The Primary Study On The Protective Effect Of Rg1 On The Damage In Cultured Hippocampal Neurons Exposed To Amyloid-β-Protein

OBJECTIVE:To explore primari ly if the effects of Rgl of protecting neurons correlate with the signal pathway of NF-κ B and discuss it' s mechanism of effects in AD cellular model which use primitive hippocampal neurons to be the deputy of cholinergic neuron and Aβ_25-35 induce apoptosis.METHODS: Primitive hippocampal neurons were culctured. Identifying the purity of the hippocampal neurons through the method of immunocytochemisty using the antibody of NSE. MTT colorimetric analysis was used to measure the survival rate of hippocampal neurons cultured with soluble beta-amyloid in oder to find out the best concentration and time point to make AD cell mode. Pretreated with the Ginsenoside Rgl for 24h, the survival rate of hippocampal neurons was measured by MTT colorimetric analysis after cultured with Ginsenoside Rgl and soluble beta-amyloid 24h for choosing the best concentration of Ginsenoside Rgl to protecting the hippocampal neurons. The Laser Scanning Confocal Microscope (LSCM) and Leica Confocal Analysis Software were performed to assay the activity of nuclear factor-kappa B in hippocampal neurons that double marked by FITC\PI. Content of NO, NOS in cultured supernatant were measured after 24h when cultured neurons were exposed toAβ_25-35. The data were expressed by x±s and analysed statistically bySPSS11.5. RESULTS:1. Hipplcampal neurons were separated and chltured from newborn(less than 24 hours) SD rats. Cultures were maintained in a CO₂ incubator with 5% CO₂, 37 ℃,95% humidity. Serum containing media were replaced with serum free media containing 2% B27 every two days. After cultured for 6 days, Hipplcampal neurons were fixed in cold acetone and assessed by neuron specific enolase(NSE)immunohistochemistry staining. The amount of positive neurons was approximately 90% of the total number of cells in culture.2. Hipplcampal neurons were observed by inverted phasecontrast microscope, and it was obviously that all the groups induced by soluble A 3 25-35 was worse than the normal group. Furthermore, the higher concentration the A 3 25-35 was, the harmer cells were. Assayed by MTT colorimetric analysis, Concentrations of A P 25-35 could decrease the survival rate of Hipplcampal neurons (P<0. 05), but on the other hand, time points couldn' t influence the vigors of Hipplcampal neurons obviously (P=0.202)analysed by two-factor ANOVA. The group of 40umol/L have a significant level compared to the group of 0y mol/L in every time point. Based on this result, 40y mol/L and 24h were selected as the stable effective concentration and time point of AP 25-35 to make AD model in hippocampal neurons.3. Observed by inverted phasecontrast microscope, all the concentrations of Ginsenoside Rgl groups are better than the modle group after pretreated Ginsenoside Rgl for 24h and subsequently co-cultured with Ginsenoside Rgl and A 3 25-35 for 24h. The living status of Ginsenoside Rgl of 2N 4 u mol/L had better than modle and K 8^ 16ymol/L groups. Assayed by MTT colorimetric analysis, all the concentrations of Ginsenoside Rgl groups could increase the survival rate of hipplcampal neurons. Ginsenoside Rgl of 2 ^ 4 y mol/L had better protective effects than modle and K 8> 16 y mol/L groups after culturing 24h, but couldn' t recovery to the level of the normal group. The best concentration was 2, 4u mol/L.4. One-Way ANOVA was used to analyse the activity of NF-k B, and it showed the activity of the model group was significantly lower than the normal group(P<0. 01). It suggested that soluble A P 25₃5 can downregulated the activity of NF-kB and induced cell apoptosis. The activity of NF-k B of the 2, 4, 8y M Rgl group were all higher than the model group(P<0. 01). It suggested that Rgl could upregulated the activity of NF-k B and inhibited the neurotoxicityOf A 0 25-35-5. TO investigate the changes of NO, NOS of cultured hippocampal nerons indued by A 3 25-35 and protective effect of ginsenoside Rgl. Content of NO, NOS in cultured supernatant increased obviously when hippocampal neurons were exposed to A{325₃5 for 24h than that of control group. After pretreated with Rgl for 24h an then exposed to A P 25-35, NO, NOS levels were lowered markedly.CONCLUSIONS: Soluble AP 25-35 can downregulate the activity of NF- k B in...