title: Pharmacological mechanism underlying anti-inflammatory properties of two structurally divergent coumarins through the inhibition of pro-inflammatory enzymes and cytokines.
abstract: 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-κB and AP-1 DNA-binding activities were assessed using an electrophoretic mobility shift assay. The docking studies were performed with Glide XP in Schrödinger 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-α) and interleukin-1 beta (IL-1β) 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-κB, 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 IKKα/β, p-IκBα and IκBα degradation as well as the nuclear translocation of the p65 subunit of pro-inflammatory transcription factor NF-κB. 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.
authors.affiliation: The Alan Edwards Centre for Research on Pain, McGill University, Montreal, QC H3A 0G1 Canada ; College of Pharmacy, Seoul National University, Gwanak-gu, Seoul 151-742 South Korea.
authors.forename: Salman
authors.initials: S
authors.lastname: Khan
authors.affiliation: College of Pharmacy, Seoul National University, Gwanak-gu, Seoul 151-742 South Korea ; Department of Pharmacy, Abdul Wali Khan University, Mardan, Pakistan.
authors.forename: Omer
authors.initials: O
authors.lastname: Shehzad
authors.affiliation: School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang, 110016 China.
authors.forename: Mao-Sheng
authors.initials: MS
authors.lastname: Cheng
authors.affiliation: School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang, 110016 China.
authors.forename: Rui-Juan
authors.initials: RJ
authors.lastname: Li
authors.affiliation: College of Pharmacy, Seoul National University, Gwanak-gu, Seoul 151-742 South Korea.
authors.forename: Yeong Shik
authors.initials: YS
authors.lastname: Kim
keywords: (3’S,4’S)-3’,4’-disenecioyloxy-3’,4’-dihydroseselin; Akt; Calipteryxin; MAPK; NF-κB; Seseli recinosum