An Algal Metabolite-Based PPAR-γ Agonist Displayed Anti-Inflammatory Effect via Inhibition of the NF-κB Pathway

In our previous study, a synthetic compound, (+)-(R,E)-6a1, that incorporated the key structures of anti-inflammatory algal metabolites and the endogenous peroxisome proliferator-activated receptor γ (PPAR-γ) ligand 15-deoxy-∆12,14-prostaglandin J2 (15d-PGJ2), exerted significant PPAR-γ transcriptional activity. Because PPAR-γ expressed in macrophages has been postulated as a negative regulator of inflammation, this study was designed to investigate the anti-inflammatory effect of the PPAR-γ agonist, (+)-(R,E)-6a1. Compound (+)-(R,E)-6a1 displayed in vitro anti-inflammatory activity in lipopolysaccharides (LPS)-stimulated murine RAW264.7 macrophages. Compound (+)-(R,E)-6a1 suppressed the expression of proinflammatory factors, such as nitric oxide (NO), inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), possibly by the inhibition of the nuclear factor-κB (NF-κB) pathway. In macrophages, (+)-(R,E)-6a1 suppressed LPS-induced phosphorylation of NF-κB, inhibitor of NF-κB α (IκBα), and IκB kinase (IKK). These results indicated that PPAR-γ agonist, (+)-(R,E)-6a1, exerts anti-inflammatory activity via inhibition of the NF-κB pathway.


Introduction
Peroxisome proliferator-activated receptor γ (PPAR-γ), which can be activated by natural or synthesized ligands, such as 15-deoxy-∆ 12,14 -prostaglandin J 2 (15d-PGJ 2 ) or rosiglitazone, respectively, is a member of the nuclear receptor superfamily [1]. Generally, inactive PPAR-γ may be localized to the cytoplasm rather than the nucleus. Activated PPAR-γ is translocated to the cell nucleus and forms a heterodimer with a second member of the nuclear receptor family, retinoic X receptor (RXR). Then, the PPAR/RXR heterodimer binds to peroxisome proliferator response element (PPRE) on target DNA to regulate the transcription of genes relevant to lipid and glucose metabolism [2]. Recent research also revealed that PPAR-γ plays a key role in the repression of inflammatory genes, especially in macrophages. PPAR-γ expressed in macrophages has been postulated as a negative regulator of inflammation [3,4]. After activation by infection, tissue damage, or exposure to endotoxin (i.e., lipopolysaccharides (LPS)), macrophages secrete a large amount of proinflammatory mediators, including inducible nitric oxide synthase (iNOS), nitric oxide (NO), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6). Some of these proinflammatory mediators are involved in systemic diseases, such as obesity, diabetes, and cancer [5][6][7]. Currently, two molecular mechanisms have been defined for anti-inflammatory actions of PPAR-γ agonist. (1) PPAR-γ agonist binds to NF-κB (nuclear factor-κB) in the nucleus to inhibit its binding to DNA gene promoter regions, resulting in the suppression of inflammatory gene transcription. Binding of active NF-κB to DNA leads to the expression of proinflammatory mediators, such as TNF-α, IL-1β, IL-6, iNOS, and COX-2 [8][9][10][11][12][13][14].
In a previous study, we isolated oxy fatty acids and prostaglandins from a red alga with substantial anti-inflammatory activity [15]. Thereafter, by incorporating the key structural motifs of these natural products, we synthesized a new class of endocyclic enone jasmonate derivatives as anti-inflammatory leads [16,17]. In a further study, we synthesized an exocyclic enone jasmonate derivative, (+)-(R,E)-6a1, as a potent PPAR-γ agonist that share the key exocyclic enone moiety with the endogenous PPAR-γ ligand, 15d-PGJ 2 [18] (See Supplementary Materials). Typical PPAR-γ ligands, such as rosiglitazone and 15d-PGJ 2 , are composed of three distinct partial structures, a polar head, linker, and a hydrophobic tail ( Figure 1). The polar head and hydrophobic tail play important roles in H-bonding and hydrophobic interaction, respectively, with the PPAR-γ LBD (ligand binding domain). The exocyclic α,β-unsaturated ketone (enone) moiety of 15d-PGJ 2 was reported to be essential for covalent bonding with Cys 285 in the PPAR-γ LBD. Similar to 15d-PGJ 2 , the enone functionality of (+)-(R,E)-6a1 may contribute to covalent bonding to the PPAR-γ LBD, and this additional covalent bonding may contribute to the activation of PPAR-γ [18][19][20]. In a continuing study, we investigated the in vitro anti-inflammatory effects of the PPAR-γ agonist, (+)-(R,E)-6a1, in RAW264.7 murine macrophages. Based on in vitro results, the possible anti-inflammatory mechanism of (+)-(R,E)-6a1 was also discussed.

