Tatsuya Oda

The potent activity of sulfated polysaccharide, ascophyllan, isolated from Ascophyllum nodosum to induce nitric oxide and cytokine production from mouse macrophage RAW264.7 cells: Comparison between ascophyllan and fucoidan

Ascophyllan isolated from the brown alga Ascophyllum nodosum is a fucose-containing sulfated polysaccharide, which has similar but distinct characteristic monosaccharide composition and entire chemical structure to fucoidan. In this study, we examined the effects of ascophyllan, fucoidan isolated from A. nodosum (A-fucoidan), and fucoidan from Sigma (S-fucoidan) as a representative fucoidan derived from other source (Fucus vesiculosus) on mouse macrophage cell line RAW264.7 cells. No significant cytotoxic effects of ascophyllan and A-fucoidan on RAW264.7 cells were observed up to 1000μg/ml, while S-fucoidan showed cytotoxic effect in a concentration-dependent manner. Ascophyllan induced extremely higher level of nitric oxide (NO) production from RAW264.7 cells than those induced by fucoidans over the concentration range tested (0-200μg/ml). Reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis revealed that expression level of inducible NO synthase (iNOS) in ascophyllan-treated RAW264.7 cells was much higher than the levels detected in the cells treated with fucoidans. Furthermore, the activities of ascophyllan to induce the secretion of tumor necrosis factor-α (TNF-α) and granulocyte colony-stimulating factor (G-CSF) from RAW264.7 cells were also greater than those induced by fucoidans especially at lower concentration range (3.1-50μg/ml). The activities of ascophyllan to induce NO and cytokine production in mouse peritoneal macrophages were also stronger than those of fucoidans. Electrophoretic mobility shift assay (EMSA) using infrared dye labeled nuclear factor-kappa B (NF-κB) and AP-1 consensus sequences suggested that ascophyllan can strongly activate these transcription factors. Marked increase in the nuclear translocation of p65, and the phosphorylation and degradation of IκB-α were also observed in ascophyllan-treated RAW264.7 cells. Analysis using mitogen-activated protein (MAP) kinase inhibitors and western blot analysis suggested that c-Jun N-terminal kinase (JNK) and p38 MAP kinase are mainly involved in ascophyllan-induced NO production.

Immunomodulatory Effects of Alginate Oligosaccharides on Murine Macrophage RAW264.7 Cells and Their Structure-Activity Relationships.

This study evaluated the immunomodulatory activities, including regulation of nitric oxide (NO), reactive oxygen species (ROS), and tumor necrosis factor (TNF)-α production in RAW264.7 murine macrophages, of alginate oligosaccharides (AOS) and investigated their structure-activity relationships. Our results revealed that unsaturated guluronate oligosaccharide prepared by enzymatic degradation (GOS-ED) induced NO production and inducible nitric oxide synthase (iNOS) expression, dose and time dependently, and stimulated ROS and TNF-α production; however, other AOS prepared by different ways or polymers showed very low and even no such effects. Moreover, GOS-ED induced macrophage activation to release the above-mentioned mediators partly involved in nuclear factor (NF)-κB and mitogen-activated protein (MAP) kinase signaling pathways. We also show that the structural characteristics of AOS, especially the unsaturated terminal structure, molecular size, and M/G ratio, play important roles in determining the macrophage-activating effects. GOS-ED could be applicable for agriculture, drug, and food industry as a potent immune-modulatory agent.

In vitro antioxidant activities of sulfated polysaccharide ascophyllan isolated from Ascophyllum nodosum.

Antioxidant activities of sulfated polysaccharide ascophyllan from Ascophyllum nodosum was investigated in vitro by various assays, and compared with those of fucoidan. A chemiluminescence (CL) analysis using a luminol analog, L-012, showed that ascophyllan scavenges superoxide, and the activity is greater than fucoidan. However, in the presence of 10μg/ml of ascophyllan or 10μg/ml and 100μg/ml of fucoidan, slightly enhanced CL-responses were observed. Since EDTA-treatment resulted in disappearance of the enhancement effects, it was suggested that metal ions especially iron ions in the polysaccharides might be involved in this phenomenon. In fact, metal element analysis revealed that ascophyllan and fucoidan inherently contain iron and other metal elements. EDTA-treatment resulted in significant increase in Fe(2+)-chelating activities of these polysaccharides. In an electron spin resonance (ESR)-spin trapping analysis in which direct UV-radiation to hydrogen peroxide was used as a source of hydroxyl radical, ascophyllan and fucoidan showed potent hydroxyl radical scavenging activity with similar extent. Reducing power of ascophyllan was stronger than that of fucoidan. Our results indicate that ascophyllan can exhibit direct and potent antioxidant activity.

