| With the development and utilization of marine resources and the increasing number of jellyfish, jellyfish stings are becoming the most common marine life injuries. Jellyfish toxins are a group of novel peptide toxins, the relative molecular weight of which are ranging from 10-600 kDa. Studies indicated that jellyfish toxins possess a variety of biological activities on cardiovascular, nerve, muscle, respiration, circulation and cytotoxicity, of which the cytotoxicity is the most common toxin in toxic jellyfish. So far, most of the reports about jellyfish toxins are focused on the local and systemic symptoms in vivo or in isolated organ, while the studies about the cellular mechanism are very rare, and require further investigations. In this paper, the in vivo and in vitro haemolytic activities of TOE are examined, respectively, and the diluted blood as a novel haemolytic detection system is comparatively studied with erythrocyte suspension. We also analyzed the of TOE on VSMCs and other tumor cells, aimed to investigate the cellular mechanism of TOE by the determination of intracellular Ca2+, ATPase, MDA and NO.First, we detected the haemolytic activity of TOE in vitro using the blood sample from SD rats, and studied the haemolytic activity in diluted whole blood and erythrocyte suspension comparatively. Factors affecting the haemolytic activity were observed in both the test systems. We tracked the haemolytic process in vivo, examined the influence of TOE on erythrocyte osmotic fragility, observed the changes in erythrocyte membrane using electron microscopy, and detected the activity of SC5b-9 before and after the TOE adminstration using a SC5b-9 assay kit. Then we compared the haemolytic activity of TOE in diluted whole blood and erythrocyte suspension used the blood samples from sheep, rabbit, mouse and human. Considering the weaker haemolytic activity in diluted whole blood, we used various aliquots of plasma and serum albumin to explore the possible mechanism of haemolysis. Besides, we used the tissue explants technique to culture vascular smooth muscle cells from SD rat aortic vascular, and compared the cell survival with a variety of tumor cells after TOE treatment by MTT assay, then detected the LDH activity of supernatant from cultured cells to further determine the cell toxicity. Morphological changes of the cells were observed under a microscope. The dynamic changes of intracellular Ca2+ after TOE treament were observed by fluorescent probe Fluo-3, the intracellular Ca2+, ATPase, MDA and NO were detected by kit assay. Results showed that the haemolytic activity of TOE was generally stronger in erythrocyte suspension than that in diluted whole blood. In the present of Mn2+, Zn2+, La3+, Cu2+ and Fe2+, the haemolytic activity of TOE was significantly inhibited. After the injection of TOE (5 mg/kg i.v.), the serum OD414 showed an acute and significant increase in the previous 10 min, then a gradually increase in the next 3 hours in the anesthetized SD rats. The maximum and minimum osmotic fragilities of erythrocyte were increased after TOE adminstration. A great many of unknown complexes were observed on a few erythrocytes with normal shapes after TOE administration under transmission electron microscopy. SC5b-9 complex were significantly up-regulated after incubation with TOE in vitro but not in vivo. Haemolytic activity of TOE in erythrocyte suspension is generally higher than that in diluted whole blood in the five kinds of blood samples tested. Antioxidants GSH and ascorbic acid can inhibit the haemolytic activity of TOE obviously but not protease inhibitor. The dose-dependent decrease of haemolytic activity of TOE was observed clearly in human erythrocyte suspension in the present of plasma and serum albumin. The obvious cytotoxicity of TOE was observed in the four kinds of cells tested by MTT assay, among which VSMCs was the most susceptible to the venom. We found higher LDH activity in the supernatant of cultured VSMCs after TOE treatment than that in the normal cells. Microscopy found the dose-dependent morphological changes of the cells, including refraction-enhanced, vacuoles, swelling, poorer refraction, and a large number of cells becoming ruptured, collapsed and died at the end. A visible increase of intracellular Ca2+ was discovered under the fluorescence microplate reader in 10 min, after a plain phase, a significant increase came again. After TOE treatment for 24 h, the levels of intracellular Ca2+were elevated obviously, while the activities of Na+-K+ATPase and Ca2+-ATPase did not change. There was no obvious change of MDA or NO level after the TOE treatment.For the first time, we utilized the diluted whole blood as well as the erythrocyte suspension with the same erythrocyte concentration to compare the haemolysis of TOE in five kinds of blood samples, and found that the haemolytic activity in the erythrocyte suspension is generally higher than that in the diluted whole blood. These consistent results suggested that the difference of haemolytic activity in diluted whole blood and erythrocyte suspension is not accidental, there were some protective factors in the plasma which may due to the albumin. The results implied that a direct, rapid and severe haemolysis takes place with a great deal of erythrocytes broken by TOE in vivo. Besides, we showed that both the maximal and minimal erythrocyte osmotic fragilities increased, further supporting partial lesion happening on erythrocyte membrane by TOE (5 mg/kg i.v.) in vivo. These results clearly elucidate that haemolysis of TOE in vivo consists of two phases:the first a rapid and direct haemolysis, and followed by a gradual haemolysis. The in vivo haemolysis is not consistent with the in vitro haemolysis and this was further illustrated that the test system erythrocyte suspension can not completely imitate the haemolysis in vivo. Compared with the tumor cells, the primary cultured VSMCs were the most sensitive to the TOE, which could further support that the target organ of jellyfish venom lies in cardiovascular system. The strong cytotoxicity of TOE could be also observed by MTT test, LDH activity detection and microscopy. The dynamic changes of intracellular Ca2+ showed an increase-balance-increased phase, indicating the increase process of intracellular Ca2+ might include two processes, it may cause the increase of the intracellular Ca2+ through the damaged cell membrane or calcium channels at first, and followed by the other indirectly mechanisms. The changes of intracellular Ca2+,ATPase,MDA,NO before and after TOE treatment were quite obscure. From all these results we can speculate that the overload of intracellular Ca2+ is the direct cause of cell death by TOE. |
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