Investigate Neuroprotective Effect And Mechanism Of Ginkgolide B Protects Fetal Hippocampal Neurons

Alzheimer's disease is the most challenging neurodegenerative disorder characterized by slowly progressive dementia and brain atrophy. It is a complex genetic disease in the global prevalence of up to 15 million people, and has close relationship with hippocampal neurons. Ginkgolide B is a well-defined plant extract which is safe in nature, effective and economic, and Ginkgolide B is a major component of Extract of Ginkgo bilobaleaves medicinal herb. However, the mechanisms underlying Ginkgolide B remain unclear especially in"Micro-environment for nerve regeneration". So we investigated the potential effectiveness of Ginkgolide B against toxicity induced by Aβ25-35 on hippocampal primary cultured cells, and further exploring the possible mechanisms concerned. We first conducted the study of the Aβ25-35-induced apoptosis characterized by the changes in morphology, cell viability, lactate dehydrogenase level, the caspase-3 activity and extracellular K+ concentration in hippocampal neurons. Moreover, the expression of Brain-derived neurotrophic factor and Nerve growth factor mRNA and the protein synthesis in neurons were detected via RT-PCR and Western-blot assay. It was found out that Aβ25-35 induced apoptosis was attenuated by Ginkgolide B. Ginkgolide B caused Brain-derived neurotrophic factor and Nerve growth factor up-regulation when cells were subjected to Aβ25-35 insults. The micro-environment for nerve regeneration also improved. Ginkgolide B attenuate Aβ25-35 induced apoptosis in hippocampal neurons by Brain-derived neurotrophic factor and Nerve growth factor pattern which play a crucial role in"Micro-environment for nerve regeneration". We consider it one of the possible mechanisms by which Ginkgolide B daunts the hippocampal neurons apoptosis.The study indicate that Ginkgolide B may significantly dampen Aβ25-35 induced apoptosis, and the neuroprotective effects may be intimately associated with Brain-derived neurotrophic factor and Nerve growth factor up-regulation caused by Ginkgolide B. These findings may demonstrate the neuroprotective effects of Ginkgolide B and offer new evidences to the possible mechanisms. The present study on the neuroprotective effects of Ginkgolide B against Aβ25-35 induced apoptosis might pave a way for further investigation of molecular mechanism underlying from protein and gene level, and also lay a foundation for clinical application in the viable molecule to treat neural degeneration diseases and nerve injury. The objective of our experiment is to study the neuroprotective effects of Ginkgolide B with a view to healing neurodegenerative disorders such as Alzheimer's disease and advancing natural medicine research, development and utilization. Objective:Investigate neuroprotective effect of Ginkgolide B protects hippocampal neurons from apoptosis induced by beta-amyloid 25-35.Methods:Primary cultures from hippocampus of pregnant 16 to 18-day Sprague Dawley rat embryos. MTT assay and living cells morphology were used to selected the best effect time and the optimal concentration of Ginkgolide B. After 3 days in vitro, these cells were put away for further experiments. Three days after planting, Ginkgolide B was added to the culture and maintained for 4 hours before being treated with 25μM Aβ25-35. Meanwhile, positive control group, the standardized Extract of Ginkgo bilobaleaves EGb761 was added to the culture, and maintained for 4 hours before being treated with 25μM Aβ25-35. Aβ25-35 was cultured in PBS for 7 days at 37℃. After the culture was treated with different treatments, cell viability was assessed through a modified MTT assay. Since the loss of MTT is not necessarily a valid indicator of cell lysis, toxicity of Aβ25-35 was further examined through an LDH-release assay. LDH activity was determined according to the protocol of an LDH kit. To investigate if Ginkgolide B can protects hippocampal neurons against Aβ25-35-induced neurotoxicity via ameliorating cell apoptosis, we did Hoechst 33342 and PI double staining experiments. In order to identify apoptotic cells, nuclei were made fluorescent by incubation with the DNA intercalating dye. NSE and Tunel staining was carried out according to the introduction of tunel staining kit. Caspase-3 activity was measured via caspase assay kit according to the manufacturer's instructions. To assess the permeability of neurons membranes being treated with Aβ25-35 in the presence or absence of Ginkgolide B, we detection of level of K+ leakage. Statistical analysis was made by using the computer software SPSS 10.0. The data was analyzed by using a two or one-way analysis of variance (ANOVA), then by Fisher's PLSD post-hoc test multiple comparisons. The data was expressed as mean±S.E.M. Statistical significance was set at