Figure 4.

Metformin inhibited p38 phosphorylation induced by HMGB1 but not that induced by HMGB1-ΔAT in RAW264.7 cells. RAW264.7 cells were incubated with 1 μg/ml full-length HMGB1, HMGB1-ΔAT, or LPS in the absence or presence of indicated concentrations of metformin at 37 °C for 1 h. HMGB1 or LPS were preincubated without or with metformin at 4 °C for 24 h before the addition to cells. Cell lysates were then analyzed by immunoblotting for phosphorylated and total p38 and for phosphorylated and total AMPK. A, typical photos of immunoblotting. Data shown are representative of four independent experiments with similar results. B and C, ratios of phosphorylated/total p38 (B) and phosphorylated/total AMPK (C) were calculated from densitometry analysis. B and C, results were analyzed as the percentage of control group (HMGB1 alone in left panels, HMGB1-ΔAT alone in center panels, and LPS alone in left panels). Data are presented as mean ± S.E. (n = 4). ***; p < 0.001 versus control group.