytic, cardiovascular, liver, kidney, nervous and dermonecrotic toxicities, amongwhich hemolytic toxicity is considered to be the most basic damage factor. Differentspecies of jellyfish venoms may induce hemolysis via different ways, and two hypothesesabout the hemolysis of jellyfish venom, including the effect of protease activity andpore-formation, have been proposed. Considering there might be enough time for jellyfishto evolve other highly efficient pathways to destroy the cell membranes, we try todetermine if lipid peroxidation is a potential mechanism underlying hemolysis in additionto pore-formation of the tentacle extract from the jellyfish Cyanea capillata in the first partof the present thesis.Severe jellyfish stings with systemic effects require immediate medical care, but mostvictims only showed dermatological symptoms, and a small proportion neededhospitalization for further treatments because of delayed multiple organ dysfunction. Ourprevious results have demonstrated that TE at a moderate dose could cause delayedjellyfish envenomation syndrome, where the kidney injury （especially the tubular epithelialcells） seemed to be more serious. So the mechanism on NRK-52E cell injury induced byTE was studied in the second part of this thesis. Based on the results of the first part, therole of lipid peroxidation, reactive oxygen species and mitochondrial dysfunction inNRK-52E cell apoptosis and necrosis was studied.MethodsPart I: Hemolytic mechanism of TE.Hemolytic activity of TE was tested in0.45%rat erythrocyte suspension, and theeffects of Ca2+and calcium antagonists （diltiazem, verapamil and nifedipine） upon thehemolytic power of TE were further studied. Using laser scanning confocal microscopy,the intracellular Ca2+concentration after TE treatment was observed. In the osmotic protection assay, polyethylenglycol （PEG） with different molecular weights was used totest the possible role of pore-formation in the hemolysis of TE. Considering lipidperoxidation being one of the main mechanisms of hemolysis, the MDA level, animportant marker of lipid peroxidation, of erythrocytes after TE treatment was measuredand the effects of the antioxidants Vc and GSH on the hemolytic activity of TE were tested.Then the effects of Vc on hemolysis of TE in vivo were further evaluated.Part II: Cytotoxicity induced by TE in NRK-52E cells.First, the survival rate of NRK-52E cells after TE treatment was assessed by MTTassay, and the morphological changes were observed under a microscope. Using AnnexinV-FITC/PI double staining, the ratio of apoptotic and necrotic cells was measured by flowcytometry. The intracellular ROS level of NRK-52E cells was detected under fluorescencemicroscope, and MDA concentration in cell homogenates was measured by thiobarbituricacid method. Using rhodamine123as fluorescent probe, the mitochondrial membranepotential was measured.ResultsPart I: Hemolytic mechanism of TE.A dose-dependent increase of hemolysis of TE was observed in rat erythrocytesuspensions and the hemolytic activity was enhanced in the presence of Ca2+but attenuatedby Ca2+channel blockers （diltiazem, verapamil and nifedipine）. Direct intracellular Ca2+increase was observed after TE treatment, and the Ca2+increase could be depressed bydiltiazem. The osmotic protectant PEG significantly blocked hemolysis at the molecularmass exceeding4000Da. The concentration of MDA increased dose-dependently in raterythrocytes after TE treatment, while in vitro hemolysis of TE was inhibited by Vc andGSH. Furthermore, in vivo hemolysis and electrolyte change after TE administration couldbe partly recovered by Vc.Part II: Cytotoxicity induced by TE in NRK-52E cells.After treatment with TE at a dose of40μg/ml for6h, NRK-52E cells were graduallydetached from the dish. TE decreased the viability of cells in a dose-and time-dependentmanner. Flow cytometry assays showed the proportions of apoptotic and necrotic cellsincreased with increasing concentrations of TE added to the NRK-52E cells. The level ofintracellular ROS and lipid peroxidation increased after TE treatment, while mitochondrialmembrane potential decreased. ConclusionThese results indicated that lipid peroxidation is another potential mechanism besidespore-formation underlying the hemolysis of TE, and both Ca2+channel blockers andantioxidants could be useful candidates against the hemolytic activity of jellyfish venoms.Mitochondrial dysfunction and ROS overproduction may contribute to cytotoxicityinduced by TE in NRK-52E cells.