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

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. 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). 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. 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.


Background
Considerable amounts of pro-inflammatory mediators and pro-inflammatory cytokines are released at injury sites during inflammation [1]. These pro-inflammatory mediators respond to numerous stimuli, including bacterial lipopolysaccharide (LPS), cytokines, and UV irradiation, which modulate to their effects by inducing the activation of NF-κB and AP-1 [2].
NF-κB activates a number of molecules involved in the inflammatory response, including iNOS, COX-2, TNF-α, IL-1β, and IL-6 [1]. The production of these mediators and cytokines through NF-κB stimulation might reflect the extent of inflammation and has been suggested as a measure to evaluate the effects of antiinflammatory agents on the inflammatory process. NF-κB signaling is activated through two diverse pathways: the canonical (classical) pathway and the non-canonical pathway [3]. NF-κB signaling through canonical or noncanonical pathways involves the expression of multiple genes [4,5]. The canonical pathway involves the IκBα kinase (IKK) complex, while the non-canonical pathway involves NF-κB inducing kinase (NIK), which recruits IKK to p100 and subsequently activates IKK [4]. In response to inflammation, the p50 and p65 subunits of NF-κB are translocated through the canonical pathway, whereas the p52-containing RelB heterodimer of NF-κB is released and translocated through the non-canonical pathway [4,5].
Mitogen-activated protein (MAP) kinases and PI3k/Akt signaling pathways play essential roles in inflammation and tissue remodeling [6,7]. Consequently, the inhibition of MAP kinases produces anti-inflammatory effects against various inflammatory diseases [6]. However, several studies have demonstrated that the activation of MAP kinases suppresses inflammatory reactions, such as bacterial LPSinduced cytokine production in macrophages.
In the present study, two structurally divergent coumarin derivatives, calipteryxin (1) and (3'S,4'S)-3' ,4'disenecioyloxy-3',4'-dihydroseselin (2) isolated from Seseli resinosum, were studied in terms of LPS-induced inflammatory signaling. The genus Seseli L. belongs to the Apiaceae family, which comprises aromatic herbs used as foods, spices, condiments and ornamentals [8]. This herb has various pharmacological applications, such as anthelmintic, carminative, stomachic and stimulant properties [8]. It has been reported that Seseli plants show significant and dose-dependent anti-inflammatory activity and analgesic effects in carrageenan-induced acute inflammation in rats [8]. However, only a few pharmacological and biological activity studies concerning the single component from Seseli have been reported. Therefore, the aim of the present study was to examine LPS-induced inflammatory signaling in murin macrophages. Moreover, a comparative investigation was performed between two coumarin type compounds.
Cells and culture medium RAW 264.7 murine macrophages were obtained from the American Type Culture Collection (Manassas, VA). These macrophages were maintained and subcultured according to a previously described procedure [5].

Western immunoblot analysis
The Western blot procedure was performed according to Khan et al., with some modifications [5].
RNA extraction and quantitative real-time (RT) PCR RT-PCR was performed with total RNA extracted using easyBlue™ according to the manufacturer's instructions (Sigma-Aldrich, St. Louis, MO). The purity and concentrations of RNA were determined using an ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE). All RNA samples were stored at −80°C until further analysis. Total RNA (1 μg) was converted to cDNA through RT-PCR (Genius FGEN05TD, Teche, England) using the iScript™ cDNA Synthesis Kit (BIO-RAD, Hercules, CA) under the following conditions: 25°C for 5 min, 42°C for 30 min and 85°C for 5 min. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed using an Applied Biosystems 7300 Real-Time PCR system and software (Applied Biosystem, Carlsbad, CA). qRT-PCR was conducted in 0.2 ml PCR tubes with forward and reverse primers and the SYBR green working solution (iTaq™ Universal SYBR Green Supermix, BIO-RAD, Hercules, CA), using customer PCR master mix under the following conditions: 95°C for 30 min, followed by 40 cycles of 95°C for 15 s, 55°C for 20 s and 72°C for 35 s. The melting point, optimal conditions and specificity of the reactions were determined. The sequences of the PCR primers were previously described [10,11]. The sense and antisense primers for iNOS were 5'CCCTTCCGAAGTT TCTGGCAGC-3' and 5'-GGCTGTCAGAGCCTCGTGG CTT-3', respectively. The sense and antisense primers for COX-2 were 5'-GGAGAGACTATCAAGATAGTGATC-3' and 5'-ATGGTCAGTAGACTTTTACA-GCTC-3' , respectively. The following primers were used for; TNF-α, sense primer, 5'-AGC ACA GAA AGC ATG ATC CG-3' and antisense primer, 5'-CTG ATG AGA GGG AGG CCA TT-3'; and for IL-1β, sense primer, 5'-ACCT GCT GGT GTG TGA CGT T-3' , and antisense primer, 5'-TCG TTG CTT GGT TCT CCT TG-3'. The sense and antisense primers for rat actin mRNA expression (used as a control for total RNA content for each sample) were 5'-TGAAGGTCGGTGTGAACGGATTTGGC-3' and 5'-CA TGTAGGCCATGAGGTCCACCAC-3', respectively.

