Figure 4

Effects of ginsenoside Rh1 combined with DEX on GR. (A-B) After pretreatment with solvent, DEX (1 μM) or Rh1 (10 μM) combined with DEX for 2 h or 24 h, TNF (20 ng/ml) was added for 8 h. (A) Western blot analysis was performed in parallel on total extracts with an anti-GR antibody. The detection of β-actin was used as a loading control. (B) Specific binding of GR was also determined in parallel on RAW 264.7 cells. RAW264.7 cells (1 × 10⁶) were incubated 3 h with 10⁻⁶ M [³H] DEX, with or without excess unlabeled DEX. Specific binding was determined. *P <0.05, **P <0.01 versus control, #P <0.01 versus DEX group. (C-D) RAW264.7 cells were treated with solvent, DEX alone or Rh1 combined with DEX for 24 h. (C) Transcription was then stopped by addition of actinomycin D (0.25, 0.5, 1 ng/ml) in the presence of Rh1. GR mRNA levels were assessed by means of quantitative PCR. β-actin was used for normalization. The left panel is the quantitative result and the right panel is RNA electrophoretic figures. *P <0. 01 versus control; #P <0.01 versus DEX group; ΔP <0.01 versus Rh1 + DEX group. (D) Protein synthesis was stopped by addition of cycloheximide (5, 2, 1 μg/ml) in the presence of Rh1. GR levels were determined by Western blot. The detection of β-actin was used as a loading control. The experiment was replicated three times and results are representative of at least two independent induction experiments. DEX, dexamethasone; GR, glucocorticoid receptor; TNF, tumor necrosis factor.