The Study On The Neuroprotective Mechanism Of The Extract Of Ginkgo Biloba Against Excitotoxicty

Background:Since the report of neurotoxicity of glutamate in the late 60's, a variety of pathologic conditions range from acute insults such as stroke and epilepsy to chronic neurodegenerative states such as amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease has been thought to be related to the damage of glutamate-induced excitotoxicity. People aimed at developing all kinds of neuroprotective drugs, but to date, the results have been disappointing. The extract of Ginkgo biloba (GBE) is most commonly prescribed as a neuroprotective drug,but the assured mechanism is unknown. The resource of GBE is most enriched in our country, but it can't compete with foreign products. The most important reason is the high concentration of ginkgolic acids, so the components isolated from the extract named Ginkgolide B (GB) and bilobalide (BB) received more and more concern.Dendritic spines is the postsynaptic parts of excitatory synapse and its important role is sensitive to the glutamate released from the presynaptic parts. Snk (serum-inducible kinase) and SPAR (spine-associated Rap GTPase activating protein) has a close relationship with the loss of mature dendritic spines. The Snk-SPAR pathway probably participates in excitotoxicity in CNS through regulation the stability of dendritic spine. The intervention given to this pathway is expected to provide the new tactics and means for the therapeutic strategies against excitotoxicity.Objectives:Our experiment observed the effect of EGb761, GB, BB against damage ofglutamate in pretreatment modes so that determine their application value and approach, as well as determine the correlation between Snk-SPAR pathway and antiexcitotoxicity of GBE and its components in pretreatment models.Method:The first part: Based on glutamate excitotoxicity to primary cultures from neonatal SD rat hippocampal, our experiment utilized trypan blue, TUNEL and LDH to study the effect of EGb761, GB, BB on neuron in different doses pretreatment modes, as well as to compare with the NMDA receptor uncompetitive antagonist-MK-801 and glutamate group. The second part: EGb761, GB, BB at the optimal doses in pretreatment models. RT-PCR was used to detect the changes in Snk/SPAR mRNA levels and compared with glutamate group.Results:The first part:1. Cell viability: The result demonstrated that neurons treated with EGb761, GB, BB were significantly different from glutamate control, and the maximal protection was achieved by pretreatment at a concentration of 100μg/ml, 100μΜ, 100μΜ, but inferior to MK-801(10μΜ). The protective effect of GB and BB is superior to EGb761.The protective effect of BB is superior to GB.2. Cell apoptosis: The normal culture showed few TUNEL-positive cells, and glutamate-exposed control culture showed an obvious increase of TUNEL-positive cells, and EGb761, GB, BB reduced apoptosis rate significantly compared with glutamate control, still inferior to MK-801. The protective effect of GB and BB is superior to EGb761.The protective effect of BB is superior to GB.3. LDH leakage: The result demonstrated that neurons treated with glutamate were significantly different from normal control.The neurons treated with EGb761, GB, BB significantly decreased LDH leakage compared with glutamate treatments. But all groups are inferior to MK-801. The protective effect of GB and BB is superior to EGb761.The protective effect of BB is superior to GB.The second part:RT-PCR was used to observe the expression of Snk and SPAR.In control group, the basal level of Snk mRNA was low, but after glutamate exposure, Snk mRNA had begun to elevate. The expression level of Snk mRNA decreased when treated with MK801, EGb761(100μg/ml), GB(100μΜ), BB(100μΜ) compared with glutamate group. The expression tendency of SPAR mRNA was contrary to Snk mRNA.Conclusions:1. Excitotoxicity could cause neuron necrosis and apoptosis at the same time.Treatment with EGb761, GB, BB could protect the neurons against glutamate-induced injury in different degree and depended on dose in certain range.2. In pretreatment models, the protective effect of GB and BB is superior to EGb761 at the optimal doses .The protective effect of BB is superior to GB. All three are inferior to MK-801.3. Pretreatment with EGb761, GB, BB at the optimal doses could induce the changes in Snk/SPAR mRNA levels, which indicated neuroprotective effects of GBE and its components against glutamate excitotoxicity was closely related to Snk-SPAR pathway.