The Mechanism Of Osthole In Adjusted Activation Of NF-κB Induced By Aβ_25-35 In AD Cell Model

OBJECTIVE:To explore the cell survival of mixed astrocytic/neuronal cultures treated with the soluble Aβ by CCK-8 assay. To study the relationship of Osthole and Aβ in regulating the activation of NF- κ B and phosphorylation of I κ B in astrocytes. METHODS:Mixed astrocytic/neuronal cultures were prepared and the morphologies were observed by inverted phasecontrast microscope. AD cell modle in mixed cultures induced by the soluble beta-amyloid was determined by CCK-8 assay measuring the survival rate for choosing the appropriate concentration of A β and cultured time. Pretreated with Osthole for 24h, cell survival rate were evaluted by CCK-8 after co-cultured with 40uM A β and Osthole for 24h again for choosing the appropriate concentration and time point of Osthole. The Laser Scanning Confocal Microscope(LSCM) and Leica Confocal Analysis Software were performed to assay the activities of NF-kB and IkB in FITC\PI double-stainedastrocytes. The data were expressed by x|-±s and analyzed by SPSS11.0statistical package. Results:1 In mixed cultures, astrocytes were distinguished by their large stellate-shaped cell bodies, abundant cytoplasm, many radiating processes and round or ovi-round karyons which leaned to one side. Hippocampies were distinguished by high survival, growth quickly, numerous and longer synapses and round or orbicular-ovate nucleus which lied in the center of cell body.2 Mixed cultures cells showed a "trophic effect" for beta-amyloid in a dose-dependent manner, which was significant up to the highest concentration(40uM) at 24h and 48h. The change of time could alse influencecell viability analyzed by using two-factor ANOVA.3 Osthole could protect astrocytes against toxicity of A 3 , especially at lower concentrations(0. OluM-luM). Compared with the modle group, cell viability of Osthole groups was higher, but the higher the concentration of osthole was, the weaker the protection from toxicity of A3 was. The protect effect of osthole showed hardly correlate with the cells cultures time,but pretreated astrocytes for 24 hours, osthole showed a significant cell protect effect.4 Treatment of astrocytes with 40uM A3 resulted in activation of NF-kB. Osthole at lower concentrations couldn' t influence activation of NF-kB in normal astrocytes, but could inhibit overactivation of NF-kB induced by A 3.5 40uM A3 could induce I k Ba overphosphorylation in astrocytes. Osthole in low concentration couldn' t effect I k B a phosphorylation in normal astrocytes, but could inhibit IkBo overphosphorylation caused by A3. CONCLUSIONS:Hippocampal neurons showed a higher survival and a faster proliferation by seeded on the confluent monolayer of astrocytes. The treatment with A3 induced a cytotoxic effect in NG108-15, astrocytes and hippocampal neurons cultures, but didn' t show toxicity effect in mixed cultures cells. A3 could stimulate IkBo overphosphorylation and lead to NF-kB overexpression in astrocytes. Osthole at lower concentrations could not influence expressiones of I k B a and NF- k B in normal astrocytes, but could upregulate I k B a expression and inhibit NF-kB overexpression caused by A3.In conclusion, our results suggest that Osthole may have a role in protecting astrocytes against A3 neurotoxic effect and delaying the development of AD.