Protective Effect Of DFGEN Against The Damage Induced By Glutamate On PC12Cells

Objective:To investigate the effect of dFGEN on the damage of PC12cells induced by glutamate in vitro and provide a new clue and theory basis for developing a drug of preventing and curing neurodegenerative diseases in the future.Methods:1.Rat adrenal pheochromocytoma monoclonal lines(PC12) were incubated with DMEM in vitro and selected in logarithmic growth phase to carry out experiments.The PC12cells were treated with different glutamate concentrations(1,5,10,15,20mmol/L) for24h in vitro.Cell morphology was observed by optical microscope. The growth and proliferation of PC12cells were detected by3-[4,5-dimethyl thiazol-2-yl]-2,5-diphenyltetrazolium bromide(MTT) assay.Cell apoptosis was determined by flow cytometry(FCM) with propidium iodide(PI) staining.The activity of lactate dehydrogenase (LDH),the activity of superoxide dismutase(SOD) and the content of malondialdelyde(MDA) were measured by kits respectively.2.Experiment groups were divided as follows:(1)blank control group;(2) DMSO control group;(3) glutamate damage group;(4) lead compound group;(5)7-difluoromethoxy-5,4’-Di-hydroxyl isoflavone group;(6) positive medicine control group.The PC12cells were pretreated with the vehicle solvent(DMSO), GEN, dFGEN and VE for30min before exposed to10mmol/L glutamate for24h. Cell morphology was observed by optical microscope. Cell growth and proliferation activity were measured by MTT assay. Cell apoptosis was determined by flow cytometry(FCM) with propidium iodide(PI) staining. The LDH activity was observed by colorimetry reaction. The activity of SOD and the content of MDA in the cells were measured by kits respectively. Acridine orange(AO) staining was used to detect characteristics of cell apoptosis.Results:1. After PCl2cells were incubated with different dose of glutamate for24hours, the proliferation of cells was reduced in a concentration dependent manner by MTT assay. Glutamate treated group cells appeared significantly changes in shape under optical microscope.As shown in FCM assay, the apoptosis of the PC12cells was increased significantly. The level of LDH and the content of MDA in glutamate groups were significantly increased in dose-dependent manner, compared with the control group. The activity of SOD in glutamate groups was decreased significantly, compared with the control group. The results above indicated that glutamate can lead to PC12cell injury.2. Then PC12cells were incubatd with different concentration of dFGEN before being injured by high glutamate level(10mmol/L). After PC12 cells were pretreated with dFGEN, experiment results showed:dFGEN group could significantly inhibit cell damage and improve cell morphology.dFGEN could improve cell growth and proliferation, suppress the apoptosis of cell, reduce the release of LDH, improve SOD activity and decrease MDA content in a concentration dependent manner. AO staining displayed that PC12cells were evidently changed in shape and apoptotic body could be observed by fluorescence microscope in glutamate treated group. The results above indicated that dFGEN group could be the most obviously protective group.Conclusion:1. dFGEN has protective effect against glutamate-induced damage in PC12cells. The protective effect was more significantly stronger than GEN in a concentration dependent manner.2. The mechanism of protective effect of dFGEN may be mainly related to its antioxidative activities.