Study Of The Signal Pathway Involved In The Protective Effects Of Gypenosides On Oxidative Nerve Injury Induced By Glutamate

Backgroud: Apart from glutamate receptor-mediated excitotoxicity pathway, inhibition of glutamate/cystine transporter was believed to be a contributive mechanism.Many research had been done about excitotoxicity, but little about oxidatitive neurotoxicity. More than 50 different GP was included in Gynostemma Pentaphyllum (Thunb) Makino. It was reported that dammarane gypenodides(GP) have many pharmacological effects. Such as inhibition to carcinoma, aging, oxidative stress etc. Although some evidences have suggested the potency of gypenosides on neuronal injury, the signal pathway involved in the protective effects of gypenosides on nerve injury still remains to be established.Objective: Glutamate toxicity causes neuronal death in neurodegenerative diseases; such as Parkinson's disease(PD), Alzheimer's disease(AD),etal. hence, there is a need for therapeutic agents rendering functional neuroprotection.In this study ,the neuroprotective activity of gypenosides was evaluated employing primary cultures of rat cortical neurons and the signal pathway responsible for the protective effects was assayed, which enriched the theory systrm applying in anti-neurotoxicity study as well as in clinical therapy research.Methods: Primary cortical neurons were prepared from embryonic day 13-14 Wistar rats. After dissociated from the cortex, cells were trypsinized to single ones and matintained in DMEM containing 10% fetal bovine serum. Experiments were performed 24 hours after the initial plating of the neurons. 1.Experiment group: Cells were divided into 3 groups (Control,Glu and GP+Glu).2.Morphological observation: Obtain morphology of cells when observing them withinverted phase contrast microscope at the right moment. 3.Cell death detection: The cell death were measured by Typan Blue stainning,Ho33342 and PI double stainning and DNA ladder. 4.Conten of NO and H2O2, activity of SOD, GSH-PX and ATPase: NO contents weredetermined by Nitrate reductase assay.The conten of H2O?,activities of superoxidedismutase (SOD) and glutathione peroxidase (GSH-Px) were detected according to thekits.5. Ultrastructure change: Observe cells ultrastucture change with electron microscop. 6.Change of mitochondrial membrane potential and activation of caspase-3: Changeof mitochondrial membrane potential and activation of caspase-3 of neurons wereanalyzed by flow cytometry. 7. Distribution of Cytc: Cellular distribution of cytochrome C was detected withimmunofluorescence. Results:1. Morphological observation:After initial plating,the embryotic cortical neurons were spherical and suspended in medium.24 hours after,most cells spreaded around and 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 borken-up fragments scattered all around.Cells pre-treated with gypenosides remained favorable growth,distinctly contrasted to the condition of glutamate-intoxicated cells.2. Typan Blue stainning indicated that 2 microM glutamate for 12 hr caused cells apoptosis,distinctly contrasted to the condition of cells in control groups and pre-treated with gypenosides groups.3. DNA fragmentation was evaluated by agarose gel electrophoresis:DNA ladder formation was detected afer exposure to high extracellular glutamate for 12h,which was not been seen in control group or protected group.lt indicated that gypenosides can protect neurons from being hurt.4.Analysis of cellular death pathways in neurons with Ho33342 and PI dual- labelingmethod: Neurons in control group were dyed into blue.The cells disposaled with Glu for 12h had two cellular death pathway:necrosis and apoptosis.Necrosisxell membrane were broken and cells were dyed into red .Apoptosisrcells were dyed into blue but their nucleus crimpled apparently and apoptotic bodies appeared.Most neurons in GP protected group were dyed into blue as cells in control group.Apoptosis and necrosis were seldom seen.5.Glutamate-induced apoptosis correlated with upregulation of GSH-PX,downregulation of SOD and ATPase .a proapoptotic change in the mitochondrial membrane potential,decreased content of H2O2, increased content of NO and increased.6.The assay of the death style of neurons through transmission electon microscope.lt showed that the normal neuron has a big nucleus with a bulk of euchrmatid.Two death pathway were showed in glutamate group:The necrotic neuron were swelled and some parts of the cell membrane region were broken with subsequent outflow of cytoplasm.The apoptotic neurons had no swelling in the cellular organelles.While its nucleus crimpled apparently and the perinuclear space was swelled visiby.Neurons in GP group were almost the same with control group.lt indicated that Gypenoside can prevent the neurons from the insults induced by glutamate.7. flow cytometry methods (FCM) results: mitochondrial membrane potential started to decline and Caspase 3 activity appeared after 12h exposure to glutamate.If pre-treated with gypenoside mitochondrial membrane potential and Caspase 3 changed little.lt indicated that Gypenoside can prevent the neurons from the insults induced by glutamate.8. Distribution of Cytc:Glutamate can cause cytochrome C content in cytoplasm arose.If pre-treated with gypenoside, cytochrome C content remained at a low level.lt also indicated that gypenoside can protected neurons.These results showed that GP exerted significant neuroprotective effects on cultured cortical cells.Therefore,GP may be efficacious in protecting neurons from oxidative damage that was produced by exposure to excess glutamate.Conclusion: Primary cortical neurons were prepared,and the oxidative neurotoxicity model was obtained by applying high dosage glutamate.GP was added before damagewas formed.Glutamate-induced oxidative stress,occurs upon glutamate inhibion of cystine uptake through a glutamate/cystine antitransporter resulting in the depletion of intracellular glutathione.Glutamate can cause neurons damaged and even to death through piling free radicals up and then leading to the reactions behind.In this study,we found that Gypenoside can prevent the neurons from the insults induced by glutamate oxidative neurotoxicity. Glutamate triggered two cellular death pathways:necrosis and apoptosis.The cells,exposed to glutamate (2 mmol/1 for 12h),showed charateristic change of damage, which could be relieved by the reatment of GP (200 u g/ml), with survial rate increasing. Activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and ATPase were protected by GP also.GP can prevent NO and H2O2 from decreasing which was induced by glutamate. The results showed that living neurons had a high mitochongdrial membrane potential and low caspase 3 activity.Glutamate caused the decrease of mitochondrial memrane potential and raised caspase 3 activity. GP can protect neurons through reincreasing them. Gypenoside can prevent the neurons from the insults induced by glutamate oxidative neurotoxicity and raise neurons survival.The function of GP may be correlated with the increased activities of antioxidant enzymes and the clearance of free radicals.The signaling pathway that involves in the protective effects afforded by GP against glutamate oxidative neurotoxicity has been suggested in this study,its mechanism had been probed into.A reserch for its signal pathway had been done.Through this research we have made sure that gypenoside has protective effects against glutamate, which contributes to the study of antioxidants to antagonize glutamate-induced neuronal degenerative disorders.