Effects of alginate oligosaccharide mixtures on the growth and fatty acid composition of the green alga Chlamydomonas reinhardtii

Alginate is a natural acidic linear polysaccharide that is produced by brown seaweeds. It is currently used in a broad range of commercial enterprises, such as the food and medical products industries. Recent evidence has demonstrated that alginate oligosaccharides may function as growth promoting agents for certain plant cells, including those of some green algae. Chlamydomonas reinhardtii is a green alga that is used as a model organism in fundamental molecular biology studies; it is also a producer of biohydrogen. In the present study, we examined effects of two types of alginate oligosaccharide mixtures (AOMs), which were prepared by either enzymatic degradation (ED) or acid hydrolysis (AH), on the growth of C. reinhardtii. Growth was significantly promoted by AOM (ED) in a concentration-dependent manner. The maximum effect was observed on day 4 of treatment. The fatty acid composition of C. reinhardtii was also influenced by AOM (ED); the levels of C16:0, C18:2 cis and C18:3 n-3 increased in treated cells. AOM (AH) and the other saccharides that we tested did not affect the growth of C. reinhardtii. The effects that we identified could promote efficient biomass production by reducing culture times and by changing cellular fatty acid levels.

Inhibitory effect of sulphated polysaccharide porphyran on nitric oxide production in lipopolysaccharide-stimulated RAW264.7 macrophages

Porphyran, extracted from an edible red alga (Porphyra yezoensis), is a sulphated polysaccharide with a wide variety of biological activities including anti-tumour, antioxidant and immuno-modulating activities. In this study, we examined the effect of porphyran on nitric oxide (NO) production in mouse macrophage cell line RAW264.7 cells. Although no significant activity of porphyran to induce NO or tumour necrosis factor-α (TNF-α) production in RAW264.7 cells was observed at the concentration range tested (10-500 µg/ml), it was found for the first time that porphyran inhibited NO production and expression of inducible nitric oxide synthase (iNOS) in RAW264.7 cells stimulated with lipopolysaccharide (LPS). In the presence of 500 µg/ml porphyran, NO production and expression of iNOS in LPS-treated RAW264.7 cells were completely suppressed. On the other hand, porphyran showed only a marginal effect on the secretion of TNF-α from LPS-stimulated RAW264.7 cells. Electrophoretic mobility shift assay (EMSA) using infrared dye labelled oligonucleotide with nuclear factor-κB (NF-κB) consensus sequence suggested that porphyran inhibited the LPS-induced NF-κB activation. The LPS-inducible nuclear translocation of p65, and the phosphorylation and degradation of IκB-α were also inhibited by the pre-treatment with porphyran. Our results obtained in in vitro analysis suggest that porphyran suppresses NO production in LPS-stimulated macrophages by the blocking of NF-κB activation.

Comparative study on antioxidative and macrophage-stimulating activities of polyguluronic acid (PG) and polymannuronic acid (PM) prepared from alginate

The antioxidant and macrophage-stimulating activities of polyguluronic acid (PG) and polymannuronic acid (PM) prepared from alginate were examined. A chemiluminescence (CL) method using a luminol analog, L-012, showed that both PM and PG scavenge superoxide produced by hypoxanthine-xanthine oxidase system in a concentration-dependent manner. At 100 μg/ml, PG showed slightly stronger superoxide scavenging activity than PM. In an electron spin resonance (ESR)-spin trapping method in which the Fenton reaction was used as hydroxyl radical generation system, we found that both PM and PG showed potent hydroxyl radical scavenging activity to a similar extent. Because PM and PG showed no chelating activity on Fe(2+), it was confirmed that PM and PG can directly scavenge hydroxyl radical. No significant scavenging activity of PM and PG toward hydrogen peroxide was observed. Interestingly, the macrophage-stimulation activity of PG as measured by nitric oxide (NO)-production from mouse macrophage cell line RAW264.7 cells was evidently stronger than that of PM. Our results suggest that RAW264.7 cells might be able to distinguish the conformational differences between PM and PG, and respond differently to them, whereas the effects of such structural differences between PM and PG on the radical scavenging activities may not be so significant.

Importance of sulfate groups for the macrophage-stimulating activities of ascophyllan isolated from the brown alga Ascophyllum nodosum

To investigate the role of sulfate groups on the macrophage-stimulating activities of ascophyllan, we prepared desulfated ascophyllan, and its effects on RAW264.7 cells were compared with native ascophyllan. The chemical structural analysis revealed that nearly 21% of sulfate groups of ascophyllan were removed by desulfation reaction, while no significant changes in the molecular mass and monosaccharide composition occurred after desulfation. NO- and cytokine- (TNF-α and G-CSF) inducing activities of the desulfated ascophyllan on RAW264.7 cells were significantly decreased as compared to native ascophyllan. Furthermore, the activity of desulfated ascophyllan to induce reactive oxygen species (ROS) generation from RAW264.7 cells decreased to almost negligible level. Our results suggest that the level of sulfate groups of ascophyllan is an important structural element responsible for the macrophage-stimulating activities. Probably, even the limited removal of sulfate residues sensitive to desulfation reaction may result in significant decrease in the bioactivities of ascophyllan.

