Experiment On The Neurotoxicity Of Amyloid Beta Protein And The Protective Action Of Panoxadiol In Primary Cultured Fetal Rat Cortical Neuron

Alzheimer's disease(AD) is a kind of degenerative disease of nervous system in senectitude and the key feature is progressive dementia. The distinctive Pathological changes in AD are the formation of senile plaques and neurofibibrillary tangles as well as loss of synapse and neuron. Amyloid beta protein(Ap), the major ingredient of senile plaques, plays a critical role in the development of AD. The investigation demonstrated that Aβ is the common iter of miscellaneous causes inducing AD.Aβ is the key factor of AD's formation and development. Aggregated Aβ_1-40 has extensive eurotoxicity to neuron,which can induces apoptosis of neuron. The utility of ginseng is great nourishment of renal qi. The investigation demonstrated that panaxoside and it's simple substance have utility of amelioration learning and study and inhibitory apoptosis of neuron, which role is like nerve growth factor. The author use Aβ to damage fatal rat cortical neuron and uses panoxadiol which is extracted from panaxoside to protect the fatal rat cortical neuron,and to investigate toxic effects of Amyloid beta protein (Aβ_1-40) on primary cultured fetal rat cortical neuron and to explore antagonism of panoxadiol to toxicity of Amyloid beta protein.Objective:To investigate protection of panoxadiol on toxic effects of β-amyloid protein(Aβ_1-40 on primary cultured fetal rat cortical neuron and to explore antagonism of panoxadiol to toxicityMethods:1. The primary cultured fetal rat cortical neuron were growth and development for 7 days and were randomization. Then treated with aggregated Aβ_1-40 with different concentration for 48h.using MTT assay and LDH kit to detect the survival rate and the changes of vitality of lactate dehydrogenase(LDH) in culture solution of primary cultured fetal rat cortical neuron, and investigating the Morphologic change of fetal rat cortical neuron.2. Primary cultured fetal rat cortical neuron were pretreated with panoxadiol for 24h and then treated with for 48h.By using the same arker, we also detect the survival rate and the changes of vitality of lactate dehydrogenase(LDH) in culture solution of primary cultured fetal rat cortical neuron, panoxadiol as a protective agent,we observed the protective efect of panoxadiol on damage induced by Aβ_1-40.Results:1. Primary cultured fetal rat cortical neuron were performed with Aβ_1-40(3, 6, 12μmol/L) for 48 hours,Survival rate of nerve cells were decreased and LDH activity was increased in culture solution,and the growth state is bad. The OD values were 0.686±0.036,0.440±0.056, 0.196±0.040 respectively in MTT experimen and LDH activity in culture solution were (265.7±25.1) U/gprot, (351.5±42.1) U/gprot,(557.8±25.8 ) U/gprot. The survival rate was decreased and LDH activity wasincreased following the increased Aβ_1-40 concentration. There was significantly diference between higher dosage group(6,12μmol/L)and control group (P<0.01),and low dosage group(3umol/L) also had statistical significance (P <0.05).2.Primary cultured fetal rat cortical neuron were pretreated with panoxadiol (10, 20, 40μg/ml) for 24h and then treated with Aβ_1-40 for 48h, the OD value were 0.239±0.027, 0.412±0.052,0.686±0.022 and LDH activity in culture solution were (549.9±17.4) U/gprot, (447.7±23.6) U/gprot, (2853±44.9)U/gprot respectively.But the group which singly proformed with Aβ_1-40 (12μmol/L) for 48h, survival rate of nerve cells and vitality of lactate dehydrogenase(LDH) in culture solution were 0.215±0.014,(569.9±18.6) U/gprot. Compared with group (12μmol/L Aβ_1-40),there was significantlydiference in panoxadiol higher dosage group(20,40mg/L) (P<0.01),and low dosagepanoxadiol group(10mg/L)also had statistical significance (P<0.05). And the state offetal rat cortical neuron is better than the group which singly proformed with Aβ_1-40(12μmol/L) 48h.The morphous of fetal rat cortical neuron is almost intact.Discussion:1 .Establishment of AD cellular modelSince Yankner 1990 first confirmed toxic effect of synthetic Aβ on hippocamp neuron in rats,cellular model was broadly used for research of mechanism of neurotoxicity due to advantages of simplification and utility. Analysis of typical indexes including morphological observation of neuron, cell survival proliferation and release of LDH. LDH is signed enzyme in cells characterized by stable chemical and biological nature. Usually leakage of LDH was little,leakage of LDH was related with integrity of cellular membrane. Vitality of LDH in nutrient solution detected indirectly reflected toxic effect of Aβ on neurocyte and anti-toxicity of drug on toxic effect of Aβ.Our results indicated:obvious neuron morphology was demonstrated in a certain concentration of Aβ_1-40, and cell state was not good, nervous process showed retraction , shedding and disintegration, decreased survival rate and increased releasing of LDH,which could indicate successful cellular model.2.Alzheimei disease and Aβ proteinPrevious research indicated: polymerized Aβ can affect special structure related with intellectual ability in brain such as Hippocampus and some cortical areas. It casused some pathological changes: considerable amyloid degeneration of nerve fibril, senile plaques and granulovacuolar degeneration, even neuronal absence .Our study indicated: panoxadiol can antagonize t toxicity of Aβ on neuron and protect nerve cell.3. Treatment of Alzheimer diseaseAlthough actual etiology is unclear,current accepted Aβ hypothesis considered Aβ triggered course of AD and played very important role in genesis and development in AD.Based on the hypothesis,researchers proposed decreasing production of AP,prevention of Aβ aggregation,formation of fibril and increasing Aβ degradation orcleaning,whatever tried to decrease toxicity of Aβ. Our study confirmed panoxadiol canantagonize morphological changes of neuron caused by Aβ and improve survival rate ofneuron, protect releasing of LDH, which indicated panoxadiol played a certain protectiverole in pathological model of neuron induced by Aβ and provided experimental supportfor panoxadiol for clinical treatment of AD.Conclusion:1. Polymerized Aβ_1-40 with a certain concentration can induce morphological changes ofcultural cortical neuron and increasing concentration of LDH in culture solution.Polymerized Aβ_1-40 with a certain concentration played a clarified toxicity on neuronand the toxicity was dose dependent.2. Panoxadiol can antagonize morphological changes of neurocyte , decreasing survivalrate and increasing releasing of LDH.