The Effect Of Neuropeptide, Soybean Flavone And C Peptide On The Expression Of Nerve Cell Protein App, Nos1 And β-secretase

Background and Purpose:Alzheimer's disease(AD),characterized clinically progressive loss of memory and cognitive function,pathologically neurofibrillary tangles(NFT) inside neuro cell and senile plaques(SPs) outside neurocell,is an age-related neurodegenerative disorder.It is indicated that the main mechanism of AD is the toxicity ofβ-amyloid(Aβ) to cholinergic neuron.So it is one of hot spots to investigate the production,degradation and clearance of Aβ.In recent years,the patho-physiological function of nitric oxide(NO) in central nervous system is one of the hot spots in neuroscience study.NO is an active material of blood vessel and nerves,acting as accommodating cerebral blood flow,promoting or inhibiting transmitter delivering,participating synaptic plasticity,neuronal excitation and inflame damage,and so on,as well relating study of remembrance. Moreover,NO possess important pathology effect,participating vascular dementia possibly.But overdose of NO can induce apoptosis by disording intracellular Ca²⁺,producing peroxidize stress,interacting brain energy metabolism,and so on.Lots of experimental data and clinical observation indicate that cerebro-hippocampus relate to remembrance.It can provoke the functional deprivation of recent memory induced by the damage of hippocampus,cerebral trigone,pars mamillaris hypothalami or marnmillo-thalarnic fasciculus and the vicinity constitution.The primary culture of hippocamal neurons is one of the most important methods for the research of neuro-cell.So,establishing the culture of invivo hippocampus cell possess great significance.In this experiment,the primary culture of hippocamal neurons is induced as aphrenia model by NO,which participates the patho-physiological damage of ischemia and oxygen deficiency.The information transduction between nerve cells is implemented by neurotransmitter.Acidic peptide is a kind of small molecular peptide isolated by our lab.It exists in animal widely and participates regulation of many functionis.It is also a good treating function to senile dementia.Soybean flavone is a kind of flavone compound.Some research have proved that soybean flavone can increase activity against oxidase so that it can promote anti-oxidation of body.This experiment explores biological mechanism to delay brain aging by the study of action of soybean flavone to protein molecules in hippocampal neurons.C peptide is another kind of peptide isolated from animal body by our lab.This experiment carries out a primary research to sdudy relationship between c peptide and neuron apoptosis.Materials and Methods:Neonatal rat hippocampi were dissected and digested with trypsin.Monoplast suspension was produced and then was innolulated,changed cultured fluid,and so on. After 10-12 days later,the biological character of cell was stable and could be used for experiment.Main procedure are arrayed as follow.1,Hippocampus tissue was dissected.2,The hippocampal neurons were cultured for 10 to 11 days in and then identified by immunohistochemistry of neurone specific enolase(NSE).The depurating rate of neurons reach to 95%,then the neurons were used for experiments.3,The primary hippocampal neurons were cultured for 10 days.And were pretreated with different concentrations of neuropeptide,soybean flavone and C peptide for 6 hours,then exposed to 50 umol/L sodium nitroprusside(SNP) for 24 hours.In this research,hippocampal neurons were randomly divided into eleven groups.Ⅰgroup;normal group.Ⅱgroup was the model of neuronal apoptosis induced by SNP;Ⅲgroup:neuronal apoptosis model +0.15mg/mL AP;Ⅳgroup: neuronal apoptosis model +0.3mg/mL AP;Ⅴgroup:neuronal apoptosis model +0.6mg/mL AP;Ⅵgroup:neuronal apoptosis model+0.15mg/mL soybean flavone;Ⅶgroup:neuronal apoptosis model+0.3mg/mL soybean flavone;Ⅷgroup:neuronal apoptosis model+0.6mg/mL soybean flavone;Ⅸgroup:neuronal apoptosis model+0.15mg/mL C peptide;Ⅹgroup:neuronal apoptosis model+0.3mg/mL C peptide;Ⅺgroup:neuronal apoptosis model+0.6mg/mL C peptide;These eleven group hippocampal neurons continue to be cultured for 24 hours,and then could be used to do experiments as follow.The experimental data were expressed and analyzed by one-way analysis of variance(ANOVA) and two-independent-samples tests.α=0.05 was the standard of test.Results:1.The model of cultured neonatal rat hippocampal neurons in vitro was established successfully and the model of neuronal apoptosis induced by SNP could be set up successfully.2.Acidic peptide,soybean flavone and C peptide could protest hippocampal neurons against SNP's neurotoxicity in a dose dependant manner.The expression of amyloid precusor protein(APP),β-secretase(BACE) and NO synzyme 1(NOS1) in neuronal apoptosis model group increased significantly compared with that of normal control group(p＜0.05).And the expression of APP,BACE and NOS1 in experimental group decreased significantly compared with that of SNP's group(p＜0.05).Conclusions:1.Neonatal rat hippocampal neurons were cultured and the model of neuronal apoptosis induced by SNP was set up successfully.2.Neuropeptide,soybean flavone and C peptide could protest hippocampal neurons against the toxicity of SNP.3.It demonstrates that the mechanism of inhibiting apoptosis is that amyloid precursor protein(APP),β-secretase and nervous system type nitricoxide synthase(NOS1) are inhibited to synthesis.