Immunostimulatory activities of the sulfated polysaccharide ascophyllan from Ascophyllum nodosum in in vivo and in vitro systems.

Splenic natural killer (NK) cell activity against YAC-1 cells increased in mice intraperitoneally injected with ascophyllan. Ascophyllan enhanced the cytotoxicity of RAW264.7 cells toward YAC-1 cells in a concentration-dependent manner. The cytotoxicity of ascophyllan-stimulated RAW264.7 cells as to YAC-1 cells was suppressed with N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME), an inhibitor of nitric oxide (NO) synthase, suggesting the involvement of NO in the cytotoxicity of ascophyllan-stimulated RAW264.7 cells.

Comparative study on modeccin- and phytohemagglutinin (PHA)-induced secretion of cytokines and nitric oxide (NO) in RAW264.7 cells.

The effects of cytotoxic lectins, modeccin and phytohemagglutinin (PHA) on mouse macrophage cell line RAW264.7 was studied by detecting the induction of inflammatory mediators. Results showed that modeccin induced the release of tumor necrosis factor-α (TNF-α) from RAW264.7 cells with a bell-shape concentration-dependent profile. PHA that showed no significant cytotoxicity on RAW264.7 cells up to 100,000 ng/ml induced much higher level of TNF-α than modeccin. PHA simultaneously induced the secretion of granulocyte colony stimulation factor (G-CSF) from RAW264.7 cells with even much higher level than that of TNF-α, whereas modeccin did not. Furthermore, PHA induced the secretion of nitric oxide (NO) in RAW264.7 cells, while no significant level of NO was detected in the modeccin-treated cells. NH₄Cl (a lysomotoropic agent) and cycloheximide (a ribosome inhibitor) strongly inhibited modeccin-induced TNF-α secretion, but no significant inhibitory effects of these reagents on the PHA-induced TNF-α secretion were observed. Contrary to modeccin-induced TNF-α secretion, even slightly increased TNF-α secretion was observed in PHA-treated cells in the presence of 10 mM NH₄Cl. In addition, the inhibition profiles of modeccin-induced TNF-α secretion by various kinase inhibitors were different from those of PHA. These results suggested that the action mode of modeccin to stimulate RAW264.7 cells leading to the secretion of inflammatory molecules, including TNF-α, is distinct from that of PHA. On the other hand, significantly increased translocation of activator protein-1 (AP-1), a crucial transcription factor involved in expression of inflammatory molecules, into nucleus was observed in RAW264.7 cells treated with PHA and modeccin.

Unsaturated guluronate oligosaccharide enhances the antibacterial activities of macrophages

Alginate from marine seaweeds is receiving continuous attention owing to its wide physiological activities. Herein, we sought to elucidate possible effects of alginate‐derived polyguluronate (PG) and unsaturated guluronate oligosaccharide (GOS) on antibacterial activities of macrophages. Our results showed that, in contrast to PG, GOS markedly increased the phagocytosis of IgG‐opsonized Escherichia coli and Staphylococcus aureus and further inhibited the survival of intracellular bacteria in macrophages. In line with this, GOS treatment resulted in the enhanced expression of Fcγ receptors on macrophages. In addition, GOS activated NF‐κB pathway, induced TNF‐α secretion, and elevated the expression of inducible nitric oxide synthase and the production of nitric oxide. Meanwhile, GOS stimulated the production of reactive oxygen species in macrophages. Moreover, guluronate trimer to hexamer (G3–G6) in GOS exhibited significant activity that increased the bacterial phagocytosis of macrophages, with the pentamer (G5), displaying the highest activity. Finally, our in vivo results further confirmed that GOS but not PG significantly improved bacterial clearance in murine acute peritonitis. In conclusion, GOS enhances antibacterial activities of macrophages via modulating signaling pathways related to innate immunity, suggesting that GOS might be a promising therapeutic candidate to improve the host defense against bacterial infection. Xu, X., Bi, D., Wu, X., Wang, Q., Wei, G., Chi, L., Jiang, Z., Oda, T., and Wan, M. Unsaturated guluronate oligosaccharide enhances the antibacterial activities of macrophages. FASEB J. 28, 2645–2654 (2014). www.fasebj.org