Electrophoretic mobility shift assay (EMSA)
EMSA was performed to investigate the inhibitory effects on NF-κB and AP-1 DNA binding, as previously described [5,7].

Computational methods
Docking studies were performed using Glide XP in Schrödinger suite (version 2013).

Statistical analysis
Unless otherwise stated, the results are expressed as the means ± standard deviations (SD) from three different experiments. One-way analysis of variance (ANOVA) followed by Dunnett's t-test was applied to assess the statistical significance of the differences between the study groups (SPSS version 10.0, Chicago, IL). A value of p <0.05 was considered statistically significant.

Discussion
Macrophages serve as a crucial link between innate and adaptive immunity and play pivotal roles in inflammatory signaling [16]. The stimulation of macrophages with bacterial exotoxins, such as LPS, occurs through the specific receptor TLR4 and triggers the recruitment of the cytoplasmic adaptor protein MyD88 and the activation of TIRAP, which subsequently stimulates down-stream signaling pathways (NF-κB and MAPKs). The LPSinduced pathways up-regulate the expression of various inflammatory mediators and cytokines involved in the pathogenesis of inflammatory responses [16]. Based on these hypotheses, the modulation of LPS-induced NF-κB and MAPK signaling or the regulation of cytokine production might constitute a therapeutic strategy in many inflammatory diseases.
Natural products have been one of the leading sources for the discovery of new anti-inflammatory agents. Coumarins are naturally isolated compounds with various remarkable pharmacological and biological properties [8]. In the present study, two different types of coumarins were investigated in terms of LPS-stimulated macrophages through the inhibition of the signaling pathways for the transcription factors NF-κB and AP-1. Initially, the effects of calipteryxin and (3'S,4'S)-3' ,4'-disenecioyloxy-3' ,4'dihydroseselin on the production of NO and on the regulatory genes for iNOSand COX-2 in LPS-stimulated RAW264.7 macrophages were examined. The LPSinduced down-regulation of the pro-inflammatory mediators through calipteryxin and (3'S,4'S)-3' ,4'-disenecioyloxy-3' ,4'-dihydroseselin were based on the suppression of the NF-κB and AP-1 signaling, leading to a therapeutic approach against inflammatory diseases.
We have previously demonstrated that LPS stimulation induces pro-inflammatory enzymes and cytokines through the activation of NF-κB and AP-1 signaling pathways, which play crucial roles in the control of cellular responses to cytokines and stresses [5,7]. NF-κB is involved in the regulation of the expression of pro-inflammatory cytokines and other mediators involved in the inflammatory response [19]. Therefore, the inhibition of this signaling might explain the potent activity of calipteryxin and (3'S,4'S)-3' ,4'-disenecioyloxy-3' ,4'-dihydroseselin as suppressors of inflammatory cytokines. Under inactive conditions, NF-κB is located in the cytoplasm as an inactive NF-κB/IκBα complex and is controlled through the inhibitory protein IκBα. The degradation of IκBα through phosphorylation releases NF-κB for translocation into the nucleus, thereby initiating the transcription of target genes [2]. Therefore, the translocation of NF-κB could be evaluated in RAW 264.7 cells based on NF-κB-DNA binding affinity. In the present study, LPS induced a marked increase of NF-κB DNA binding, while treatment with the coumarin derivatives significantly inhibited NF-κB-DNA binding activity. To explore a more in-depth mechanism of calipteryxin and (3'S,4'S)-3' ,4'-disenecioyloxy-3' ,4'-dihydroseselin, MAPKs and Akt signaling pathways were examined. The results of this study demonstrated that calipteryxin and (3'S,4'S)-3',4'-disenecioyloxy-3',4'-dihydroseselin block the activation of MAPKs and Akt during early LPS stimulation. This significant inhibition might reflect the potential anti-inflammatory effect of calipteryxin and (3'S,4'S)-3' ,4'-disenecioyloxy-3' ,4'-dihydroseselin. Experiments demonstrating the molecular docking of NF-κB inducing kinase (NIK) further supported the molecular analysis data [4]. NIK is a key regulator of inflammation through the non-canonical NF-κB pathway. In response to inflammation, the molecular docking molecule (NIK) in the non-canonical pathway recruits IKKα to p100 for subsequent phosphorylation, ubiquitination and degradation, resulting in the release and translocation of NF-κB heterodimers [4]. The docking simulation revealed that calipteryxin and (3'S,4'S)-3' ,4'-disenecioyloxy-3' ,4'-dihydroseselin form two hydrogen bonds with LYS517 and SER476 residues. The secondary structure of the protein is shown as a solid ribbon (gray). Key residues are displayed in line style (blue). Calipteryxin and (3'S,4'S)-3' ,4'-disenecioyloxy-3' ,4'-dihydroseselin are displayed in stick style (carbon atoms in cyan).

Additional file
Additional file 1: Figure S1. Time optimization of coumarins treatment at various time point by stimulating LPS in RAW 264.7 cells.