Sulfated Glucosamine Research Articles

Therapeutic effect of San Bi Tang combined with glucosamine sulfate capsules in cold-dampness-type knee osteoarthritis.

Cold-dampness-type knee osteoarthritis is a common middle-aged and elderly disease, but its pathogenesis is not fully understood, and its clinical treatment has limitations. Glucosamine sulfate capsules are commonly used for treating arthritis, and San Bi Tang is a classic formula of traditional Chinese medicine (TCM) that has the effects of warming yang, dispelling dampness, relaxing muscles, and activating collaterals. This research hypothesized that the combination of modified San Bi Tang and glucosamine sulfate capsules could enhance the clinical efficacy of treating cold-dampness-type knee osteoarthritis through complementary effects. To analyze the clinical efficacy of San Bi Tang combined with glucosamine sulfate capsules when treating cold-dampness-type knee osteoarthritis. A total of 110 patients with cold-dampness-type knee osteoarthritis were selected as research subjects and randomly divided into a control group and an experimental group of 55 cases each. The control group received only treatment with glucosamine sulfate capsules, while the experimental group received additional treatment with modified San Bi Tang for a duration of 5 wk. The patients' knee joint functions, liver and kidney function indicators, adverse reactions, and vital signs were evaluated and analyzed using SPSS 26.0 software. Before treatment, the two groups' genders, ages, and scores were not significantly different, indicating comparability. Both groups' scores improved after treatment, which could indicate pain and knee joint function improvement, but the test group had better scores. The TCM-specific symptoms and the clinical efficacy of the treatment in the test group were higher. Before and after treatment, there were no abnormalities in the patients' liver and kidney function indicators. The combination of modified San Bi Tang and glucosamine sulfate capsules is superior to treatment with sulfated glucosamine alone and has high safety.

Efficacy of Chondroprotective Food Supplements Based on Collagen Hydrolysate and Compounds Isolated from Marine Organisms.

Osteoarthritis belongs to the most common joint diseases in humans and animals and shows increased incidence in older patients. The bioactivities of collagen hydrolysates, sulfated glucosamine and a special fatty acid enriched dog-food were tested in a dog patient study of 52 dogs as potential therapeutic treatment options in early osteoarthritis. Biophysical, biochemical, cell biological and molecular modeling methods support that these well-defined substances may act as effective nutraceuticals. Importantly, the applied collagen hydrolysates as well as sulfated glucosamine residues from marine organisms were strongly supported by both an animal model and molecular modeling of intermolecular interactions. Molecular modeling of predicted interaction dynamics was evaluated for the receptor proteins MMP-3 and ADAMTS-5. These proteins play a prominent role in the maintenance of cartilage health as well as innate and adapted immunity. Nutraceutical data were generated in a veterinary clinical study focusing on mobility and agility. Specifically, key clinical parameter (MMP-3 and TIMP-1) were obtained from blood probes of German shepherd dogs with early osteoarthritis symptoms fed with collagen hydrolysates. Collagen hydrolysate, a chondroprotective food supplement was examined by high resolution NMR experiments. Molecular modeling simulations were used to further characterize the interaction potency of collagen fragments and glucosamines with protein receptor structures. Potential beneficial effects of collagen hydrolysates, sulfated glycans (i.e., sulfated glucosamine from crabs and mussels) and lipids, especially, eicosapentaenoic acid (extracted from fish oil) on biochemical and physiological processes are discussed here in the context of human and veterinary medicine.

Systematic Analysis of Pharmaceutical Preparations of Chondroitin Sulfate Combined with Glucosamine.

Glycosaminoglycans are carbohydrate-based compounds widely employed as nutraceuticals or prescribed drugs. Oral formulations of chondroitin sulfate combined with glucosamine sulfate have been increasingly used to treat the symptoms of osteoarthritis and osteoarthrosis. The chondroitin sulfate of these combinations can be obtained from shark or bovine cartilages and hence presents differences regarding the proportions of 4- and 6-sulfated N-acetyl β-d-galactosamine units. Herein, we proposed a systematic protocol to assess pharmaceutical batches of this combination drug. Chemical analyses on the amounts of chondroitin sulfate and glucosamine in the batches were in accordance with those declared by the manufacturers. Anion-exchange chromatography has proven more effective than electrophoresis to determine the type of chondroitin sulfate present in the combinations and to detect the presence of keratan sulfate, a common contaminant found in batches prepared with shark chondroitin sulfate. 1D NMR spectra revealed the presence of non-sulfated instead of sulfated glucosamine in the formulations and thus in disagreement with the claims declared on the label. Moreover, 1D and 2D NMR analyses allowed a precise determination on the chemical structures of the chondroitin sulfate present in the formulations. The set of analytical tools suggested here could be useful as guidelines to improve the quality of this medication.

