The Effects Of Small Molecular Acidic Peptide On The Expression Of Nerve Cell Apoptosis Relate Genes Bcl-2 And Bax

Background and Purpose:Alzheimer's disease(AD), characterized clinically by progressive loss of memory and cognitive function, pathologically by senile plaques(SP), neurofibrillary tangles(NFT), and neuronal loss, is an age-related neurodegenerative disorder. The primary pathogenesis and cause of AD is not yet ciear. The morbidity of AD is high among the elderly. In developed countries AD has already been the fourth leading cause of death after heart disease, cancer and stroke for lack of effective early diagnostic and therapeutic methods. So it's very important and urgent to actively study AD. Recent research have found that apoptotic death of neurons is a notable feature of AD neuronal loss and abnormal apoptosis induce a great of AD neuronal loss. So researching the molecular mechanism of neuronal apoptosis have great meaning for enucleating the pathogenesis of AD.NO is an unstable but multifunction gas molecule which can diffuse freely through the membrane of the cell. In central nervous system NO is synthesized from L-arginine by neuronal NO synthase (nNOS) and is a neuronal messenger molecule. However its overproduction has been implicated in the pathogenesis of neural degeneration such as AD. Some research showed that No can induce apoptosis of neurons through many pathways.The culture of neonatal rat hippocamal neurons is the primary method to researchmorphology , function and damage of neurons. Hippocampal is the main pathological changes area of Ad and correlate strongly to memory and cognitive function. So , the model of neuronal apoptosis induced by Sodium nitroprusside SNP could be established successfully.Neuropeptides are some important substances which have functions of neuronal signal transmission and regulation. They distribute widely in the central neural system, participating in many kinds of functional regulation. An Yuhui has isolated some small neuropeptides from bovine brains and found a new peptide: bovine acidic neuropeptidel (AP), small molecular peptide without antigenicity. Animal experiments have proved that ①it can regulate neural system by increase amino acid of brain and affecting the contents of cGMP of brain, ② by increasing the synthesis of protein and the antioxidants defences of brain and improving the energy metabolise of brain and decreasing the synthesis of No in brain ,AP have good treatment on model of AD rats, but the methanism of AP on molecular level remains unknown, so, we study the effect of acidic peptide on expression of apoptosis relate genes bcl-2 and bax.In this study, the new born hippocampal neurons were cultured in serum free medium. And the effects of AP on apoptotic neurons were observed by setting up the neuronal apoptosis model, in which SNP was added into medium. The mechanism of the protective function of AP to apoptotic neurons was studied by MTT assay, fluorescent staining, DNA agarose gel electrophoresis analysis and Western blot which was used to investigate the expression of bcl-2, bax. Materials and Methods:Neonatal rat hippocampi were dissected and diested with 0.125% trypsin. Tissue was dissociated by repeated trituration and cells were seeded at a density of 0.8 ×106 cells /Ml.. All experiments were performed in 10-12-day-old cultures.1. Neuron' s identification: The hippocamal neurons were cultured for 10 days in vitro. And the cultured hippocamal neurons were purified with B27, then were identificated with IHC of Neurofilament protein.2. The prime hippocamal neurons were cultured for 11 days in vitro. And thecultured hippocamal neurons were pretreated with different concentrations of AP for six hours , then exposed to 50 μ mol/L SNP for 24 hours . In this research, hippocamal neurons were randomly divided into five groups. I group was normal group; II was the model of neuronal apoptosis induced by SNP;Ⅲ, Ⅳ, Ⅴ group was neuronal apoptosis model + AP 0.0375mg/mL,0.075mg//mL ,015mg/mL respectively. Normal control and model group hippocamal neurons had not been treated . Cells were harvested at same periods and then MTT assay were used to test the survival rate of neurons, agarose gel electrophoresis assay used to analysis DNA ladder, fluorescont staining were used to study the morphologic change of apoptosis such as chromatin condensation, shrinkage, cleavage ,Western blot was used to investigate the expression of apoptosis related gene bcl-2, bax in translation level to explain the mechanism of the protective function of AP to apoptotic neurons induced by SNP. The experimental data were expressed by and analyzed by one-way analysis of variance (ANOVA). a =0.05 was the standard of test. Results:1. The model of cultured neonatal rat hippocampal neurons in vitro was established successfully.2. The model of neuronal apoptosis induced by SNP could be set up successfully. 3.AP improved the morphologic changes elicited by SNP.4. AP can significantly increase the survival rate of neurons against SNP in a dose dependent manner. The neurons survival rate in neuronal apoptosis model group decreased to 58.9% ,which showed significant different compared with that of normal control group (P<0.01) . The neurons survival rate in AP exprimental group rose, and there appeared significant different between AP exprimental group and neuronal apoptosis model group (P<0.05) .5. AP inhibited the DNA ladder. DNA agarose gel electrophoresis analysis did not show a ladder in normal group and AP exprimental group , but there was a ladder in neuronal apoptosis model group.6. AP protected the neuronal neclei from shrinkage condensation and cleavage triggered by SNP.7. AP up-regulated the expression of Bcl-2 and down-regulated the expression of Bax in a concentration dependent fashion. Conclusions:1. The model of neuronal apoptosis induced by SNP could be set up successfully. 2 .AP can inhibit the neurotoxicity of SNP to neurons and significantly increase the survival rate of neurons in a dose dependent manner, and can inhibit the apoptosis of cultured hippocamal neurons.3. The mechanism of AP neuronal protective function is likely involved in regulating the expression of apoptosis related gene Bcl-2, Bax, Which played a pivotal role in survival of cell. AP can accelerate the expression of Bcl-2 and restrain the expression of Bax,4. The effects of AP are associated with its concentration in this experiment.