P≤0.05.Results:Living cells morphology: When Ginkgolide B was added, the apoptosis of the neurons caused by Aβ25-35 were improved obviously. The amount of cell debris decreased, refractivity in their bodies was promoted, and networks were formed by those neurite linking with each other. Hoechst 33342 and PI double staining: When Ginkgolide B was added, the apoptosis of the neurons caused by Aβ25-35 were improved obviously. The number of apoptotic nuclei decreases significantly, the number of surviving nuclei increase significantly. NSE and Tunel double staining: When Ginkgolide B was added, the apoptosis of the neurons caused by Aβ25-35 were improved obviously. The number of apoptotic nuclei decreased significantly, the state of cell bodies improved significantly. Effects of Ginkgolide B on cell viability, LDH release, Caspase-3 activity and Kalium ion concentration of embryonic hippocampal neurons treated with Ginkgolide B (GKB, 40μg / mL) and / or Aβ25-35 (25μM) for 48 h. The concentration of Extract of Ginkgo bilobaleaves (EGB) is 150μg / mL. The comparison on cell viability, LDH release, Caspase-3 activity and Kalium ion concentration of embryonic hippocampal neurons indicates that Ginkgolide B can protect hippocampal nerves, and Ginkgolide B has neuroprotective effect of hippocampal neurons from apoptosis induced by beta-amyloid 25-35. These figure show control group vs. Aβ25-35 group, P < 0.01; Aβ25-35 group vs. Ginkgolide B+Aβ25-35 group, P < 0.01; Aβ25-35 group vs. Ginkgolide B+I+Aβ25-35 group, P < 0.05; Ginkgolide B+Aβ25-35 group vs. Ginkgolide B+I+Aβ25-35 group, P < 0.05; Ginkgolide B+Aβ25-35 group vs. EGB+Aβ25-35 group, P > 0.05; Ginkgolide B group vs. EGB group, P > 0.05, it shows that Ginkgolide B and EGB have a similar capability of neuroprotection. Ginkgolide B and EGB both have neuroprotective effect of hippocampal neurons from apoptosis induced by Aβ25-35.Conclusions:1. Ginkgolide B has a clear role in promoting neuronal growth on normal hippocampal neurons.2. Ginkgolide B has neuroprotective effect of hippocampal neurons from apoptosis induced by beta-amyloid 25-35.PartⅡ: The mechanism of neuroprotective effect on Ginkgolide BObjective:Investigate neuroprotective mechanism of Ginkgolide B protects fetal hippocampal neurons apoptosis induced by beta-amyloid 25-35.Methods:Primary cultures hippocampus neurons. Aβ25-35 was cultured in PBS for 7 days at 37℃. After the culture was treated with the below different treatments: (A): control group; (B): Aβ25-35 group; (C): Ginkgolide B+Aβ25-35 group; (D): EGB+Aβ25-35 group; (E): Ginkgolide B group; (F): EGB group. Respectively use Western-blot and RT-PCR methods to detect changes in the expression of BDNF and NGF. Statistical analysis was made by using the computer software SPSS 10.0. The data was analyzed by using a two or one-way analysis of variance (ANOVA), then by Fisher's PLSD post-hoc test multiple comparisons. The data was expressed as mean±S.E.M. Statistical significance was set at P≤0.05.Results:Western-blot and RT-PCR analysis of hippocampal neurons cells revealed that Ginkgolide B and Extract of Ginkgo bilobaleaves have a similar capability of neuroprotection mechanism, and both can be achieved by the up-regulation of BDNF and NGF expression. Meanwhile, it seemed that the cytotoxicity effect could not be found in Ginkgolide B group and Extract of Ginkgo bilobaleaves group, but Ginkgolide B and Extract of Ginkgo bilobaleaves could promote neuronal growth in cultured embryonic rat hippocampal neurons, compared to the control group. Furthermore, the same changes of BDNF protein and NGF protein could be detected in different groups of hippocampal neurons cells by Western-blot. Thus, BDNF and NGF were inhibited in damaged hippocampal neurons cells and the effects could be partly recovered by Ginkgolide B. The micro-environment for nerve regeneration also improved. The figure show control group vs. Aβ25-35 group, P < 0.01; Aβ25-35 group vs. Ginkgolide B+Aβ25-35 group, P < 0.01; Ginkgolide B+Aβ25-35 group vs. EGB+Aβ25-35 group, P > 0.05; Ginkgolide B group vs. EGB group, P > 0.05. The data indicates that Ginkgolide B and Extract of Ginkgo bilobaleaves have a similar capability of neuroprotection mechanism. Conclusions:1. Ginkgolide B and Extract of Ginkgo bilobaleaves have a similar capability of neuroprotection mechanism, and both can be partly achieved by up-regulation the expression of BDNF and NGF.2. Ginkgolide B has neuroprotective effect of hippocampal neurons from apoptosis induced by beta-amyloid 25-35. The neuroprotective mechanism of Ginkgolide B is via up-regulation the expression of BDNF and NGF, improve the micro-environment for nerve regeneration.3. Ginkgolide B is the extract of ginkgo bilobaleaves, the natural medicine has wide variety of sources, stable and easy to use, it is worth further research, development and utilization.