Figure 8: Chrysophanol inhibited iNOS and COX-2 expression and MMP-9 activation in activated microglia.

(a) Microglial BV2 cells were pretreated with the vehicle (DMSO) or chrysophanol (0.5, 1, 2, and 5 μM) and stimulated with LPS (150 ng/ml) for 24 hours. iNOS and COX-2 expression in cell lysates was evaluated using western blot. All the Western blotting experiments were performed under the same conditions. After transferring the blots onto nitrocellulose membranes, we immediately cropped the targeted blots according to referenced indicating markers (Supplementary Fig. S3), and then targeted proteins were immunoblotted with its specific monoclonal antibody. (b) Primary microglia cells were pretreated with the vehicle (DMSO) or chrysophanol (5, 10, 20, and 50 μM) and stimulated with LPS (150 ng/mL) for 24 hours. MMP-9 activation in cell supernatants was evaluated using zymography. All the zymographic experiments were performed under the same conditions. After gelatin staining, we immediately cropped the gelatinolytic regions in the gels (Supplementary Fig. S3). The data are presented as the mean ± SEM of at least 3 experiments in each group. R: resting; V: vehicle. ^#p < 0.001 compared with the control group treated with vehicle, *p < 0.01 compared with the LPS-exposed group treated with vehicle, **p < 0.001 compared with the LPS-exposed group treated with vehicle.