| Alzheimer's disease (AD) is a hazardous and severe nerves degenerative disease. One of the typical pathological changes of AD is the massive senile plaques, whose main component is amyloid beta-protein. Most researchers considered that the accumulation and deposition of Aβ in brain tissue is the common pathway where AD be induced by various reasons, as well as the key element in forming and developing of AD. Present findings indicate that the dysfunctional deposition of Aβ can switch on the series cascade reaction participated by sorts of proteins, induce neurotoxicity, injure the nervous system and result in loss of cognition function. However, how many and which proteins and enzymes have participated in the neurotoxicity damages induced by AP? How these protein molecules affect each other? The underlying molecular mechanism of Aβ neurotoxicity is remaining unclear.To explore the mechanism of A β neurotoxic effects and its primary target site molecules, primary cultured hippocampal neurons were treated with various concentrations of A P 1.40 (0.1, 1, 10, 20, 30 μ mol/L),cell survival rate was measured by using MTT method, and AD-damaged neural cell model was established by selecting suitable concentrations of Aβ1-40.SELDI-TOF and protein array technology was combined and applied into the using of detecting the protein expression of intracellular fluid so as to search for the differencial expression proteins associated with induction of Aβ1-40.Information on differencial proteins was searched in Swiss Protein Database to define their characters initially. Semi-quantitation RT-PCR was applied in detecting the variation of differential proteins in cultivating hippocampus neuromime on the level of mRNA transcription. Simultaneously, protein expression changes, induced by Aβ in cells, were verified on the level of genetic transcription and protein expression.Protein expression changes induced by Aβ in cells was analyzed to find the possible pathway between the induced neurotoxicity patricipated with Ap and the cell damages.Same experiment condition was reduplicated to display whether its pathway unlocked or enhanced by detecting hippocampal neurons in vitro culture induced by APmo. MeanwhiIe,Bak Foong Pills(BFP), estrogenic-alike drug which was confirmed as effective nerves protective dose, was supplied to interfere and observe the changes of its pathway. In addition, the expressions of correlated proteins, aopE and LRP, were detected in AD cell model in this study. Effects on removing exogenous Ap^oin medium by interaction of apoE and LRP were explored as well. The experimental results show that:1. Significant cytotoxicity was observed after incubated with 10 P mol/L A P 1.40 (P<0.0\) , while the cytotoxicity was more mild at 1 u mol/L A 3 mo CP<0.05) , Then the two doses were used to set up AD cells model.2. No protein just only expressed in neurons after A P treated was determinated in proteins profiles ranged from 4000 to 20000 Da. Overall, nine differential expressive proteins were found (PO.01)3. One of different expressive protein identified in Swiss-Prot Protein knowledgebase was close to SKP1A "S-phase kinase-associated protein 1A (Cyclin A/CDK2-associated protein pi 9)" from mouse, which up- regulated only after treated with lOumol/LA 3 mo-4. Using semi-quantitated RT-PCR, the level of SKP1A mRNA increased after treated with 10 u mol/L A P 1-40 at 3h and the progress lasted till 48h. However, the level of SKPIA mRNA just increased at 3h transitorily after treated with 1 y mol/L A P M0-5. Incubated with 10 y M A P 1.40, bcl-2 expression was down-regulated, while bax expression was up- regulated. The ratio of bcl-2/bax was low, which enhanced the expression of caspase-3. Apoptosis was observed in neurons after incubated with lOumol/LAP M0.6. Interfered with BFPC100 u g/ml), the expression of bcl-2 induced by A P mo was resisted partly while no marked effects were found in the expression of bax, and the apoptotic index was reduced. The ratio of bcl-2/bax was higher and the expression of caspase-3 was reduced.7. ApoE expression was up-regulated after incubated with 1 u mol/L A & |.4O, while down-regulated with 10umol/L A P ho. LRP expression was up-regulated at both A P 1.40 concentrations. Extracellular 1 u mol/L A P 1.40 can be up-taked by neurons themselves, and then can reduce A P 1-40 concentration in culture medium. But it's reverse if 10 u mol/L A P 1-40 in culture medium.Summarily, abnormal expressions of multiple proteins induced by Ap excessive deposition out of hippocampal neurons was investigated by applying techniques and devices in proteomics and molecular biology. SKPlA,a protein molecule associated with cell cycle regulation, which was persistently and highly expressed under noxious dosage of Ap, co-acted with multiple molecules including cyclin A, leading to the disorder of hippocampus neurons cell cycle, the disproportion of apoptosis controlling gene bcl-2/bax,and the activation of caspase-3.The neurons was led to excessive apoptosis, displaying evident cytotoxicity. One of the mechanisms in Ap injurying nervous system was thus found. Bak Foong Pills, which has the neuroprotective effects, can block the excessive apoptosis of hippocampus neuron by regulating the ratios of bcl-2/bax and reducing the gene expression ofcaspase-3.
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