Figure 6

AICAR down-regulates Aβ ± LPS- or sphingomyelinase-induced generation of reactive oxygen species (ROS) and expression of iNOS, COX-2 and MnSOD. Figure A shows LPS ± Aβ- (25–35) induced ROS generation in glial cell cultures. Pre-treatment of glial cells with AICAR inhibits LPS- and Aβ-mediated ROS generation. Cells were pre-incubated in the culture medium with 1 mM AICAR for 4 h prior to treatment with LPS (0.125 μg/ml) and/or 7.5 μM Aβ (25–35). ROS generation was measured by incubating the cells with the fluorescent dye Hydro Ethidene (HE) as described under Methods. Columns where AICAR was added have been shown diagramatically for brevity. Figure B: anti-oxidants (NAC and vitamin E) mediated inhibition of LPS, Aβ-stimulated nitric oxide release in glial cultures. Cells were pretreated with either AICAR (1 mM), vitamin E (20 μM) or N-acetyl cysteine (NAC) (10 mM) 4 h prior to stimulation with 125 ng/ml LPS and Aβ 7.5 μM (25–35). Nitric oxide released into the medium was measured as described under methods. Figure C shows AICAR mediated inhibition of both SMase- and Aβ-stimulated nitric oxide release (top) as well as the expression of iNOS, COX-2 and MnSOD in astrocyte enriched glial cultures (bottom). Cultures were pre-incubated with AICAR 4 h prior to stimulation with SMase (5 units/ml) ± 7.5 μM Aβ (25–35). Nitric oxide produced in the culture medium was measured using 'Greis reagent'. The cell lysates were western-immunoblotted for iNOS, COX-2 and MnSOD expression.