The carrageenan-induced paw edema model in rats, one of the well-established acute inflammatory models in vivo, was used to test the anti-inflammatory activity of neorogioltriol. In the control group, the intraplantar injection of 100 μL of carrageenan (0.6%) into the rat’s hind paw induced a significant increase in paw edema (Table 1). This observation indicates the development of an inflammatory response and paw swelling (edema). The edema was present as early as one hour after carrageenan injection, progressed rapidly and persisted for at least 5 h after treatment. Both dexamethasone and ASA, used as positives controls, inhibited the oedematogenic response evoked by carrageenan in rats at all assessment times. However, as previously reported [17], the kinetics of the effect of both components was different: whereas the dexamethasone-associated effect was rapid and produced a 42% reduction of the edema after the first hour, the ASA-induced effect was slower (only 17% in the first hour), but reached a 59% inhibition by the fifth hour. Treatment of rats with the neorogioltriol molecule at an intraperitoneal dose of 1 mg/kg significantly reduced paw swelling at all time points tested (Table 1). This reduction was about 28% after the first hour and reached a maximum of 58% at 3 h post injection. Moreover, a 1 mg/kg dose of neorogioltriol proved as effective as 300 mg/kg of ASA in reducing inflammation.

Using the carrageenan-induced paw edema model in rats, our results showed that the red algae-derived natural molecule neorogioltriol was able to reduce the formation of edema in a concentration- and time-dependent manner.

Cytotoxic potential of neorogioltriol on Raw264.7 cells was tested using the MTT assay. Our result shows that in the presence of up to 250 μM of neorogioltriol, Raw264.7 macrophages viability was not significantly lower than in non-treated cells. We find cytotoxicity only at the highest neorogioltriol concentration (500 μM) which causes an optical density (OD) reduction of about 50%.

Carrageenan-induced rat paw edema has been fully characterized and shown to be associated with the production of several inflammatory mediators [18,19] including TNFα, prostaglandins and nitric oxide release [20–22].

We first analyzed the effects of neorogioltriol in vitro, on different inflammatory molecules starting with TNFα. The release of TNFα was measured using ELISA assay in the supernatant of LPS-stimulated Raw264.7 cells, pre-treated or not with neorogioltriol for 30 min. The treatment of resting Raw264.7 cells with LPS increased the production of TNFα. For all the concentrations tested, neorogioltriol significantly inhibited TNFα production (Figure 2).

Cyclooxygenase-2 (COX-2) is the key enzyme regulating the production of prostaglandins, the central mediators of inflammation. On the other hand, iNOS activation induces massive NO production at the site of inflammation. We thus investigated the effect of our molecule on LPS-induced COX-2 expression by Western blot and on NO production by measuring the released nitrite in the culture medium by Griess reaction.

Our results show that in murine Raw264.7 cells, LPS treatment (100 ng/mL for 24 h) induces the expression of COX-2 (Figure 3A) and increases NO production 30-fold, as compared to controls. After neorogioltriol pretreatment, the LPS-induced nitrite release was inhibited. At concentrations ranging from 0.125 μM to 12.5 μM, neorogioltriol significantly reduced the level of NO production. However, for neorogioltriol concentrations above 25 μM the curve of NO release turns upward and at 62.5 μM (25 μg/mL) of neorogioltriol, NO release is almost similar to untreated cells (Figure 3A).

The expression of COX-2 was also tested in cells treated with 12.5 μM, 25 μM and 62.5 μM neorogioltriol prior to LPS stimulation. Consistent with the inhibitory effect on NO release, the results (Figure 3B) show that neorogioltriol up to 25 μM inhibited the LPS induced COX-2 expression. However, at 62.5 μM the COX-2 expression is partially restored.

Taken together, our data show that, whereas neorogioltriol has no effect by itself (data not shown), this molecule inhibits the expression of TNFα and COX-2 and the production of NO induced by LPS stimulated Raw264.7 cells. Neorogioltriol however, shows a biphasic effect since this molecule fails to inhibit the expression of key inflammatory mediators, such as COX-2 and NO at the highest concentrations.