Compound (+)-(R,E)-6a1 Promoted PPAR-γ Translocation to Cell Nuclei
Generally, after ligand binding, the activated PPAR-γ will translocate to the nucleus and bind to NF-κB to repress the gene expression of proinflammatory mediators. In our previous study, the PPAR-γ agonistic activity of (+)-(R,E)-6a1 was evaluated by luciferase assay by using the PPRE-luciferase reporter plasmid [18]. Herein, we used Western blot to assess the protein level of the translocated PPAR-γ at the nucleus due to activation by (+)-(R,E)-6a1 in RAW264.7 cells. The result showed that the endonuclear PPAR-γ protein level was significantly increased by (+)-(R,E)-6a1 treatment in a concentration-dependent manner, and the activity was comparable to the standard PPAR-γ agonist rosiglitazone ( Figure 2). Since PPAR-γ expressed in macrophages can downregulate inflammatory responses [21], we investigated the expression of proinflammatory mediators.

(+)-(R,E)-6a1 Inhibited LPS-Induced Expression of Proinflammatory Factors in RAW264.7 Cells
In order to verify the in vitro anti-inflammatory effect of (+)-(R,E)-6a1, the protein levels of the proinflammatory factors, iNOS and COX-2, were examined in RAW264.7 cells by Western blot. As expected, LPS stimulation markedly increased iNOS and COX-2 protein levels, but this increase could be diminished in a dose-dependent manner by pretreatment with (+)-(R,E)-6a1 ( Figure 4). Notably, at the concentration of 30 µM, (+)-(R,E)-6a1 significantly decreased the protein levels of iNOS and COX-2 with a potency comparable to that of 10 µM dexamethasone. Dexamethasone was employed as a standard anti-inflammatory drug for comparison. Meanwhile, as an inflammatory mediator, high levels of NO are produced in response to inflammatory stimuli and mediation of inflammatory effects. iNOS is a family of enzymes that catalyze NO production from L-arginine. Thereby, it was hypothesized that the suppression of iNOS by (+)-(R,E)-6a1 ( Figure 4) would lead to decreased NO production in macrophages, and thus, we examined NO levels in RAW264.7 cell supernatant using the Griess reagent. Compound (+)-(R,E)-6a1 significantly decreased the NO production in a concentration-dependent manner ( Figure 5A). In addition, the amount of TNF-α and IL-6 that was produced was analyzed by enzyme-linked immunosorbent assay (ELISA). TNF-α and IL-6 are inflammatory cytokines that provide a host of defensive effects during the inflammatory response and maintain normal cellular conditions [22]. Proinflammatory mediator levels were markedly increased when murine macrophages RAW264.7 were exposed to LPS. However, these increases in TNF-α and IL-6 were inhibited by (+)-(R,E)-6a1 in a dose-dependent manner ( Figure 5B,C), suggesting that (+)-(R,E)-6a1 participates in a signaling pathway activated by LPS in macrophages. This result is consistent with that of other studies, which have shown that treatment of macrophages with various concentrations of PPAR-γ agonists reduces the production of proinflammatory cytokines [23,24].  