Correlation Between The Neuroprotective Effect Of Ginkgo Biloba Extract Against Excitotoxicity And The Expression Of Amyloid Precursor Protein, Presenilin-1

Background and objectiveThe glutamate-induced excitotoxicity has been implicated in the etiology of many CNS diseases such as stroke, epilepsy, Alzheimer's disease and amyotropic lateral sclerosis. N-methy-D-aspartate (NMDA) subtype of ionotropic glutamate receptors is proved to play a critical role in excitotoxicity. Accordingly, a significant effort had been made to develop therapeutic strategies against excitotoxicity, but successful therapy in humans was limited by many factors. The study on strategy against excitotoxicity needs to be found new target.Amyloid precursor protein (APP) is an integral membrane protein. APP is cleavage byα-Secretase(ADAM10,TACE and ADAM9),β-Secretase (BACE1) andγ-Secretase(Presenilin-1,Presenilin-2,Nicastrin,Aph-1 and Pen-2 complexes).Cleavage of APP releases secreted APP (sAPPα, sAPPβ) and amyloidβ-protein(Aβ) and other metabolic products. It is well established that Aβplays a central role in the pathogenesis of Alzheimer's disease (AD). Abnormal metabolism of Aβand APP is the highlight of AD research for a long time. However this recognition will be changed by some findings: APP and it's metabolic products have correlation with process of excitation: sAPP may have the property of anti-excitotoxicity; APP could increase activity of excitatory amino acid transporters. So the damage of excitotoxicity and dysfunction of reparation may have correlation withmetabolism of APP and it's secretases. Intervention of APP may have extensive significances besides AD.Ginkgo biloba extract (GBE) contains multiple compounds such as flavonoids and terpenoids, and it has many neuroprotective effects such as removing free radicle, protecting neurons against apoptosis induced by ischemia injury, inhibiting the accumulation of Aβ, and influencing the transport of neurotransmitters. GBE has been therapeutically used for several decades to increase peripheral and cerebral blood flow as well as for the treatment of cognitive speed, dementia, and aging damages, but the mechanism still remains elusive.The objective of this study is to observe the relationship between excitotoxicity in cultured rat hippocampal neurons and expression of APP and Presenilin-1(PS1), and to explore the effect of GBE on damages and reparation of excitotoxicity, furthermore discuss the molecular mechanism of neuroprotective effect of GBE, and hope to find a new strategy for neuroprotection and neurogenerative regulation. MethodsThe primary hippocampal cultures from neonatal rat were treated with 25μM glutamate and 5μM glycine at 12 day in vitro. Trypan blue staining, MTT assay, Hematoxylin-Eosin staining, Nissl's staining were used to study the glutamate-induced injury in neurons. Observed the neuroprotective effect of GBE and MK-801 on neurons. Double immunofluorescent staining was used to observe the glutamate-induced expression and distribution changes of APP and PS1 in cultured hippocampal neurons, and effects of GBE and MK-801 on this process. Results1. Glutamate could induce marked damage in cultured hippocampal neurons.2. Survival rate demonstrated by trypan blue staining of MK-801 group and GBE pretreatment group is 83.8±2.4% and 61.0±2.9% respectively,OD value demonstrated by MTT assay of MK-801 group and GBE pretreatment group is 0.313±0.009 and 0.249±0.015 respectively. HE staining and Nissl's staining show that both MK-801 and GBE pretreatment have protective effects on the glutamate induced damage on morphous and no obviouslosses of Nissl body. The protective effect of GBE posttreatment is not obvious.3. Expressive location of APP and PS1 is uniform, both of them are expressed on nuclei, soma and processes of neurons. 18h after glutamate exposure,the level of APP is 169.5% times compared with normal group, the level of PS1 is 47.9% times compared with normal group. Both MK-801 and GBE pretreatment could inhibit expressive changes of APP and PS1 induced by glutamate.Conclusions1. GBE pretreatment can protect neurons against glutamate-induced excitotoxicity, but this effect is weak compared with MK-801.The effect of anti-excitotoxicity of GBE posttreatment is not obvious.2. In protein levels, the expression of APP and PS1 was changed in glutamate-induced exicitotoxicty. It proves that the excitotoxicity induced by glutamate is related to APP and PS1 closely.3. GBE could inhibit expressive changes of APP and PS1. This suggests that the neuroprotective effect of GBE is relevant to APP and it's metabolism.