Myricitrin Modulates NADPH Oxidase-Dependent ROS Production to Inhibit Endotoxin-Mediated Inflammation by Blocking the JAK/STAT1 and NOX2/p47^phox Pathways

Figure 2

Noncytotoxic level of myricitrin inhibited LPS-induced inflammatory-associated cytokine and mediator production. RAW264.7 cells were treated with myricitrin (100, 200, and 400 μg/ml) or vehicle for 2 h. Next, cells were stimulated with LPS (100 ng/ml) for 16 h. (a) The supernatants were taken, and the amounts of NO were measured by Griess reagents. (b–e) The levels of PGE2, TNF-α, IL-6, and MCP-1 were measured in the culture medium by ELISA kits. (f) RAW264.7 cells were treated with indicated concentration of myricitrin for 24 h. Cell viability was evaluated using the CCK-8 assay, and the results were expressed as percentage of surviving cells over the control group. RAW264.7 cells were incubated with 100, 200, and 400 μg/ml of myricitrin or vehicle for 2 h and then were stimulated with LPS (100 ng/ml). (g–h) After incubation of 16 h, cell lysates were prepared and subjected to western blotting by using anti-iNOS and anti-COX-2 antibodies. GAPDH was the internal control. After incubation of 8 h, total RNA was isolated and iNOS and COX-2 mRNA were determined by RT-PCR (i) and qRT-PCR (j). Each bar represents the mean ± SD of three independent experiments. and versus LPS-stimulated groups.

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)