The anti-inflammatory potential of terpenes has been linked to their capacity to suppress NF-κB signaling [4,23] which coordinates the expression of proinflammatory enzymes and cytokines including iNOS, COX-2 and TNFα [24–26]. Neorogioltriol was thus evaluated in vitro for its ability to inhibit LPS-mediated NF-κB transcriptional activity. Raw264.7 cells, stably transfected with pNF-κB-luc plasmid, containing three NF-κB target sequences linked upstream to the luciferase reporter gene, were either stimulated with LPS or treated with different concentrations of neorogioltriol prior to LPS stimulation. Our result shows that LPS induces NF-κB activation. The pre-treatment with neorogioltriol prior to LPS stimulation significantly decreased LPS induced NF-κB transactivation (Figure 4). This result shows that the anti-oedematogenic effect of neorogioltriol correlates with the suppression of NF-κB activation.

However, despite reducing NF-κB activity, high concentrations of neorogioltriol fail to inhibit the expression of certain NF-κB-dependent genes that are relevant to the inflammatory process, such as COX-2. These results suggest that the observed loss of anti-inflammatory efficacy at high doses of neorogioltriol was independent of NF-κB or indirectly dependent on NF-κB inhibition.

The mitogen-activated protein (MAP) kinases play a key role in the regulation of cellular response to cytokines and stresses and are also known to be important for NF-κB activation. We thus tested whether the observed loss of anti-inflammatory efficacy may be mediated through MAPK activation.

We first studied the effect of neorogioltriol on LPS-induced MAPK activation. Our results show that at the highest concentrations used (25 μM and 62.5 μM), the neorogioltriol molecule fails to interfere with LPS-dependent ERK activation and only slightly inhibited the p38 MAPK phosphorylation (Figure 5). Moreover, the inhibition of MAPK pathways by PD98059 or SB203580 treatment did not alter the capacity of neorogioltriol to inhibit the LPS-induced NF-κB transactivation (data not shown).

MAPKs have been reported to be involved in the LPS-induced iNOS expression signaling pathway [27] which regulates the production of NO which, in turn, may enhance the expression of COX-2. On the other hand, at the highest concentrations used, neorogioltriol does not display a significant inhibitory effect on MAPK phosphorylation. Using SB203580 (or PD98059), we thus tried to see whether or not MAPK activity may explain the recovery of NO release by the cells treated with the highest concentrations of neorogioltriol.

Raw264.7 cells were incubated for one hour with SB203580 (or PD98059) prior to neorogioltriol treatment and LPS stimulation. Our results show that the use of p38MAPK (or ERK1/2) inhibitor does not inhibit the recovery of NO production observed with the highest concentration of neorogioltriol (Figure 6) suggesting that abrogation of NO inhibition in the neorogioltriol treated cells is not dependent on MAPK activation.

Taken together, these results show that the effect of neorogioltriol at high concentration is independent of MAPK and NF-κB. This effect may however be indirectly dependent on NF-κB inhibition. Indeed, some non steroid anti-inflammatory drugs (NSAIDs) are known to activate COX-2 through signaling pathways independent of NF-κB and MAPK and involving the nuclear factor PPARγ. On the other hand, LPS has been shown to drive down PPARγ expression through the activation of NF-κB [28]. This may suggest that the repression of NF-κB by neorogioltriol inhibits the negative loop of NF-κB on PPARγ, which may allow the latter to induce the expression of NOS2 and COX-2. We are currently investigating this hypothesis.

The collection of Laurencia glandulifera and the extraction and isolation procedure of neorogioltriol have been already described [16].

The anti inflammatory activity on carrageenan-induced paw edema was determined according to the method described by [29]. Naïve rats were randomly allocated to four groups and pretreated 1 hour before intraplantar administration of 100 μL of 0.6% of carrageenan. (i) Control group: received 2.5 mL/kg of vehicle (physiological solution (0.9% NaCl)) used to re-suspend the different drugs (ii) Standard group 1: received 1 mg/kg of dexamethasone (2.5 mL/kg); (iii) Standard group 2: received 300 mg/kg of asprin (2.5 mL/kg); and (iiii) Test group: received 1 mg/kg or 0.5 mg/kg of neorogioltriol (2.5 mL/kg). The drugs were administrated by intraperitoneal injection. The edema volume was determined using a plethysmometer (model 7150, Ugo Basile, Italy) prior to and 1, 3 and 5 h after carrageenan injection. The percentage of inhibition in treated rats versus control was calculated using the following formula: EI (%) = ([(C_t − C₀) control − (C_t − C₀) treated]/(C_t − C₀) control) × 100 where C_t correspond to edema volume after carrageenan injection and C₀ correspond to edema volume before carrageenan injection.

