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

Figure 4

Noncytotoxic level of myricitrin inhibited LPS-induced JAK activation, which was required for LPS-induced STAT phosphorylation and nuclear translocation as well as inflammation-related substances. (a) RAW264.7 cells were treated with LPS (100 ng/ml) for 6 h in the presence or absence of 10 μM ruxolitinib for 2 h. Phosphorylation of STAT1 and STAT3 was measured by western blotting analysis using specific antibodies. (b) RAW264.7 cells were pretreated with 10 μM ruxolitinib or 20 μM NAC for 2 h and stimulated with 100 ng/ml LPS for 16 h. Expression levels of iNOS and COX-2 protein were determined by immunoblotting. RAW264.7 cells were pretreated with the indicated concentrations of myricitrin for 2 h before incubation with LPS (100 ng/ml) for 6 h. (c) The nuclear and cytoplasm proteins were extracted. Equal amounts of protein were subjected to immunoblot analysis with antibodies against STAT1 and STAT3. GAPDH was used as the cytoplasmic internal control and TBP was used as the nucleus internal control. (d) Immunostaining was performed with STAT1 (in red) and nuclei were stained with DAPI (in blue). Scale bars: 10 μm. Nuclear translocation of STAT1 was observed under a fluorescence microscope. (e) Nuclear extracts were analyzed for STAT1 activity by EMSA in the presence or absence of excess amounts of cold probe, mutant probe, or STAT1 antibody. (f) RAW264.7 cells were treated with ruxolitinib (10 μM) for 2 h. Next, cells were stimulated with LPS (100 ng/ml) for 16 h. Levels of TNF-α, IL-6, MCP-1, and PGE2 in culture supernatants were determined by ELISA. The amounts of NO were measured by Griess reagents. The data obtained from three different areas were mean ± SD. One of the representative data obtained from three individual experiments was shown. and were compared with the LPS group.

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