Niranjan Rajapakse

Inhibition of inducible nitric oxide synthase and cyclooxygenase-2 in lipopolysaccharide-stimulated RAW264.7 cells by carboxybutyrylated glucosamine takes place via down-regulation of mitogen-activated protein kinase-mediated nuclear factor-κB signaling

Glucosamine (GlcN) has been reported to possess several biomedical properties, and currently a great deal of attention has been focused on improving the functional properties of GlcN for different applications. Therefore, this study was conducted to introduce a carboxybutyryl functional group to GlcN and to find out the inhibitory mechanism of a novel GlcN derivative, carboxybutyrylated GlcN (CGlcN), on the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2) in bacterial lipopolysaccharide (LPS)‐induced mouse macrophages (RAW264.7 cells). In the initial experiments, the production of NO and prostaglandin E2 (PGE2) was inhibited by CGlcN pretreatment and suggested the possibility of down‐regulating their respective genes, iNOS and COX‐2. Reverse transcription‐polymerase chain reaction and Western blot analysis revealed that CGlcN can affect both transcriptional and translational levels of iNOS and COX‐2 expression. The data from the nuclear factor‐κB (NF‐κB) promoter gene transfection experiment supported the idea that inhibition of iNOS and COX‐2 is caused by the down‐regulation of their transcription factor, NF‐κB. Following stimulation with LPS, p38 mitogen‐activated protein kinase (p38 MAPK) and c‐Jun N‐terminal kinase (JNK) present upstream of NF‐κB signaling were also inhibited by CGlcN treatment. However, the protein level of another MAPK, extracellular signal‐regulated kinase (ERK), remained unaffected. Moreover, following treatment with CGlcN, the protein expression of I‐κB kinase (IKK) clearly confirmed that its down‐regulation directly inhibited the degradation of IκB and release of NF‐κB. Therefore, it can be concluded that CGlcN is capable of inhibiting iNOS and COX‐2 expression in LPS‐induced RAW264.7 cells via attenuation of NF‐κB signaling by p38 MAPK and JNK, but not by ERK.

An in vitro cellular analysis of the radical scavenging efficacy of chitooligosaccharides.

Despite extensive study on biological activities of chitosan and chitooligosaccharides (COS), there is no experimental evidence available as to COS mediated inhibition of free radical damage in cellular oxidizing systems. In this study, radical scavenging efficacies of different molecular weight bearing COS were assessed and their intracellular radical scavenging effects were tested employing B16F1, murine melanoma cell line. The results exhibited appreciable suppression in occurrence of intracellular radical species in the presence of low molecular weight bearing COS (<1 kDa) confirming low molecular weight is important for observed activities in biological systems. However, DNA oxidation carried out in the presence of COS clearly exhibited that COS exert protective effect on oxidative damage of purified genomic DNA regardless of molecular weight. Low molecular weight bearing COS was observed to be successively participated in suppression of NF-kappaB gene promoter activity suggesting its capability to prevent oxidative stress related disease complications. Moreover, induction of intracellular glutathione (GSH) level in the presence of COS promoted the effectiveness of COS to act against cellular oxidative stress.

Nutritional and digestive health benefits of seaweed.

Seaweed is a famous delicacy in some parts of the Asia and also a well-known source of important food hydrocolloids, such as agar, alginates, and carrageenan. In addition to the food value of seaweed, several health benefits have also been reported to be present in this valuable food source. It is presumed that the unique features of the marine environment, where the seaweeds are grown, are mainly responsible for most of its properties. Among the functional effects of the seaweed, nutritional and health-related benefits have been widely studied. Compared to the terrestrial plants and animal-based foods, seaweed is rich in some health-promoting molecules and materials such as, dietary fiber, ω-3 fatty acids, essential amino acids, and vitamins A, B, C, and E. In this chapter, the nutritive value of seaweed and the functional effects of its soluble fiber are discussed with a special reference to the digestive health promotion of human.

Anti-inflammatory effect of Ishige okamurae ethanolic extract via inhibition of NF-κB transcription factor in RAW 264.7 cells

Nowadays, much attention has been paid to the development of anti‐inflammatory agents from marine natural resources. As a result of screening anti‐inflammatory agents from marine algae using immunoassay, we found for the first time that ethanolic extract of Ishige okamurae (IO) classified into brown algae was effective in inhibiting the production of inflammatory mediators, such as tumor necrosis factor‐α, interleukin‐1β, interleukin‐6 and prostaglandin E2, in RAW264.7 cells stimulated by lipopolysaccharide, compared with dexamethasone and aspirin used as positive control in this study. Moreover, transcriptional activation of NF‐κB transcription factor that regulates the expression of these inflammatory mediators was also examined using reporter gene assay and western blot analysis. It was observed that IO extract exerted anti‐inflammatory effect via inactivation of NF‐κB transcription factor in macrophages. In addition, the expression and activity of matrix metalloproteinase‐2 and 9 that play an important role in chronic inflammation were decreased in dose‐dependent manner in the presence of IO extract in HT1080 cells. The above results suggest that IO extract can inhibit inflammation through inactivation of NF‐κB transcription factor in macrophage. Copyright

The inhibitory mechanism of a novel cationic glucosamine derivative against MMP-2 and MMP-9 expressions.

