Evaluation of anti-nociceptive and anti-inflammatory activities of a heterofucan from Dictyota menstrualis

Abstract

Fucan is a term that defines a family of homo- and hetero-polysaccharides containing sulfated l-fucose in its structure. In this work, a heterofucan (F2.0v) from the seaweed, Dictyota menstrualis, was evaluated as an antinociceptive and anti-inflammatory agent. F2.0v (20.0 mg/kg) inhibits 100% of leukocyte migration into the peritoneal cavity after chemical stimulation. However, F2.0v does not alter the expression of interleukin-1 beta (IL-1β) and interleukin-6 (IL-6), as well as tumor necrosis factor alpha (TNF-α). F2.0v (20.0 mg/kg) has peripheral antinociceptive activity with potency similar to dipyrone. On the other hand, it had no effect on pain response on the hot plate test. Confocal microscopy analysis and flow cytometry showed that F2.0v binds to the surface of leucocytes, which leads us to suggest that the mechanism of action of anti-inflammatory and antinociceptive F2.0v is related to its ability to inhibit the migration of leukocytes to the site of tissue injury. In summary, the data show that F2.0v compound has great potential as an antinociceptive and anti-inflammatory, and future studies will be performed to further characterize the mechanism of action of F2.0v.

Figure 1

(A) Elution profile of F2.0v in gel filtration chromatography (A). Gel filtration chromatography fractions of a 1 mL/tube were collected and checked by the method of phenol/sulfuric [18], as well as by the presence of fucose [19]; (B) F2.0v representative agarose gel electrophoresis in 1,3-diaminopropane acetate buffer, stained with toluidine blue.

Figure 2

FTIR spectra of F2.0v from D. menstrualis.

Figure 3

The effect of F2.0v in the migration of leukocytes in mice with induced peritonitis peptone. The bars show the media of the number of leukocytes per mL of peritoneal fluid, 6 h after intra-peritoneal administration of peptone. Peptone (10%) and dexamethasone (1.0 μg/g) are the negative and positive control, respectively. The effect of F2.0v when compared to the positive control (* p < 0.05; ** p < 0.001). ∆: There are no significant differences when compared to each other.

Figure 4

F2.0v binds leukocytes. Equal number of cells from peritoneal lavage were treated with phosphate buffered saline (PBS) (A), C11 antibody (B), biotinylated F2.0v (C) or both (D) and analyzed by flow cytometry. Similar results were obtained in three independent experiments.

Figure 5

F2.0v binds leukocyte cell surface. (A) Biotinylated F2.0v is shown in red; (B) Nuclei are shown in blue, stained with DAPI; (C) WGA-lectin conjugated with FITC is shown in green; (D) Merge of the three images and the yellow color indicates colocalization of F2.0v and WGA-lectin. Similar results were obtained in two independent experiments.

Figure 6

Endothelial cells exposed to biotinylated fucan; the cells were grown for three days and exposed to biotinylated fucan. The image was obtained by confocal microscopy. Barr = 20 μm. Similar results were obtained in two independent experiments. (I) The cells were exposed to biotinylated fucan and revealed with streptavidin conjugated with Texas Red; (II) Fibronectin revealed with anti-fibronectin conjugated with FTIC; (III) Nucleus stained in blue with DAPI; (IV) Superposition of images A and B. Arrows indicate the cell nucleus stained with DAPI.

Figure 7

Biotinylated fucan does not bind to the endothelial cell surface; the biding of fucan on the endothelial cell surface was analyzed by flow cytometry.WGA: FITC conjugated WGA-lectin; C: endothelial cell; FB: endothelial cell exposed to biotinylated fucan. A representative out of three experiments yielding identical results is shown.

Figure 8

The effect of administration of F2.0v in the number of abdominal contractions in acetic acid-induced mice. Data are expressed as the mean ± S.E.M. of six animals for each group. * p < 0.001 indicates significant difference from the saline group. ∆: There are no significant differences when compared to each other.

Figure 9

The effect of administration of F2.0v in the reaction time of the hot place test. Data are expressed as the mean ± S.E.M. of six animals for each group. * p < 0.001 indicates significant difference from the saline group.