Fractal analysis of extra-embryonic vessels of chick embryos under the effect of glucosamine and chondroitin sulfates

Like heparan sulfate proteoglycans, some monosaccharides and glycosaminoglycans, such as sulfated glucosamine (GS) and chondroitin (CS), integrate the vascular extracellular matrix and may influence vascular endothelial cell growth. To assess the effects of these substances on blood vessel formation, we used the chick yolk sac membrane (YSM) model and fractal geometry quantification, which provided an objective in vivo method for testing potential agents that promote vasculogenesis and angiogenesis. An image processing method was developed to evaluate YSM capillary vessels after they were implanted in a methylcellulose disk of GS or CS at a concentration between 0.001–0.1mg/disk (performed on 2-day old embryos). This method resulted in a binary image of the microvascular network (white vessels on a black background). Fractal box-counting (DBC) and information (DINF) dimensions were used to quantify the activity of GS and CS in vasculogenesis and angiogenesis. YSM treated with GS (0.001–0.1mg) and CS (0.03–0.1mg) showed an increase in fractal dimensions that corresponded to vitelline vessel growth compared to the control group (vehicle), with GS displaying higher fractal dimension values.

Anharmonic simulations of the vibrational spectrum of sulfated compounds: application to the glycosaminoglycan fragment glucosamine 6-sulfate.

Mid-infrared spectroscopy coupled with mass spectrometry is an appealing tool for the sequencing and structural elucidation of functional modifications in biopolymers, as it offers direct spectroscopic identification of the functionality where the traditional mass spectrometric approach is insufficient. Whereas the gas phase vibrational spectroscopy of peptides (and to a lesser extent saccharides) has been widely investigated, sulfation has attracted much less attention, despite its prevalence in natural polymers. The simulation of the vibrational spectra of such functionalized compounds is however notoriously challenging, which impairs the interpretation of spectroscopic data in terms of structure. Driven by a striking case of such a failure for a sulfated glycosaminoglycan fragment, we elaborate on an original hybrid GVPT2 anharmonic approach. This strategy offers a significantly improved accuracy in the description of the sulfate modes, without the recourse to empirical scaling factors, and with a greatly reduced computational cost which is otherwise prohibitive for molecules of this size. Alternatively, we propose a selection of reasonably accurate harmonic methods with adequate scaling factors optimized on a set of benchmark compounds.

Quantitative Phosphoproteomics Analysis Reveals Broad Regulatory Role of Heparan Sulfate on Endothelial Signaling

Heparan sulfate (HS) is a linear, abundant, highly sulfated polysaccharide that expresses in the vasculature. Recent genetic studies documented that HS critically modulates various endothelial cell functions. However, elucidation of the underlying molecular mechanism has been challenging because of the presence of a large number of HS-binding ligands found in the examined experimental conditions. In this report, we used quantitative phosphoproteomics to examine the global HS-dependent signaling by comparing wild type and HS-deficient endothelial cells that were cultured in a serum-containing medium. A total of 7222 phosphopeptides, corresponding to 1179 proteins, were identified. Functional correlation analysis identified 25 HS-dependent functional networks, and the top five are related to cell morphology, cellular assembly and organization, cellular function and maintenance, cell-to-cell communication, inflammatory response and disorder, cell growth and proliferation, cell movement, and cellular survival and death. This is consistent with cell function studies showing that HS deficiency altered endothelial cell growth and mobility. Mining for the underlying molecular mechanisms further revealed that HS modulates signaling pathways critically related to cell adhesion, migration, and coagulation, including ILK, integrin, actin cytoskeleton organization, tight junction and thrombin signaling. Intriguingly, this analysis unexpectedly determined that the top HS-dependent signaling is the IGF-1 signaling pathway, which has not been known to be modulated by HS. In-depth analysis of growth factor signaling identified 22 HS-dependent growth factor/cytokine/growth hormone signaling pathways, including those both previously known, such as HGF and VEGF, and those unknown, such as IGF-1, erythropoietin, angiopoietin/Tie, IL-17A and growth hormones. Twelve of the identified 22 growth factor/cytokine/growth hormone signaling pathways, including IGF-1 and angiopoietin/Tie signaling, were alternatively confirmed in phospho-receptor tyrosine kinase array analysis. In summary, our SILAC-based quantitative phosphoproteomic analysis confirmed previous findings and also uncovered novel HS-dependent functional networks and signaling, revealing a much broader regulatory role of HS on endothelial signaling.

