| Background: 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 and Huntington's disease may been caused at least by excitotoxicity. Several clinical trials have been performed to evaluate the potential of antiglutamate drugs, but to date, the results have been disappointing. The extract of Ginkgo biloba (GBE) is most commonly prescribed as a neuroprotective drug and the resource of GBE is most enriched in our country. In recent decades, GBE has been widely explored and utilized, but it can't compete with foreign products. The most important reason is the high concentration of ginkgolic acids, and the assured mechanism is unknown. So it has theory and practical importance to explore more neuroprotective effect of GBE, to produce achieving international standard product, and to determine what the possible mechanism.Objectives: we adopt CO2-supercritical fluid extraction (CO2-SFE) to prepare nGBE and GB. This technique not only increases pharmaceutical ingredients, but also avoids the contamination of traditional organic solution. Content of ginkgolic acids is under 5ppm, exceeding the international corresponding criterion. Our experiment observed the effect of nGBE and GB against damage of glutamate in different modes so that determine their application value and approach, as well as determine the possible mechanism of nGBE and GB antiexcitotoxicity in pretreatment.Method:The first part: Based on glutamate excitotoxicity to primary cultures from neonatalWistar rat hippocampal, our experiment utilized trypan bluem TUNEL and LDH to study the effect of nGBE and GB on neuron in different doses between pre-treatment and post-treatment administration modes, as well as to compare with the NMDA receptor uncompetitive antagonist-MK-801. Effect of nGBE and GB at the optimal doses between pre-treatment, acute-treatment and post-treatment was observed then.The second part: Based on the first part, we introduced glutamate receptor phosphorylation and glycosylation inhibitors. Trypan blue and LDH were used to compare the antiexcitotoxicity effect of inhibitors alone or in combination with nGBE and GB.Results:The first part: (1) Cell viability: The result demonstrated that neurons treated with nGBE and GB were significantly different from glutamate control, and the maximal protection was achieved by pretreatment with nGBE at a concentration of 150ug/ml and with GB at a concentration of 100uM. The protection effect of GB is superior to nGBE, but inferior to MK-801. (2) CelF apoptosis: The normal culture showed few TUNEL-positive cells, and glutamate-exposed control culture showed a obvious increase of TUNEL-positive cells, and nGBE and GB reduced apoptosis rate significantly compared with glutamate control. (3) LDH efflux: The result demonstrated that neurons treated with nGBE and GB significantly decreased LDH efflux compared with glutamate treatments. (4) Effect of nGBE and GB in different administration modes at the optimal doses: The benefit of pre-treatment preceded acute-treatment and post-treatment distinctly, but the difference between acute-treatment and post-treatment had no prominence.The second part: (1) Cell viability: PKC inhibitor chelerythrine and protein synthesize inhibitor cycloheximide could protect the neurons against glutamate induced injury in different degree, both of which showed a synergistic effect in combination with nGBE and GB, respectively; However, glycosylation inhibitor tunicamycin and tyrosine kinase inhibitor genistein alone or in combination with nGBE and GB had no effect on cell viability. (2) LDH efflux: There is no prominent difference on LDH efflux between tunicamycin or genistein and glutamate control, nor in combination with nGBE and GB. However, chelerythrine and cycloheximide could decrease LDH efflux, especially in combination with nGBE and GB.Conclusions:(1) Excitotoxicity could cause neuron necrosis and...
|
PDF Full Text Request