ERK1/2 antibodies and p38 MAPK antibodies were from Ozyme (Ozyme, Saint Quentin en Yveline, France). COX-2 antibody was from Becton Dickinson (BD Biosciences, France). PD098059, SB203580, LPS and griess reagent were purchased from Sigma (Sigma-Aldrich, Taufkirchen, Germany).

The mouse macrophagic cell line Raw264.7 cells were obtained from American Type Culture Collection and were grown at 37 °C, in RPMI 1640, supplemented with 5 mM L-glutamine, 10% heat inactivated fetal calf serum (endotoxin-free; HyClone), penicillin (100 U/mL) and streptomycin (100 μg/mL).

The MTT assay was used to test the potential toxic effects of neorogioltriol on the mouse macrophagic cell line Raw264.7 cells. Briefly, Raw264.7 cells were incubated with various concentrations of neorogioltriol for 24 h. MTT solution was added to cell cultures at the final concentration of 0.5 mg/mL and cells were incubated for an additional 2 h. Thereafter, medium was removed and plates were thoroughly washed with PBS. Cells were finally lysed in DMSO. The absorbance of each well at 540 nm was read by an automatic microplate reader. The average OD measured at different concentrations of neorogioltriol was compared using one-way analysis of variance (ANOVA). Proliferation of the cells exposed to the drugs was compared to the negative control. P < 0.05 was considered statistically significant.

Raw264.7 cells were stably transfected by electroporation with an NF-κB luciferase reporter construct containing three NF-κB target sequences in tandem fused to the luciferase reporter gene (3X-NF-κB-Luc), a gift from Dr J. Pierre (Faculté de pharmacy, Chatenay-Malabry, France). Cells were either left untreated or were treated with neorogioltriol at different doses for 30 min, they were then washed and stimulated with LPS (100 ng/mL) for an additional 6 hours. Protein concentration was determined by microBCA assay. Luciferase activity was determined using a luminometer TD-20/20 (Promega, charbonnieres, France).

Cell extracts were obtained by adding 25 μL of lysis buffer containing 10 mM Tris-HCL, pH 7.5, 50 mM NaCl, 50 mM sodium fluoride (NaF), 2 mM EDTA, 1 mM ethylene glycol-bis (β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 2% Nonidet-P40 (NP-40), 0.75% sodium deoxycholate (DOC), 1 mM orthovanadate, 1 μg/mL aprotinine, 1 mM PMSF, 1 mM DTT. After 30 min incubation on ice, the extracts were centrifuged at 15,000 rpm for 20 min. 25 μg of whole cell lysates were resolved by electrophoresis in a 12% SDS-polyacrylamide gel. Resolved proteins were electrophoretically transferred onto PVDF sheets (Hybond-P; Amersham) and membranes were blocked by incubation in 3% non-fat milk and 0.1% Tween in PBS for 1 h at room temperature followed by incubation with COX-2 antibody in 0.1% Tween-PBS. Bound antibody was detected by incubation with horseradish peroxidase-coupled secondary antibody (Amersham Pharmacia Biotech., Buckinghamshire, UK). To ensure equal loading, the blots were then stripped (62.5 mM Tris, pH 6.8), 0.1 M β-mercaptoethanol, 2% SDS) and re-probed with ERK1/2 antibody.

Macrophage cells were seeded in 24 wells, treated with different concentrations of neorogioltriol or vehicle for 30 min and then incubated with 100 ng/mL of LPS for 24 h. NO production in culture supernatant was spectrophotometrically evaluated by measuring nitrite, an oxidative product of NO. Nitrites were measured by Griess reaction by mixing 50 μL of culture supernatant with 50 μL of griess reagent. Absorbance was measured in microplates at 540 nm against a calibration curve with sodium nitrite standard. Supernatants were also analysed for TNFα content using BD Pharmingen (BD Biosciences, France) antibodies. The assays were performed according to the manufacturer’s instructions.

For TNFα production, NO release and NF-κB activation, the Kruskal-Wallis test was used to analyze significant differences between groups. The comparison between the two groups (presence or absence of SB203580) was performed using the Mann-Whitney test. For all statistical comparisons a p value <0.05 was defined as significant.