| ObjectiveThe present study was designed to investigate the neurotoxicity and mechanism ofβ-amyloid peptide(Aβ)as well as the neuroprotective effect and mechanism of Soybean isoflavone(SIF),folic acid(FA)and their coadmistration against the damage induced by Aβ,which provide theoretical basis for the prevention and control of Alzheimer's disease. Methods1.vivo study Aβ1-40,Aβ25-35 or Aβ31-35 were injected into lateral ventricles of rats to establish a learninig and memory impairment rat model.Wistar rats were randomly divided into control group,model group,isoflavone(SIF,160mg/kg.bw.day)solo-treated group,folic acid (FA,0.7mg/kg·bw·day)solo-treated group and their coadmistration group,15 rats in each group.SIF and/or FA was administrated by gavage two weeks before surgery and last for three weeks.One week after surgery morris water maze test was designed to evaluate the learning and memory abilities of rats which lasted for five days.After the rats were sacrificed,organs such as brain,heart,lung were sampled and weighted immediately.The pathological change in rats'cerebral cortex and hippocampus was measured by immunohistochemical method.2.vitro study Cerebral cortex of newborn Wistar rat was dissected,then cells were dissociated and digested by trypsin,planted at a density of 2×10~5 cells/ml.After two days Aβ25-35 or Aβ31-35 were added into neurons to establish a vitro damage model.There were five groups:control group,model group,genstein(Gen,27μg/ml)solo-treated group,folic acid (FA,40μg/ml)solo-treated group and their coadmistration group.Gen or/and FA were applied in two hours before adding Aβ.Mitochondrial membrane potential(MMP)was performed to investigate the alteration of mitochondrial structure and function.MTT assay was performed to mesure the neurons'viability.DNA damage was measured by single cell gel electrophoresis (SCGE).The expressions of bcl-2,bax and p53 gene were measured by RT-PCR.Each experiment was repeated 3 times. Results1.vivo study 1)Compared to control group,the mean escape latency and total distance of Aβtreated group were significantly prolonged(p＜0.05),while the mount of Aβpositive cells increased significantly(p＜0.01).2)The mean escape latency and total distance of SIF and/or FA treatedgroup were significantly abbreviated(p＜0.05),while the mount of Aβpositive cells decreased significantly(p＜0.05).2.vitro study 1)In control group,cell membrane and nucleus were integrated.In Aβ-treated group,there were many morphological characteristics of apoptosis including cell shrinkage,nucleus pycnosis,and chromatin margination or crescent-shaped.And apoptotic bodies also could be seen.The percentage of comet ceils and tail length were increased(p＜0.01)while viabilities of neurons as well as their MPP were decreased(p＜0.01).The expression of bcl-2 gene was decreased while the expressions of bax and p53 gene were increased(p＜0.05).2)After Gen and/or FA treatment,The number of cells with morphological characteristics of apoptosis were reduced.The percentage of comet ceils and tail length were decreased(p＜0.01)and viabilities of neurons as well as their MPP were increased(p＜0.01).The expression of bcl-2 gene was increased(p＜0.05)while the expressions of bax and p53 gene weredecreased(p＜0.05).ConeludesionThe results showed that:1)Aβhad definite neurotoxicity and could induce learning and memory impairment of rats in vivo;2)Aβcould induce oxidative damage and apoptosis of cultured cortical neurons in vitro;3)SIF and FAplayed an important protective role against Aβ-induced neurotoxicity.The probable mechanism is that SIF and FA could change the anti-oxidative state and increased the anti-oxidative level.Besides,SIF and FA could also up-regulate the anti-apoptosis genes expression and down-regulate the pro-apoptosis genes expression.Eventually SIF and FA and reduce deposition of Aβand improvethe learning and memory function of rats.
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