For example, macrophages rely on NF-κB for the secretion of proinflammatory cytokines [25]. The conventional anti-inflammatory mechanism of PPAR-γ ligands is known via the inhibition of NF-κB [8][9][10][11][12][13][14]. PPAR-γ ligands, such as rosiglitazone, activate PPAR-γ, and activated PPAR-γ is translocated into the nucleus to bind with NF-κB. The NF-κB-PPAR-γ complex cannot bind to the promotor region of DNA; thereby, gene expression of proinflammatory mediators is suppressed (Figure 6, path A). Meanwhile, an endogenous PPAR-γ ligand, 15-deoxy-∆ 12,14 -prostaglandin J 2 (15d-PGJ 2 ), was reported to exert anti-inflammatory activity via additional mechanism by inhibiting the activation and nuclear translocation of NF-κB ( Figure 6, path B). Recently, Rossi et al. indicated that IKK is the critical target of 15d-PGJ 2 in the NF-κB activation pathway. The study showed that the exocyclic enone moiety in 15d-PGJ 2 can form Michael adducts that covalently modify Cys 179 of IKK, thus leading to the inhibition of IKK phosphorylation, and subsequent interference with the downstream NF-κB activation events [9,11]. Figure 6. The speculative anti-inflammatory mechanism of (+)-(R,E)-6a1 on the nuclear factor-κB (NF-κB) signal pathway in RAW 264.7 cells. Path A: Conventional anti-inflammatory mechanism of peroxisome proliferator-activated receptor γ (PPAR-γ) ligands via binding with NF-κB in the nucleus, and thereby blocking it from binding to DNA promotor regions. Path B: Alternative anti-inflammatory mechanism of 15d-PGJ 2 via the inhibition of NF-κB activation and endonuclear translocation.
Since (+)-(R,E)-6a1 share the same exocyclic enone moiety with 15d-PGJ 2 , we investigated the effect of (+)-(R,E)-6a1 on the alternative pathway of 15d-PGJ 2 involving the inhibition of IKK and subsequent inhibition of NF-κB. As shown in Figure 7, the phosphorylation levels of NF-κB significantly increased after LPS treatment for 30 min, but pretreatment with (+)-(R,E)-6a1 obviously decreased the NF-κB p65 phosphorylation in a dose-dependent manner ( Figure 7A). As a result, the phosphorylated protein level of NF-κB in the nucleus was also significantly decreased by (+)-(R,E)-6a1 treatment ( Figure 7B). IKK and IκB activate NF-κB, because phosphorylated IκBα by IKK releases the active NF-κB for translocation into the nucleus [26]. As expected, LPS stimulation markedly increased phosphorylated protein levels of IKK and IκBα. However, the phosphorylation of IKK and IκBα was decreased ( Figure 7C,D), and IκBα degradation was prevented in a dose-dependent manner ( Figure 7E) by (+)-(R,E)-6a1 treatment. At the same time, the immunofluorescence assay showed that the LPS-stimulated translocation of NF-κB into the nucleus was moderately prevented by (+)-(R,E)-6a1, especially at the concentration of 30 µM ( Figure 7F). Our findings suggested that (+)-(R,E)-6a1 may exert anti-inflammatory activity not only by the conventional inhibition of NF-κB from DNA binding like typical PPAR-γ ligands ( Figure 6, path A), but also by inhibition of the activation and endonuclear translocation of NF-κB ( Figure 6, path B) in the same manner as the 15d-PGJ 2 , which shares the same exocyclic enone moiety. (F) Immunofluorescence assay, NF-κB p65 is viewed as green fluorescence, and cell nuclei are viewed as red fluorescence by PI staining using confocal microscopy. The results shown are representative of three independent experiments. ## p < 0.01, ### p < 0.001 compared with the control group; * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the LPS-stimulated group.

