| Objective:Alzheimer's disease (AD), as the most common form ofdementia, is a progressive central nervous system disorder which can causeincreasingly severe impairment in cognitive and me mory deterioration. AD ischaracterized extracellular deposition of of β-amyloid (Aβ) as senile plaques,as well as intracellular accumulation of hyperphosphorylated, aggregated tauas neurofibrillary tangles. In recent studies, Aβ deposition is thought to beprimary mechanism of AD pathgenesis. Preliminary studies found thatneurosteroids progesterone (PROG) and dehydroepiandrosterone (DHEA) canregulate amino acid neurotransmitter systems and affect the nervous systemexcitability via inhibition of neuronal glutamate (GLU) release and promotionof γ-aminobutyric acid (GABA) release. Hence, PROG and DHEA play animportant role in protecting neuron. PROG also can inhibit the Aβ inducedneurotoxicity with a concentration-dependent manner, and consequentlyprotect nervus. PROG might be able to regulate the excitability of the aminoacid neurotransmitter systems to against Aβ-induced neurotoxicity, thusimproving the symptoms of AD.In this study, the cerebral cortex of SD ratscultured in vitro and applied Aβ active fragment Aβ25-35induced neuronaldamage to establish the AD cell model. When PROG was administrated in themodel, determine the GLU and GABA's concentration in cell culture mediumusing high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS); detect neurons amino acid receptor subunit NMDAR2B(NR2B) and GABAR2(GBR2)receptor's expression by immunocytochemistry.This way could help observe the PROG effects on amino acidneurotransmitter release and its receptor's expression in the AD cellular modeland experimentally support the mechanism of PROG protecting neuronsagainst the Aβ neurotoxicity. Thus, provide new ideas for prevention and treatment of AD.Methods:1Primary culture of rat cerebral cortical neurons and observation ofmorphologicalCerebral cortical neurons were obtained from new born SD rats bytrypsin digestionand and mechanical dissection. The cells were seeded on theglass coverslip pre-coated with poly-L-lysine in the DMEM containing10%fetal bovine serum at the density of1.0×109L-1in the culture plates.Thecells were cultivanted at37oC,5%CO2and saturation humidity in anatmosphere containing. The neurobasal medium were changed24hours afterculture and followed by changing half medium every2days. Experimentationwith the neurons of living10days. Morphological changes of neurons wereobserved with inverted microscope.2Grouping and drug treatment programs2.1The establishment of Aβ25-35injury neuron modelOn10-day in culture, the neurons were divided into control group, GLUgroup(1mmol·L-1GLU) and Aβ-125-35-L,H,G groups (0.1μmol·L,1μmol·L-1,10μmol·L-1) randomly (n=6). Morphology of neurons were observed, and MTTassay was used to evaluate the cell viability after being treated24h.2.2The effects of PROG on Aβ25-35injury neuronOn10-day in culture, the neurons were divided into randomly with6ineach group, including control group, model group (1μmol·L-1Aβ25-35) andtreated groups (cotreated with1μmol·L-1Aβ25-35and PROG0.001μmol·L-1,0.01μmol·L-1,0.1μmol·L-1respectively). Morphology of neuronswere observed and MTT assay was used to evaluate the cell viability afterbeing treated24h.2.3The determination of GLU,GABA concentration and NR2B,GBR2expressionNeurons were divided into5groups and treated the same as2.2.GLU,GABA concentration were determined by HPLC-MS/MS. NR2B,GBR2expression were determined by immunocytochemistry. 3Cell viability determinitionThe MTT assay was used to detect the viability of the neurons cultured in24-well plates.The tenth day,20μL MTT (3.6mmol·L-1)was added to theculture medium after the drug's treatment. After4hours, the culture mediumwas removed and300μL DMSO was added to dissolve the crystallizations.The absorbance of the solution was detected at570nm on a ultravioletspectrophotometer and the cell viability was calculated by compare to blankcontrols.4Determinition of the GLU and GABA's concentration in cell culturemedium using HPLC-MS/MSThe cell culture medium(1ml) and3ml methanol were mixed in the10mlcentrifuge tube to precipitate for getting the supernate. Theleavings,prouduction of the desiccated supernate,were dissolved by100μLmorphine (MOR,the internal standard) with a final concentration of50ng·ml-1.Then, the samples were determinated using high performance liquidchromatography-tandem mass spectrometry (HPLC-MS/MS). The analyticalcolumn was ZIC-HILIC column(2.1mm×50mm,3.5μm) with a Zorbax SBC18guard column(2.1mm×12.5mm,5μm). The mobile phase, pumped in200μL·min-1, consists of acetonitrile and water. The column temperature was30℃. Five microliters of sample was analyzed. ESI ionization was used inselected ion monitoring mode. The positive quasi-molecular