Fig. 1. Effect of taheebo on the viability of RAW264.7 cells. RAW264.7 cells (1 × 10⁶ cells/ml) were treated with taheebo (0–400 μg/ml) for 24 h. Cell viability was determined by the MTT assay.

Fig. 2. Effect of taheebo on the production of PGE₂ and the expression of COX-II in LPS-activated RAW264.7 cells. (A) RAW264.7 cells (1 × 10⁶ cells/ml) were stimulated with taheebo (0–400 μg/ml) in the presence or absence of LPS (2 μg/ml) for 24 h. Supernatants were collected and the PGE₂ concentration from the supernatants was determined by EIA. (B) The mRNA levels of COX-II and TNF-α from RAW264.7 cells (1 × 10⁷ cells/ml) was determined by semi-quantitative RT-PCR under the same conditions. Relative intensity (RI) was calculated by a ratio of the GAPDH band intensity. **p < 0.01 represents significant difference compared to LPS alone.

Fig. 3. Effect of taheebo on the production of NO, the expression of iNOS and cytoprotective effect in LPS-activated RAW264.7 cells. (A) RAW264.7 cells (1 × 10⁶ cells/ml) were stimulated with taheebo (0–400 μg/ml) in the presence or absence of LPS (2 μg/ml) for 24 h. Supernatants were collected and the NO concentration from the supernatants was determined by the Griess assay. (B) The mRNA level of iNOS from RAW264.7 cells (1 × 10⁷ cells/ml) was determined by a semi-quantitative RT-PCR under the same conditions. Relative intensity (RI) was calculated by a ratio of the GAPDH band intensity. (C) RAW264.7 cells (1 × 10⁶ cells/ml) were stimulated by LPS (2 μg/ml) with taheebo (0–400 μg/ml) for 24 h. Cell viability was determined by the MTT assay. **p < 0.01 represents significant difference compared to LPS alone.

Fig. 4. Effect of taheebo on the activation of LPS-induced signaling pathways in RAW264.7 cells. (A) RAW264.7 cells (5 × 10⁶ cells/ml) were stimulated with taheebo (0–400 μg/ml) in the presence or absence of LPS (2 μg/ml) for 30 min or 2 h. After immunoblotting, the phosphorylation or total levels of ERK, JNK, p38, Akt, IκBα and β-actin were identified by the corresponding phospho-specific or non-phospho antibodies. (B) RAW264.7 cells (1 × 10⁶) were treated with various inhibitors (U0126: 20 μM; SB203: SB203580, 10 μM; SP600: SP600125, 10 μM; LY294: LY294002, 20 μM and BAY11-7082: BAY, 20 μM) in the presence or absence of LPS (2 μg/ml) for 24 h. Supernatants were collected and the PGE₂ concentration from the supernatants was determined by ELISA. *p < 0.05 and **p < 0.01 represent significant difference compared to LPS alone. (C) RAW264.7 cells (5×106 cells/ml) were stimulated with various inhibitors (U0126: 20 μM; SB203: SB203580, 10 μM and SP600: SP600125, 10 μM) in the presence or absence of LPS (2 μg/ml) for 5 or 30 min. After immunoblotting, the phosphorylation or total levels of IκBα and β-actin were identified by the corresponding phospho-specific or non-phospho antibodies.

Fig. 5. Effect of taheebo on arachidonic acid- and croton oil-treated mouse ear edema. (A and B) ICR mice were orally administered with taheebo (100 mg/kg) for 7 days. Arachidonic acid (2%) (A) and croton oil (2.5%) (B) solution were topically applied (25 μl/ear) to the left ear of ICR mice (n = 7). The thickness of the edema was measured with a constant-pressure thickness gauge 1 or 4 h (arachidonic acid) or 3 h (croton oil) after treatment. The ear thickness of the inducer alone is represented by 100%. *p < 0.05 represents significant difference compared to inducer alone.

Sequences of primers used in a RT-PCR analysis