Cell Culture and Cell Viability
RAW264.7 murine macrophages were purchased from the Korean Cell Line Bank (KCLB ® , Seoul, Korea); rat liver Ac2F cells and human oral epidermoid cancer cells (KB) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were cultured at 37 • C in a 5% CO 2 humidified incubator and maintained in high-glucose Dulbecco's Modified Eagle Medium (DMEM, Nissui, Tokyo, Japan) containing 100 mg/mL streptomycin, 2.5 mg/L amphotericin B, and 10% heat-inactivated fetal bovine serum (FBS). Suspensions of tested cell lines (cal. 1.0 × 10 4 cells/well) were seeded in 96-well culture plates, cultured for 12 h, and then treated with various diluted concentrations of (+)-(R,E)-6a1 for 24 h, 48 h, and 72 h, respectively. Control cultures were treated with culture medium alone. The tested compounds were evaluated at twice-fold dilutions, and the highest concentration was 50 µM. Cell viability was evaluated using water soluble tetrazolium (WST) reagent (EZ-CyTox, Daeil Lab Service Co., Ltd., Seoul, Korea), which was added to each well (10 µL) and incubated at 37 • C for 1 h. Absorbances were read using an iMark Microplate Absorbance Reader (Bio-Rad Laboratories, Hercules, CA, USA) at a wavelength of 450 nm. Cells in the exponential phase were used for all experiments.

Production Levels of NO and Cytokines Released into the Medium
RAW264.7 macrophages (cal. 1 × 10 4 cells/well) were seeded in a 96-well culture plate and cultured for 12 h. Cells were pretreated with various concentrations of drug for 1 h and then co-incubated with 25 ng/mL of LPS for 24 h. NO concentrations in medium were determined using a Griess assay. Griess reagent (80 µL) was added to media supernatants (80 µL) and then incubated at 37 • C for 15 min in the dark. Absorbance was measured at 520 nm using an iMark Microplate Absorbance Reader (Bio-Rad Laboratories, Hercules, CA, USA). NO concentrations were calculated using 0-100 µM sodium nitrite standards. TNF-α and IL-6 expression levels in culture medium were quantified using a sandwich-type ELISA kit (Biolegend, San Diego, CA, USA). Absorbance was measured at 450 nm.

Immunofluorescence Staining of NF-Kb P65 in RAW264.7 Cells
Cells were grown on confocal dish and treated with compound treatment for 24 h. After treatment, cells were fixed in 10% formalin solution for 15 min, washed with phosphate buffer saline (PBS) thrice, treated with 0.5% (v/v) Triton X-100/PBS for 15 min, washed with PBS thrice, and then blocked at room temperature for 30 min in 10% FBS/PBS. Cells were incubated with rabbit anti-NFκB-p65 antibody (Cell signaling technology, Danvers, MA, USA) at 4 • C overnight, washed thrice with PBS, incubated for 30 min at room temperature with secondary antibody anti-rabbit Alexa 488 (Cell signaling technology, USA) as a molecular probe, washed thrice with PBS, and then incubated with PI/Rnase (10 µg/mL) at room temperature for 20 min. The location of NFκB-p65 was viewed with a confocal microscopy FluoView FV10i (Olympus, Australia) using an excitation wavelength of 488 nm and an emission wavelength of 537 nm.

Western Blot Assay
RAW264.7 cells were harvested and suspended in lysis buffer containing protease and phosphatase inhibitor cocktails. The concentration of proteins was determined using a bicinchoninic acid (BCA) protein assay (Thermo Scientific, Rockford, IL, USA). Equal amounts of proteins were resolved by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes, which were then blocked in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) and 5% skimmed milk for 1 h at room temperature. Then, the membranes were incubated with specific primary antibodies (Cell Signaling Technology, Danvers, MA, USA) overnight at 4 • C. Anti-rabbit IgG-HRP was used as the secondary antibody. Signals were developed using the ChemiDoc™Touch Imaging System (Bio-Rad Laboratories, Hercules, CA, USA).

Statistical Analysis
The significance of intergroup differences was determined by ANOVA. Results are expressed as the mean ± SDs of indicated numbers of independent experiments. Values of p < 0.05 were considered statistically significant.

Conflicts of Interest:
The authors declare no competing financial interest.