FIGURE 3

FIGURE 3

Emodin regulates the Nur77/c-Jun pathway in LPS-stimulated RAW264.7 cells by inhibiting the JNK pathway. (A) Emodin regulates the Nur77/c-Jun pathway. RAW264.7 cells were pretreated with emodin (40 μM) for 2 h followed by exposure to LPS (100 ng/ml) for 18 h. The expression levels of p-Nur77 (Ser351), Nur77, p-c-Jun (Ser73), and c-Jun were measured by Western blotting. (B) The functional verification of SP600125 and anisomycin. RAW264.7 cells were treated with SP600125 (10 μM) for 22 h or anisomycin (18 nM) for 24 h. The expression levels of p-JNK (Thr183/Tyr185), JNK, p-Nur77 (Ser351), Nur77, p-c-Jun (Ser73), and c-Jun in total cell lysates were detected by Western blotting. (C) Anisomycin blocks the regulatory effects of emodin on the Nur77/c-Jun pathway. RAW264.7 cells were pretreated with SP600125 (8 μM) for 2 h or anisomycin (18 nM) for 4 h, and then treated with emodin (40 μM) for 2 h, followed by exposure to LPS (100 ng/ml) for another 18 h. The expression levels of p-Nur77 (Ser351), Nur77, p-c-Jun (Ser73), and c-Jun in total cell lysates were detected by Western blotting. (D) Emodin increases the transcriptional activity of Nur77 via the JNK pathway. HEK293.7T cells were transfected with plasmid Nur77-luciferase (1.6 μg/ml) and pRL-TK (1.6 μg/ml) in the presence or absence of JNK1 plasmid (3.2 μg/ml). After 24 h transfection, cells were treated with emodin for 8 h, and then luciferase assay was performed. Relative luciferase activity was expressed as the ratio of firefly to Renilla luciferase. Data are presented as mean ± SEM, n = 3. **p < 0.01 compared with the control group; ^###p < 0.001 compared with the emodin group. (E) Emodin increases the protein expression of Nur77 via the JNK pathway. HEK293.7T cells were transfected with JNK1 plasmid (3.2 μg/ml). After 24 h transfection, cells were treated with emodin for 8 h, and then the expression levels of p-JNK (Thr183/Tyr185), JNK, p-Nur77 (Ser351), Nur77, p-c-Jun (Ser73), and c-Jun in total cell lysates were assessed using Western blotting.