Protective Effect Of Gypenosides-containing Serum Against Oxidation Neurotoxicity Induced By Glutamate

Backgroud:Glutamate is known to be a potent neurotoxin when presents in excess at synapses. Both glutamate excitotoxicity mediated by glutamate receptors and oxidative toxicity via the inhibition of glutamate/cystine antiporter have been shown to contribute to neural degeneration disease. Gynostemma Pentaphyllum (Thunb) Makino is a widespread herb in South China. The molecular components responsible for it's actions are gypenosides(GP). At present, In vitro test it has proven that GP has the protective effect against oxidation neurotoxicity induced by glutamate. But there are no studies that can identify if it has the same ability in vivo tests. The conception of "serum pharmacology" is that give the drug to animal, then collect the serum and use the serum on the test in vitro. We use the serum containing gypenosides to study if GP has the protective effect against oxidation neurotoxicity induced by glutamate in vivo by way of seropharmacology .Objective:Our studying groups have proved GP has the protective effect against oxidation neurotoxicity induced by glutamate in vitro. We study further the protective effect of GP-containing serum against oxidation neurotoxicity induced by glutamate , Further more ,we also analyzed the possible signal pathway for the protective efects, which afford the theory system for gypenosides as one clinical therapy.Methods:Collect the sera containing gypenosides by way of seropharmacology; Primary cultures cortical neurons(from embryonic day 14～15 Wistar rat fetuses) were studied. Cells were divided into different groups, then the experimental agents were added. 1. Collect sera containing GP:Give rats different concentration of GP by ig (intra gastric) (give control rat water) for 4 days; When give rat GP the last time for 1 hours, collect blood from heart of rat. Then get sera by centrifudge.2. MAP2 immunofluorescence staining identify the pure neural cells.3. Cells were divided into 3 groups :â‘ Control,â‘¡Glu ,â‘¢GP-containing sera . When cells were cultured for 24 hours , put control rat sera toâ‘ â‘¡groups and GP GP-containing sera toâ‘¢group, the last sera concentration in cell culture fluid was 5%. After 12 hours , put Glu (2mM) toâ‘¡â‘¢groups. After another 12 hours,do the follow assy.4. Morphology of cells observing ; Obtain morphology of cells when observing them with inverted phase contrast microscope at the right moment.5. The morphology of cells were measured by acridine orange (AO) staining,6. Using the MTT method assayed cell viability, which to make sure the best GP containing sera concentration.7. The way of cell death was assyed by Ho33342 and PI double staining .8. Intracellular GSH content and SOD activity were measured according to the kits.9. Changes of intracellular calcium concentration and change of mitochondrial membrane potential were examined by flow cytometry.10. caspase 3 was determined by western blot.Results:GP-containing serum protects Primary Cortical Cultures From Glutamate-induced Oxidative Toxicity-Primary cultured immature cortical neurons underwent a cell death 12hr after glutamate exposure. This glutamate-induced oxidative toxicity has been extensively used as a model for oxidative stress in neurons. In this study, we found that glutamate-induced oxidative toxicity could be significantly abrogated in cortical cultures by administration of GP-containing serum.1. MAP2 immunofluorescence staining observation: results showed that 90% cells were positive to MAP2.2. Morphological observation: After Plating 48 hours, the embryotic cortical neurons most were spread around adhered to the dishes with a triangular or polygonal plump body. After exposure to high extracellular glutamate for 12h, almost all of the synapses between neurons were destroyed with broken-up fragments scattered all around. Cells pre-treated with gypenosides-containing serum (400 mg·kg^-1·d^-1 GP ig) remained favorable growth, distinctly contrasted to the condition of glutamate-intoxicated cells.3. Acridine orange (AO) staining: neurons nucleus which were dyed into flavo-green in control group were bigger and preserve ellipse or spherical morphous, and had cytoplasm which were dyed into jacinth surrounding it. Neurons nucleus in Glu group were smaller and broken into fragments, and there were nearly no cytoplasm surrounding it. Most neurons in GP-containing serum (400 mg·kg^-1·d^-1GP ig ) protected group were similar with neurons in control group.4. Cell viability was assayed using the MTT assay: GP-containing serumcould significantly attenuated glutamate-induced neutotoxicity in primary cultured cortical neuron. Contrast with Glu group(34.67%), GP-containing serum could preserve cells at a high viability(up to 89.83%). The optimized cell viability was obtained by a 12hr pretreatment with ig GP 400 mg·kg^-1·d^-1 containing serum.5. Analysis of cellular death pathways in neurons with Ho33342 and P1 dual-labeling method : In the Ho33342 and P1 dual-labeling method, Neurons in control group were dyed into blue. The cells disposaled with Glu for 12h had two cellular death pathways: necrosis and apoptosis. Necrosis: cell membrane was broken and cells were dyed into red. Apoptosis: cells were dyed into blue but their nucleus crimpled apparently and apoptotic bodies appeared. Most neurons in GP-containing serum (400 mg·kg^-1·d^-1GP ig ) protected group were dyed into blue as cells in control group. Apoptosis and necrosis were seldom seen.6. Intracelluer GSH and SOD assaying: Contrasted to the depletion of GSH content and the decrease of SOD activity induced by glutamate, GP-containing serum (400 mg·kg^-1·d^-1GP ig) preserved intracellular glutathione content and SOD activity at a high level.7. Flow cytometry methods (FCM) results: mitochondrial membrane potential started to decline and intracellular calcium concentration started to increase after 12h exposure to glutamate. If pre-treated with GP-containing serum (400 mg·kg^-1·d^-1 GP ig ), mitochondrial membrane potential and intracellular calcium concentration changed little.8. Intracelluer caspase3 content assayed by western bolt: glutamate (2mM, 12h) could increase intracelluer caspase3 content, If pre-treated with GP-containing serum (400 mg·kg^-1·d^-1GP ig) intracelluer caspase3 content changed little.Conclusion:1. Get a vitro test model of studying GP-containing serum against oxidation neurotoxicity induced by glutamate.2. Make sure that GP-containing serum has a distinct protective effect against oxidation neurotoxicity induced by glutamate.3. Glu results in the depletion of intracellular GSH and SOD, GP-containing serum plays its protective effects by enhancing of GSH and SOD.4. GP-containing serum can protect neurons by high mitochongdrial membrane potential, low intracelluar Ca²⁺ concentration and caspase3 content.5. Make sure that GP has the similar protective effect against oxidation neurotoxicity induced by glutamate in vivo.