Anti-inflammatory action of sulfated glucosamine on cytokine regulation in LPS-activated PMA-differentiated THP-1 macrophages

The aim of this study was to evaluate the effect of sulfated glucosamine (SGlc) on the regulation of inflammatory cytokines and profiles involved in immunological activities. Changes in the inflammatory profiles of lipopolysaccharide (LPS)-activated phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophage models were investigated following SGlc treatment. Human THP-1 macrophages were used to evaluate anti-inflammatory profiles. The cytokine secretion levels were measured by enzyme-linked immunosorbent assay (ELISA). Effects of SGlc on the regulation of mRNA and protein levels were determined using RT-PCR and Western blot analysis. The effect of SGlc on the activation of mitogen-activated protein kinases (MAPKs) and NF-κB protein was also determined by Western blot analysis. Treatment of THP-1 cells with SGlc inhibited the production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6. In addition, SGlc suppressed the mRNA and protein expression levels of inflammatory mediators such as inducible nitric oxide synthase, cyclooxygenase-2, TNF-α, IL-1β, IL-6, 5-lipoxygenase and cytoplasmic phospholipase A(2) in LPS-activated THP-1 macrophages. Furthermore, we confirmed that the LPS-activated transcriptions of MAPKs and NF-κB were inhibited by SGlc treatment in PMA-differentiated THP-1 macrophages. These results suggest that SGlc can be considered as a potential anti-inflammatory supplement.

Anti-obesity effect of sulfated glucosamine by AMPK signal pathway in 3T3-L1 adipocytes

In this study, we investigated the effect of sulfated glucosamine (SGlc) on adipogenesis of 3T3-L1 adipocytes during differentiation of preadipocytes into adipocytes by measuring lipid accumulation and adipogenesis related factors. Treatment with SGlc reduced the triglyceride content in Oil-Red O staining and enhanced glycerol secretion in adipocytes in a dose-dependent manner. In addition, SGlc induced the down-regulation of adipogenesis related factors and adipocyte specific gene promoters. Moreover, treatment of 3T3-L1 adipocytes with SGlc activated the phosphorylated adenosine monophosphate-activated protein kinase (AMPK) α and β along with their substrate, acetyl-CoA carboxylase (ACC). These results suggest that inhibitory effect of SGlc on adipocyte differentiation might be mediated through the up-regulation of AMPK pathway.

Sulfated glucosamine inhibits oxidation of biomolecules in cells via a mechanism involving intracellular free radical scavenging

Although, several effects of glucosamine and its sulfated form (sulfated glucosamine) have been proposed for the suppression of osteoarthritis, their exact mechanisms have not been completely elucidated. This study explains the novel possibility of involvement of sulfated glucosamine in improving cellular antioxidant potential and thereby controlling oxidative damage that could be effective for its therapeutic potential in osteoarthritis. Treatment with sulfated glucosamine to human chondrocytes and macrophages inhibited radical simulated oxidation of membrane lipids, proteins and DNA in a dose-dependent manner. Moreover, detection of reactive oxygen species by electron spin resonance (ESR) spectroscopy and 2′,7′-dichlorodihydrofluororescein diacetate (DCFH-DA) fluorescence probe clearly confirmed effective radical scavenging potential of sulfated glucosamine in cellular and non-cellular systems. More importantly, NF-κB reporter gene assay and western blot analysis revealed that sulfated glucosamine inhibits radical mediated expression and activation of nuclear factor κB (NF-κB) proteins (transcription factor involves in expression of a number of genes related to osteoarthritis). Further, sulfated glucosamine enhanced reduced glutathione (GSH) level in oxidatively stressed human chondrocytes improving cellular redox balance. In conclusion, it is suggested that potential effects of sulfated glucosamine in controlling osteoarthritis might be partly via mechanisms involving direct scavenging of cellular radical species and alteration of oxidation mediated destructive events.

