AP-1/IRF-3 Targeted Anti-Inflammatory Activity of Andrographolide Isolated from Andrographis paniculata
Figure 5
The effect of AG on AP-1 activation. (a) RAW264.7 cells cotransfected with AP-1-luciferase and β-gal constructs (as a transfection control) were treated with AG in the absence or presence of LPS (1 μg/mL) for 12 h. Luciferase activity was determined by luminescence as described in Section 2. (b) RAW264.7 cells ( cells/mL) were incubated with AG in the absence or presence of LPS (1 μg/mL) for the indicated times. After preparing whole cell lysates, levels of total or phospho-forms of upstream signaling enzymes (ERK, p38, and JNK) were identified by immunoblotting. ((c), left panel) HEK293 cells co-transfected with AP-1-luciferase and -gal constructs were treated with MAPK inhibitors (U0126, SB203580, and SP600125) or the JAK2 inhibitor AG490 in the absence or presence of PMA (100 nM) for 12 h. Luciferase activities were determined by luminescence. ((c), right panel) A direct ERK enzyme assay was performed with immunoprecipitated phospho-ERK prepared from RAW264.7 cells ( cells/mL) incubated with LPS (1 μg/mL) for 30 min in the presence or absence of AG or U0126 for 30 min, using an ERK kinase assay kit. Relative intensities of phospho-MBP level were calculated with a DNR Bioimaging system (Gelquant software version 2.7). Data are presented as the mean ± SD of an experiment done with 6 biological replicates () per treatment ((a) and (c), left panels) or 3 different blots ((c), right lower panel). Other data (b) are representative images of three different experiments that had similar results. and compared to control group.
