| Background:Alzheimer’s disease (AD), a devastating neurodegenerative disease, is the most common form of dementia. Currently, the "amyloid hypothesis" is the most widely accepted explanation for the pathogenesis of AD. According to this hypothesis, amyloid-β (AP) peptide, the major component of senile plaques, is considered to play a crucial role in the pathological cascade invoved in the etiology of AD. Accumulation of Aβ in the specific encephalic region can lead to synaptic dysfunction, disrupting neural connectivity and neuronal death, which give rise to the clinical symptoms of AD, progressive loss of memory and cognitive impairment.At present, treatment of AD research strategy includes three main areas:first is the development of low-toxicity of new drugs intended for choline; second is the application of neurotrophic substances, including direct intraventricular perfusion and neurotrophic factor gene-modified cell transplantation in the brain; and the third is stem cell transplantation therapy. But these therapys is just to slow down the degeneration of neurons or accelerate the genesis of neurons, and fundamentally don’t prevent of disease in AD patients, their long term treatment should not be optimistic. Clinical Chinese medicine research showed that Acorus prescription is a mainly prescription in treating AD infrastructure, first frequency single drug in using, and the effect is curative. However, Acorus’ rivalry on AD and anti-AD "Aβ" mechanism has not yet been clarified, which affect the further improvement of the Acorus to anti-AD effect.Our result showed that the volatile oil of Acorus has a significant effect on learning and memory ability of mice in AD model, roles’ mechanisms relatied to its antagonistic expression of apoptosis-related factors. These results suggest that essential oils could be used for prevention and treatment of AD. Yoshifumi’s studies proved that the main component of volatile oil of Acorus in protecting brain cells is β-asarone and eugenol. β-asarone and eugenol can be resistant to apoptosis, but its molecular mechanism is not clear. To explore β-asarone and eugenol in vitro AD cell model is also has Neuroprotective effects and mechanism and why, first of all, we use Aβ1-42on primary cultured cortical neurons for the establishment of a neuron injury model induced by Aβ1-42oligomers. The aim of this study was to determine the rivalry function of β-asarone and eugenol on the synthesis and release of anti-apoptosis-related factors, and further more, to study the protective function of β-asarone and eugenol on the neuron cell Models of Alzheimer’s Disease by Amyloid-p.Objective1. To observe β-asarone and eugenol on the impact of morphology and cell viability on primary cortical neurons and PC12cell in normal state, in order to get started with acquaintance of neuroprotective effect of β-asarone and eugenol;2. To observe β-asarone and eugenol on the protection of primary cortical neurons and PC12cell models of Alzheimer’s Disease by Amyloid-β, and discuss on its mechanism.MethodsExperiment one1. First take of fetal rat cortical tissue for cortical neurons in culture, then identification of immunocytochemistry staining after sixth days’culture;2. To prove the establishment of model of cortical neuron injury induced by Aβ, using ELISA detect related factors about the release level; 3. Using MTT to observe cell viability on primary cortical neurons with the effect of β-asarone and eugenol;4. After sixth day of primary cortical neurons culture, establish a neuron injury model induced by Aβ1-42oligomers, then use β-asarone and eugenol24hours later on the cells. To use western blotting to detect the extraction of protein of apoptosis-related factors three days later;Experiment two5. Under normal state, Acorus main active ingredient β-asarone and eugenol on impact of morphology and cell viability of PC12cell;6. To establish of model of PC12cells injury induced by Aβ1-42oligomers;7. After sixth day of PC12cells culture, establish PC12cells injury model induced by Aβ1-42oligomers, then use β-asarone and eugenol24hours later on the cells. To use RT-PCR to detect the extraction of mRNA of apoptosis-related factors three days later;8. To study the protection mechanism of β-asarone and eugenol on Aβ1-42induced PC12cells model through Tunel method.Results:Experiment one1. After sixth days’culture, primary cortical neurons’dendritic was obvious, immunocytochemistry staining showed that neuronal markers is positive;2. The level of TNF-alpha detected by ELISA, showed that the establishment of model of cortical neuron injury induced by Aβ was set up;3. Compared with the normal cultivation of neurons, the third day of cortical neurons and10-10-10-5M of β-asarone and eugenol co-incubated for24hours, showed that10-6M β-asarone and10-/M eugenol survive cells in the highest;4. Western blotting detection of apoptosis-related protein, showed that Aβ1-42oligomers effects on cortical neurons after6h, promoting the expression of apoptotic protein Bax, to24h reaches a higher level;On the other hand, expression of anti-apoptosis protein Bcl-2as a prolonged Beta1-42oligomers effects gradually reduced; 5.10-10-10-5M of β-asarone and eugenol can be obvious antagonism of Bax protein upregulation and Bcl-2expression of anti-apoptotic proteins declined induced by Aβ1-42oligomers;Experiment two6. Compared with the normal cultivation of PC12cells,10-10-10-5M of β-asarone and eugenol co-incubation24h respectively with the PC12cells, showed that10-6M β-asarone and10-/M eugenol survive cells in the highest;7. RT-PCR for detection of apoptosis-related genes, showed that Aβ1-42oligomers effects on PC12cells, promote the expression of apoptotic mRNA Bax, gradually reducing expression of anti-apoptosis mRNA Bcl-2;8.10-10-10-5M of β-asarone and eugenol co-incubation can be obvious antagonism of Aβ1-42oligomers inducing apoptosis of PC12cells induced by Bax gene upregulation and anti-apoptotic gene expression of Bcl-2declined;9. TUNEL detection of apoptosis of PC12cells:compared with the control group, cultivation of β-asarone, eugenol and its mixture incubated with PC12cells induced by Aβ1-42oligomers, significantly reduced the number of positive cells.mixed-incubation group better than the separate effects of monomers of β-asarone and eugenol.Conclusions:1. Aβ1-42oligomers can significant inhibition of neuronal regeneration, this toxic effects may be related to promoting the expression of apoptosis-related factors.2. Aβ1-42oligomers, which might be related to inhibiting expβ-asarone and eugenol apparently antagonistic the neurons (PC12cells) apoptosis induced by ression of apoptosis-related factors.3. For Aβ1-42oligomers intervention in primary cortical neurons and the impact of PC12cells,β-asarone and eugenol mixed-incubation group better than the separate effects of monomers.This study provides a scientific basis of Acorus for anti-Alheimer’s disease drug research. |
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