A number of recent researches have demonstrated the therapeutic effects of glucosamine (Glc) in a range of human diseases including arthritis. For the first time, we identified that a novel Glc derivative having quaternized amino functionality (QAGlc) suppresses MMP-9 and MMP-2, gelatinases in HT1080, human fibrosarcoma cells at 40microg/ml, following stimulation with PMA. Reporter gene assay results revealed that, the mechanism of suppression involves decreased transcriptional activation of MMP-9 and MMP-2 via transcription factors NF-kappaB and AP-1. However based on western blot results, QAGlc did not attenuate the nuclear translocation of both NF-kappaB and AP-1. Apparently, phorbol myristate acetate (PMA) stimulated expressions of ERK, JNK and p38 were altered in the presence of potent tumour inducer, phorbol myristate acetate QAGlc, suggesting their suppression also contributes to QAGlc-mediated inhibition of MMP-9 and MMP-2. Moreover, the ability of QAGlc to inhibit gelatinases was confirmed by its ability to act against invasiveness of HT1080 cells through extracellular matrix components.

Suppression of cytokine production in lipopolysaccharide-stimulated mouse macrophages by novel cationic glucosamine derivative involves down-regulation of NF-κB and MAPK expressions

Exposure of macrophages to bacterial lipopolysaccharide (LPS) induces release of proinflammatory cytokines that play crucial roles in chronic inflammation. Glucosamine has reported to possess anti-inflammatory properties and currently is the oral supplement of choice for the management of inflammation related complications including osteoarthritis. In this study, quaternized amino glucosamine (QAGlc), a newly synthesized cationic glucosamine (Glc) derivative was found to inhibit LPS-stimulated production of IL-1beta, IL-6, TNF-alpha, and PGE(2) in RAW264.7, mouse macrophages more potently than its starting material Glc. Since production of cytokines is regulated mainly via activation of NF-kappaB and regulation of mitogen-activated protein kinases (MAPKs), we examined if QAGlc could be responsible for the suppression of NF-kappaB pathway and MAPKs. We used reporter gene assay and Western blotting to examine the effects of QAGlc on activation and translocation of NF-kappaB. Further, QAGlc-mediated inhibition of NF-kappaB was accompanied with a suppression of its translocation. Apparently, QAGlc was shown to attenuate LPS-induced activation of p38 MAPK and JNK in RAW264.7 cells suggesting that inhibition of MAPK-mediated LPS signaling also contribute to suppression of cytokine production following stimulation of macrophages with LPS.

Effect of spongin derived from Hymeniacidon sinapium on bone mineralization

Marine sponges have been known to provide a source of novel bone and cartilage replacements because of their secondary metabolites and specific skeleton structures. In particular, it has been reported that spongin as a component of fibrous skeleton, pseudokeratin, neurokeratin, horny protein, and collagen-like protein in sponges can be used in several biomedical applications including osteoarthritis (OA). However, the pharmacological mechanism of action of spongin remains obscure. In this study, it was investigated whether spongin derived from Hymeniacidon sinapium can promote bone mineralization of osteoblast-like MG-63 cells. Our present study provides the first evidence that spongin is effective in activating bone mineralization. Furthermore, spongin increased ALP activity, collagen synthesis, and osteocalcin secretion in addition to bone mineralization in osteoblastic cells in vitro. In addition, it was demonstrated that spongin exerted the inhibitory effect on production of inflammatory mediators such as TNF-alpha, IL-1beta, and PGE(2) in macrophage, RAW264.7 cells. These results suggest that the anti-inflammatory effect of spongin derived from Hymeniacidon sinapium can play a critical role in bone mineralization of osteoblast-like MG-63 cells.

Chitosan Nanoparticles: Preparation, Characterization, and Applications

Chitosan, a polysaccharide obtained by partial deacetylation of chitin from the shells of crustaceans, is one of the most often studied natural biopolymers, having the potential for numerous industrial applications. The development of micro- and nanoparticles of chitosan and its derivatives paved a path for applying these biomolecules in a more effective and economical manner, and expanding their applications in more diverse fields than those expected. Similar to all the other types of nanoparticles developed from different materials, chitosan nanoparticles also possess their own physical, chemical, and morphological characteristics that finally determine their applications. The methods of preparation of chitosan nanoparticles are significantly responsible for their bioactivities and behavioral characteristics in different systems and applications. The main methods of preparation of chitosan nanoparticles include emulsion cross-linking, emulsion-droplet coalescence, coacervation/precipitation, ionotropic gelation, reverse micelles, template polymerization, and molecular self-assembly. All these methods have their own advantages as well as drawbacks, in relation to the properties of the nanoparticles. However, careful preparation of chitosan nanoparticles could provide a higher affinity for negatively charged biological membranes and site-specific targeting in vivo, enabling their application as encapsulating materials of drugs, enzymes, and DNA, used in controlled release systems and as coatings of wound dressings to accelerate healing. Chitosan nanoparticles-based films are used in the food industry to control microorganisms in food and to enhance shelf life while strengthening the mechanical properties and stability of the food-packing materials. Although the chitosan nanoparticles appear to be safe in some of their applications, knowledge on the risks imposed in these food and pharmaceutical applications needs to be strengthened further.