Toll-like receptors (TLRs) play an important role in the induction of innate immune responses recognizingconserved microbial structural molecules. The microbial pathogens trigger the activation of two downstream signaling pathways of TLRs; myeloid differential factor 88 (MyD88)-and toll interleukin-1 receptor domain containing adapter inducing interferon-β (TRIF)-dependent pathways leading to the activation of nuclear factor-κB (NF-κB) and interferon regulatory factor 3 (IRF3). Saussureae Radix has been used for centuries to treat a variety of diseases. Costunolide, one of the active ingredients in Saussureae Radix, has been used to treat many chronic diseases. However, the mechanism of costunolide's beneficial effects is largely unknown. Here, we report biochemical evidence that costunolide inhibits NF-κB activation and cyclooxygenase-2 expression induced by the TLR3 agonist polyriboinosinic polyribocytidylic acid (poly[I : C]) or the TLR4 agonist lipopolysaccharide (LPS). These results suggest that costunolide can modulate the immune responses regulated by TLR signaling pathways.

Toll-like receptor 4 (TLR4) recognizes LPS and triggers the activation of the myeloid differential factor 88 (MyD88)- and toll-interleukin-1 receptor domain-containing adapter, inducing interferon-β (TRIF)-dependent major downstream signaling pathways. Previously, we presented biochemical evidence that 1-[4-Fluoro-2-(2-nitrovinyl)phenyl]pyrrolidine (FPP), which was synthesized in our laboratory, inhibits NF-κB activation induced by LPS. Here, we investigated whether FPP modulates the TLR4 downstream signaling pathways and what anti-inflammatory target in TLR4 signaling is regulated by FPP. FPP inhibited LPS-induced NF-κB activation by targeting TLR4 dimerization. These results suggest that FPP can modulate the TLR4 signaling pathway at the receptor level to decrease inflammatory gene expression.