| ObjectiveRed tide induced by fulminating proliferation of planktons (mainly alga) in ocean can lead to allochroism and stale of the water, which severely aggravates the environment of ocean, disrupt the ocean fishing industry and tourism, and, further more threatens the health condition of human being. Over the past few years, the aggravation of water bloom has resulted in the proliferation of alga. Some of the alga can produce algae toxin, most of which can be solved by water and pollute the body of water directly. Toxin alga can poison fishes and shellfishes through food-chain. Human food poisoning (even death) may occur from eating contaminated fishes and shellfishes. Domoic Acid (DA), a relatively scarce neural toxicity amino acids and mainly from diatom, can cause Amnesic Shellfish Poisoning (ASP). Recent studies suggest that DA is a neural toxin and there may be three possibilities of its toxic mechanism: First, the neural toxin exert through activating the receptor of glutamic acid; Second, by overloading cellular free Ca2+; Third, by disorder of energy metablism.Lately, researchers believe that neuroglial cells construct the micro-environment of brain, affect the transmission of synapses and advanced integration function of brain. In addition, neuroglial cells excrete many growth factors which can prompt the differentiation and proliferation of neuron. In a word, neuroglial cells act as an important part in the nerve growth and development, especially in CNS.Current studies further proved that excess oxygen free radical can cause lipid peroxidation. There are enormous unsaturated fatty acid (PUFA) at membrane which can be easily injured by oxygen free radical. HO-1 is protective protein induced by oxidative stress. Cells will produce more HO-1 when confronting stress such as hypoxia, oxidative stress, oxided low density lipoprotein, heavy metal and endotoxin. In general, HO-1 play an important role in anti-inflammation, anti-apoptosis, and anti-proliferation. In various tissues HO-1 can be generated by different stresses. The amplify expression of HO-1 gene can resist oxidative stress and apoptosis.This research is to study the effect of domoic acid on cell toxicity, oxidative injury and membrane injury using neuroglial cell model.Methods1. The primary culture of glial cells and immunocytochemistry staining were applied to identify neuroglial cells.2. MTT was used to assess the effect of DA on the survival rate of neuroglial cells.3. Chemistry colorimetry was applied to measure the impact of DA on the content of MDA and the activity of SOD in neuroglial cells.4. The expression of HO-1 protein was measured by immunofluorescence cytochemistry after the treatment of DA in neuroglial cells.5. The effect of DA on expression of mRNA of HO-1 in neuroglial cells was evaluated by RT-PCR.6. Chemistry colorimetry was used to measure the role of DA on the activity of Na+-K+-ATPase和Ca2+-Mg2+-ATPase in neuroglial cells.7. The influence of DA on membrane fluidity in neuroglial cells was detected by fluorospectrophotometry.8. Flow cytometry was applied to measure the effect of DA on membrane permeability in neuroglial cells.Results1. After immunocytochemistry staining, a large amount of Glial Fibrillary Acidic Protein (GFAP) immunoreactive cells were checked. More than 90 % of total checked cells showed positive (GFAP). Therefore the primary cultured cells can be used as the vitro model of neuroglial cells.2. The MTT experiment showed that Domoic Acid had cellular toxicity, and with the rising of doses and the extending of time, the survival rate generally decreased. Besides, a dose-time-effect relation was observed.3. Results of DA on MDA and SOD indicated that, after exposed to DA 24 h, the amount of MDA rised, however, the activity of SOD was inhibited in neuroglial cells. The difference between middle dose, high dose groups and control group was significant. Finding of this study demonstrated that DA have oxidative injuring effect on neuroglial cells.4. The essay of immunofluorescence cytochemistry prompted such result: we demonstrated the content of HO-1 expression with the average fluorescence intensity and then we found DA could induce more protein expression of HO-1 in glial cells.5. The expression of HO-1 mRNA was demonstrated by the relative value of optical density between GADPH and HO-1. Results demonstrate that DA can boost expression of HO-1 mRNA. There were significant difference between the three treatment groups and control group.6. Through chemistry chromatometry, we found after 24h treatment of DA, the activity of Na+-K+-ATPase and Ca2+-Mg2+-ATPase was severely inhibited in neuroglial cells.7. We applied the fluorospectrophotometry to measure the effect of DA on membrane fluidity in neuroglial cells. The membrane fluidity was often specified by coefficient of viscosityη, the smaller theηwas, the larger the fluidity was. After exposed to DA 24 h, coefficient of viscosity of the three doses treatment groups were much higher than that of the control group. It suggested that the fluidity of membrane significantly degraded and the function of cell membrane was damaged.8. When cell membrane is damaged, its integrity will be injured which triggers an increased intake of PI. Therefore we measured the percentage of PI flowing into the cells to reflect the change of membrane permeability. By using flow cytometry and comparing with the control group, the percentage of PI flowing into cells significantly increased in all treatment groups, which indicated that DA could elevate the membrane permeability of neuroglial cells.ConclusionsDomoic Acid (DA) is an excitability neurotoxin which may lead to the increase of ROS and thus produce the oxidative stress. Thereafter, oxidative stress induces the over-expression of HO-1. Concurrently oxidative stress can also result in the impairment of membrane function. In this research, DA showed cytotoxicity to neuroglial cells in following aspects: 1. depressing the survival rate of cells; 2. obvious oxidative injury; 3. inducing the over-expression of protein and mRNA of HO-1; 4. inhibiting the activity of ATPase; 5. lowering the fluidity of cellular membrane; 6. raising the permeability of membrane.
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