Protective effect of 7-difluoromethoxy-5,4’-Di-hydroxyl isoflavone against the damage induced by glutamate in PC12 cells

Abstract

7-difluoromethoxy-5,4'-Di-hydroxyl isoflavone (dFGEN), prepared by the difluoromethylation of genistein, is an active chemical entity. In this study, our main purpose was to investigate whether dFGEN had an effect on glutamate-induced apoptosis in cultured PC12 cells. The PC12 cells were treated with different glutamate concentrations for 24 h in vitro. The PC12 cells impaired by glutamate were used as the cell model of excitability. Cells were incubated for 30 min with genistein, dFGEN, vitamin E, and exposed to 10 mM glutamate for 24 h. Cell morphology was observed by light microscopy. The growth and proliferation of PC12 cells were detected by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Cell apoptosis was determined by flow cytome-try (FCM) with propidium iodide (PI) staining. The activity of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and the content of malondialdelyde (MDA) were measured by kits, respectively. Acridine orange (AO) staining was used to detect characteristics of cell apoptosis. When PC12 cells were incubated with glutamate for 24 h, cells appeared to have significant changes in shape. The cellular viability was reduced and the apoptotic rate was increased. The levels of LDH and the content of MDA were increased. The activity of SOD was decreased. After PC12 cells were pretreated with dFGEN, dFGEN significantly improved cell morphology, cell growth and proliferation, suppressed apoptosis of cells, reduced the release of LDH, improving SOD activity and decreased MDA content in a concentration-dependent manner. AO staining displayed that apoptosis was decreased. These results suggested that dFGEN has a protective effect against glutamate-induced damage in PC12 cells. dFGEN seemed to have a better protective effect than the lead compound genistein in a concentration-dependent manner. The mechanism of protective effect of dFGEN may be mainly related to its antioxidative activity.