The Study Of Ginsenoside Rg1 On The Activation Of NF-κB In Astrocytes Which Induced By Aβ And Its Inflammation Mechanism

Objective:To explore the changes of the survival rate of astrocyte using the soluble beta-amyloid and Ginsenoside Rgl; To study the relationgship of Ginsenoside Rgl and soluble beta-amyloid to regulating the activation of nuclear factor kappa B(NF-kB) and its inflammation mechanism in the rat primary astrocytes.Methods: The primary rat astrocytes were cultured and the morphologies were observed by inverted phasecontrast microscope. The purity of the astrocytes was identified through the method of immunocytochemisty using the antibody of GFAP. AD cell modle in astrocyte induced by the soluble beta-amyloid was determined by MTT colorimetric analysis measuring the viability for choosing the best concentration and time point. Pretreated with the Ginsenoside Rgl for 24h, the survival rate of astrocytes 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 astrocytes. The Laser Scanning Confocal Microscope(LSCM) and Leica Confocal Analysis Software were performed to assay the activity of nuclear factor-kappa B in astrocytes which double marked by FITC\PI. The supernatant were collected at the last time point, then the expression of IL-6 protein was assayed by enzyoned-1inked immunosorbent assay(ELISA). The datawere expressed by x±s and analysed statistically by SPSS11.0.Results:1. Almost pure cells were obtained after primary culture, subculture and differential cell adhesiveness. They coincided with the characters of astrocyte forms: unregular shapes, big cell bodies, abundant cytoplasm, many longer apophyses and round or ovi-round karyons which leaned to one side. Immunohistochemical staining demonstrated that nearly 95%of the cells were positive.2. Astrocytes were observed by inverted phasecontrast microscope, and it was obviously that all the groups induced by soluble Aβ 25-35 had been worse than the normal group. Furthermore, the higher concentration of the A β 25-35was, the harmer the cells were. Assayed by MTT colorimetric analysis, concentration of A β 25-35 could influence the survival rate of astrocytes (P<0. 05), but on the other hand, time points couldn' t influence the viability (P=0. 4)analyzed by two-factor ANOVA. The groups of 40μmol/L were significantly different than the groups of 0μ mol/L in three time points (P<0. 05). At last, 40μmol/L and 24h were selected as the stable effective concentration and time point of A β induced in astrocytes.3. Observed by inverted phasecontrast microscope, all the concentrations of Ginsenoside Rgl groups were better than the modle group after pretreated Ginsenoside Rgl for 24h and subsequently co-cultured with Ginsenoside Rgl and Aβ 25-35 for 24h. The living status of Ginsenoside Rgl of 1 2, 4μmol/L were better than the modle group and 8,16 μ mol/L groups. Assayed by MTT colorimetric analysis, all the concentrations of Ginsenoside Rgl groups could increase the viability of astrocytes. Ginsenoside Rgl of l,2,4μ mol/L had better protective effects than the modle group and 8, 16 μ mol/L groups after culturing 24h, but couldn' t recovery to the level of the normal group.4. One-Way ANOVA was used to analyse the activation of NF- k B, and it showed the activation of the model group was significantly higher than the normal group(P<0. 05).It suggested that soluble A β can upregulate the activity of NF-kB. The activation of NF-k B in the groups of 2, 4, 8 μ mol/L Rgl were lower than the model group (P<0. 01). 16 μ mol/L Rgl group has no significance compared to the model group(P>0.05). It suggested that soluble Aβ could stimulate the activation of NF- k B in astrocytes, and Ginsenoside Rgl of lower concentrations could downregulate the activation of NF-k B better.5. Cell supernatant were assayed by ELISA to assess the secreting of IL-6. It was shown that the protein expression of IL-6 in the modle group had been much higher than the normal group(P>0.05). Ginsenoside Rgl groups could inhibite the secreting of IL-6 protein in certain degree. The IL-6 protein of the groups in 2, 4μmol/L Rgl was lower than the model group(P<0. 05) but didn' t achieve nomal level. 8, 16μ mol/L Rgl groups had no statistically significant compared with the modle group(P>0. 05). The highest is model group, and the lowest is normal group. It suggested that Ginsenoside Rgl of low...