21-O-Angeloyltheasapogenol E3, a Novel Triterpenoid Saponin from the Seeds of Tea Plants, Inhibits Macrophage-Mediated Inflammatory Responses in a NF-κB-Dependent Manner

Figure 3

Effect of ATS-E3 on the transcriptional activation of RAW264.7 cells during TLR signaling. (a) The level of iNOS mRNA in RAW264.7 cells treated with ATS-E3 (0 to 10 μM) in the presence or absence of LPS (1 μg/mL) for 6 h was determined by real-time quantitative RT-PCR. ((b) and (c)) HEK293 cells cotransfected with NF-κB-Luc and β-gal (as a transfection control) plasmid constructs were treated with ATS-E3 under the cotransfection conditions with FLAG-MyD88 or Myc-Syk (1 μg/mL each) for 12 h. Luciferase activity was determined using luminometery, as described in Section 2. (d) RAW264.7 cells (5 × 10⁶ cells/mL) were incubated with LPS (1 μg/mL) in the presence or absence of ATS-E3 for the indicated times. After preparing the nuclear fractions, the translocated levels of total transcription factors (p65, p50, and lamin A/C) were identified using immunoblotting. Relative intensity was calculated using total levels by the DNR Bio-Imaging System. compared to control group.

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