ions monitoredfor samples were m/z148â†'m/z130(GLU), m/z104â†'m/z87(GABA), m/z285â†'m/z200(MOR), respectively.5Determinition of the NR2B and GBR2recrptor subunit's expression byimmunocytochemistryOn11-day in culture, the neurons were treated by PROG andAβ25-35.Then, the neurons, growth on the glass coverslip,were poached by0.9%Nacl(4oC) and fixed by4%paraform(4oC). NR2B and GBR2recrptorwere drum dyeing by ABC immunocytochemistry. MOD was represented theexpression of recrptor which determined by a soft of Image-Pro Plus5.1. Results:1Neurotoxicity induced by Aβ25-35in primary rat cerebral corticalneurons1.1Morphological changement of neuronsObserved under inverted microscopy, neurons of control group, most ofprimary cerebral cortical neurons aggregated with their branching dendritesinterconnected as netwok. Their bodys were well-stacked.Compared with control group,24h after1mmol·L-1Glu treatment,numbers of neurons decreased obviously. On neuron's morphological, theirbodys were swelling, with reduced refraction and granuliform pieces.24hafter Aβ25-35treatment, with their increased concentrations, Neuons numberwas found a decreased trend. Number of1μmol·L-1Aβ25-35treatment groupwas roughly same as GLU group.1.2Influence on neurons viabilityCompared with control group, neurons were obviously damaged in GLUgroup which viability was (50.6±3.8)%(P<0.01). Aβ25-35treatment (0.1μmol·L-1,1μmol·L-1and10μmol·L-1) decreased neurons viability in adose-dependent manner with the Pearson correlation coefficient was-0.982(P<0.01). Cell survival rate of the group of1μmol·L-1Aβ25-35was(51.1±3.7)%, who was no different with GLU group(P>0.05).2Influence of PROG treatment on damagement neuron induced by Aβ25-35Observed by inverted microscope, compared with control group,numbers of neurons decreased obviously in model group (1μmol·L-1Aβ25-35).when add elevatory concentrations of PROG treatment, numbers of neuronshad an ascending trend. Massured by MTT assay, neurons were obviouslydamaged in model group which viability was (50.2±3.3)%(P<0.01) comparedwith control group. Compared with model group, PROG treatment (0.001μmol·L-1,0.01μmol·L-1and0.1μmol·L-1) increased neurons viability in adose-dependent manner with the Pearson correlation coefficient was0.980(P<0.01). The neuron viability was (100.2±2.6)%in0.1μmol·L-1PROG group, who was no different with control group(P>0.05).3The identification of amino acids assay and the effection of PROG treatment on GLU and GABA release in Aβ25-35induced AD modelUnder the assay, the linear calibration curves of GLU and GABA wereboth obtained in the range of5500ng·mL-1. Inter-day and intra-day precisionwere respectively less than7.4%and8.6%. The extraction recovery of themwere both above of89%. LLOQ of them was5ng·mL-1.Compared with control group, the level of GLU increased obviously andGABA decreased obviously in model group.The value of them were48.8%(GLU,P<0.01) and30.3%(GABA,P<0.01). Compared with modelgroup, the level of GLU was depressed by35.9%,43.1%and51.3%(P<0.01),GLU in0.1μmol·L-1PROG group was equivalent to controlgroup (P>0.05);Meanwhile,the level of GABA was enhanced by11.7%,23.4%and41.8%(P<0.01orP<0.05), GABA in0.1μmol·L-1PROG group wasno different with control group(P>0.05). PROG treatment (0.001μmol·L-1,0.01μmol·L-1and0.1μmol·L-1) decreased level of GLU and increased level ofGABA in a apparente dose-dependent way and the Pearson correlationcoefficient were-0.951(P<0.01) and0.844(P<0.01).4Effection of PROG treatment on the expression of NR2B and GBR2recrptor subunit in Aβ25-35induced AD modelCompared with control group, both of the expression of NR2B andGBR2recrptor subunit decreased significantly in model group. Theyrespectively loss40.8%(P<0.01) and44%(P<0.01). Compared with modelgroup, the expression of NR2B increased after24.4%,33.9%,59.6%(P<0.01)by different concentrations of PROG treatment (0.001μmol·L-1,0.01μmol·L-1and0.1μmol·L-1); meanwhile GBR2recrptor subunit wasenhanced41.5%,48.3%,65.9%(P<0.01). The expression of NR2B andGBR2recrptor subunit in0.1μmol·L-1PROG group were both having nodifference with control group.Conclusions:1The research successfully establish a cell AD model whose damagementinduced by Aβ25-35on primary rat cerebral cortical neurons. Aβ25-35treatment decreased neurons viability had no different with1mmol·L-1 GLU group. And Aβ25-35treatment cut down neurons viability in adose-dependent manner.2PROG treatment inhibited the declination of cell viability induced maybeby Aβ25-35in a dose-dependent way. Cell survival rate of the group of0.1μmol·L-1PROG was no different with control group.3PROG protected neuron against injury by decreasing the GLU level andincreasing the GABA level.4PROG protected neuron against injury by enhancing the expression ofneuron NR2B and GBR1receptor subunit. |
PDF Full Text Request