Figure 1.

Caspase inhibitor Z-VAD-FMK reduces LPS-induced cytokine release, HMGB1 translocation, and NF-κB activation in RAW 264.7 cells and reduces sepsis-induced serum cytokine levels in mice. (A) Mouse macrophage-like RAW 264.7 cells were stimulated with 200 ng/ml LPS in the presence of Z-VAD-FMK or control peptide (Z-FA-FMK) at doses indicated for 16 h. Levels of HMGB1, TNF, and IL-6 in conditioned media were measured, and data are expressed as percentages of stimulation by LPS alone. *, P < 0.05 versus LPS + control group; n = 7. (B) RAW 264.7 cells were stimulated with 100 ng/ml LPS in the presence of 1 or 10 μM Z-VAD-FMK or control peptide for 6 h at 37°C. Nuclear extracts were prepared, and EMSA was performed using biotin-labeled nucleotides to measure NF-κB content. *, P < 0.05 versus LPS alone; n = 5–7 experiments. (C) BALB/c mice underwent CLP and received either Z-VAD-FMK or control peptide (0.5 mg/mouse) injected intraperitoneally at 90 min and 12 h after CLP. Mice were killed 24 h after CLP surgery. Serum levels of HMGB1, IL-6, KC, and MIP-2 were measured. *, P < 0.05 versus CLP group; n = 7–11 mice per group. Error bars represent SEM. (D) RAW 264.7 cells were incubated with 100 ng/ml LPS alone or in the presence of 1 μm Z-VAD-FMK or control peptide for 16 h. Cells were then incubated with polyclonal anti-HMGB1 antibodies followed by FITC-labeled anti–rabbit antibodies and viewed by fluorescent confocal microscopy. Note that LPS caused the export of HMGB1 into the cytosol, whereas Z-VAD-FMK (but not control peptide) prevented its cytoplasmic translocation and preserved HMGB1 in the nucleus. Data are representative of four separate experiments.