Abstract: Objective To investigate the dual role of daphnetin in suppressing high mobility group box-1 protein (HMGB1)
release and blocking HMGB1-induced inflammatory response. Methods Murine macrophage RAW264.7 cells were cultured in
the presence of daphnetin, lipopolysaccharide (LPS), or both. HMGB1 release from the cells was determined using ELISA, and
phosphorylations of JAK1/2 and of STAT1 were detected by Western blotting. Human monocytic THP-1 cells exposed to
daphnetin, rhHMGB1, or both were examined for NO production using a NO detection kit, for the release of tumor necrosis
factor-α (TNF-α), interleukin-6 (IL-6), and prostaglandin E2 (PGE2) using ELISA, and for expressions of iNOS, COX-2 and
phosphorylated p38, ERK, and JNK with Western blotting. Results Daphnetin dose-dependently reduced the release of
HMGB1 in RAW264.7 cells and suppressed rhHMGB1-induced iNOS and COX-2 expressions and release of TNF-α, IL-6,
PGE2, and NO in THP-1 cells. Western blotting revealed that daphnetin significantly down-regulated the phosphorylations of
JAK-STAT1 pathway in LPS-stimulated RAW264.7 cells but did not suppress the phosphorylations of MAPKs signaling
pathway induced by rhHMGB1 in THP-1 cells. Conclusion Daphnetin can reduce the release of HMGB1 and suppress
HMGB1-induced inflammatory response. In RAW264.7 cells, daphnetin inhibited LPS induced HMGB1 release is at least
partly mediated by suppressing JAK-STAT1 signaling pathway activation.