Pharmacological mechanism underlying anti-inflammatory properties of two structurally divergent coumarins through the inhibition of pro-inflammatory enzymes and cytokines

Background: The aim of the present study is to investigate the effects of two structurally divergent coumarins, calipteryxin (1) and (3'S, 4'S)-3', 4'-disenecioyloxy-3', 4'-dihydroseselin (2) from Seseli recinosum, in lipopolysaccharide (LPS)-stimulated murine macrophages. Methods: The nitrite production was evaluated using Griess reagent. The protein and mRNA expression levels were investigated through Western blot and quantitative real time-PCR analyses. The NF-kappa B and AP-1 DNA-binding activities were assessed using an electrophoretic mobility shift assay. The docking studies were performed with Glide XP in Schrodinger suite (version 2013). Results: The results of the present study revealed that calipteryxin (1) and (3'S,4'S)-3',4'-disenecioyloxy-3',4'-dihydroseselin (2) treatment showed potent inhibitory effects on pro-inflammatory enzymes and cytokines associated with molecular signaling pathways. Treatment with calipteryxin and (3'S, 4'S)-3',4'-disenecioyloxy-3',4'-dihydroseselin also decreased the production of nitric oxide (NO), tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) in a dose-dependent manner. Additionally, both coumarins inhibited the LPS-induced protein and mRNA expression levels of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in RAW264.7 cells. To explore the potential mechanisms underlying the inhibitory activity of coumarin derivatives, the protein signaling pathways for NF-kappa B, mitogen-activated protein kinase (MAPK) and Akt were examined. Calipteryxin and (3'S,4'S)-3',4'-disenecioyloxy-3',4'-dihydroseselin markedly reduced the LPS-stimulated phosphorylation of IKK alpha/beta, p-I kappa Ba and I kappa Ba degradation as well as the nuclear translocation of the p65 subunit of pro-inflammatory transcription factor NF-kappa B. In addition, calipteryxin and (3'S,4'S)-3',4'-disenecioyloxy-3',4'-dihydroseselin) considerably inhibited the LPS-induced expression of ERK, c-Jun N-terminal kinase (JNK), p38 and Akt proteins. Furthermore, both coumarins significantly inhibited c-Jun expression in the nucleus. Conclusions: Taken together, these results support the therapeutic potential and molecular mechanism of calipteryxin and (3'S,4'S)-3',4'-disenecioyloxy-3',4'-dihydroseselin associated with inflammatory diseases.