Figure 2. Effects of 17β-estradiol (E₂) on LPS-induced TNF-α production and activation of MAPKs in BMMs.

(A) Cells were treated with LPS (1 µg/ml) alone or in combination with different concentrations of E₂ for 24 h, and the culture media were collected to measure TNF-α concentrations using ELISA. The relative expression of TNF-α was evaluated with the results obtained from LPS-stimulated macrophages. The results are expressed as the means ± the SEMs of three independent experiments. (B) Cells were treated with E₂ (1 nM) for different periods. Protein extracts were subjected to western blotting to detect the phosphorylated and total forms of three MAPK molecules, p38 MAPK, ERK1/2 and JNK. Stimulation of the cells with anisomycin (AN, 10 µg/ml, 30 min) was used as a positive control. (C) Cells were stimulated with LPS (1 µg/ml) alone or in combination with E₂ (1 nM) for 15 min. Protein extracts were subjected to western blotting to detect the phosphorylated and total forms of p38 MAPK. The relative activation of p38 was densitometrically evaluated. Representative blots are shown, and the results were verified by at least three independent experiments. *P<0.05 compared to the control and LPS.