Sulfated glucosamine inhibits MMP-2 and MMP-9 expressions in human fibrosarcoma cells

In the present study, sulfated glucosamine (SGlc) that has been reported to relieve joint pain and inflammation in many arthritis patients was studied for its inhibitory effects on MMP-2 and MMP-9 in human fibrosarcoma cells. Expression and activity of above MMPs studied using gelatin zymography suggested SGlc as a potent MMP inhibitor. Further, transfection of promoter genes of MMPs and their transcription factors clearly exhibited that inhibition of MMP-2 and MMP-9 was due to down-regulation of transcription factor, NF-κB. However expression of activator protein-1 (AP-1), another important transcription factor of MMPs, was not affected by SGlc treatment. In addition, protein expression results of Western blot analysis were also in agreement with the results of gene transfection experiments. Moreover, down-regulation of NF-κB resulted in production of low levels of both NF-κB p50 and p65 proteins and directly affected activation process of MMP-2 and MMP-9 expressions. Since MMPs involve in joint inflammation, it can be presumed that inhibition of MMP-2 and MMP-9 can be one of the mechanisms of SGlc to be an effective drug in relieving the symptoms of osteoarthritis.

Glucosamine Coating for Inhibiting Bacterial Adhesion to Titanium Surfaces

It is known that glucosamine/chitosan derivatives have the ability of inhibiting bacterial adhesion to tooth and biomaterial surfaces. The hypotheses of this article included 1) the inhibition effects of different (chemically) glucosamine products are different and 2) more water-soluble glucosamine preparation(s) may have a superior inhibition effect. The basic testing systems are in vitro static adhesion models and the amount of the adhesion is evaluated by direct counting using epifluorecense microscopy. The test material is a smooth surfaced commercially pure titanium (cp-Ti). The bacterium tested is Staphylococcus aureus. The results showed that compared to the control samples (without glucosamine or chitosan coating) the sulfated p-GlcNAc and Sigma glucosamine HCl significantly inhibited S. aureus adhesion to titanium surfaces (94% and 78% respectively)

Quantitative proteomic analysis of fibroblast nuclear proteins after stimulation with mitogen activated protein kinase inhibiting heparan sulfate

Certain structures of heparan sulfate (HS) inhibit cell proliferation of fibroblasts. Whether this inhibition is dependent on inhibition of mitogenic signaling pathways or nuclear translocation of HS is unknown. In this study we investigated possible mechanism(s) and structural requirements by which antiproliferative glycosaminoglycans exert their effects on mitogen-activated protein kinase (MAP kinase) phosphorylation, a key intermediate in cell signaling, followed by quantitative proteomic analysis of nuclear proteins by stable isotope coded affinity tags, multidimensional chromatography and tandem mass spectrometry. Serum starved human lung fibroblasts were stimulated with serum, platelet derived growth factor (PDGF-BB) or epidermal growth factor (EGF) in the presence of structurally different glycosaminoglycans. Antiproliferative heparan sulfate with a high content of 2- O-sulfated iduronic acid (IdoA-2SO 4) and heavily sulfated glucosamine, and the structurally related glycosaminoglycan heparin inhibited significantly serum stimulated MAP kinase phosphorylation, by at least 80% when stimulated by serum and HS6. We hypothesized that the inhibition of the MAP kinase pathway will have effect in the nuclear proteome. Therefore an isotope coded affinity tag (ICAT™) reagent labeling of nuclear proteins and tandem mass spectrometry was applied, resulting in the identification and quantification of 206 proteins. Several nuclear proteins were found to be induced or repressed due to HS stimulation, where the repression EBNA-2 co-activator and the induction of PML protein were of special interest. These results show that heparan sulfate with high content of (IdoA-2SO 4) and heavily sulfated glucosamine specifically inhibits MAP kinase activation with a subsequent change in the nuclear proteome of the fibroblast.

Order Out of Chaos: Assembly of Ligand Binding Sites in Heparan Sulfate

Virtually every cell type in metazoan organisms produces heparan sulfate. These complex polysaccharides provide docking sites for numerous protein ligands and receptors involved in diverse biological processes, including growth control, signal transduction, cell adhesion, hemostasis, and lipid metabolism. The binding sites consist of relatively small tracts of variably sulfated glucosamine and uronic acid residues in specific arrangements. Their formation occurs in a tissue-specific fashion, generated by the action of a large family of enzymes involved in nucleotide sugar metabolism, polymer formation (glycosyltransferases), and chain processing (sulfotransferases and an epimerase). New insights into the specificity and organization of the biosynthetic apparatus have emerged from genetic studies of cultured cells, nematodes, fruit flies, zebrafish, rodents, and humans. This review covers recent developments in the field and provides a resource for investigators interested in the incredible diversity and specificity of this process.

Sequencing of 3-O sulfate containing heparin decasaccharides with a partial antithrombin III binding site.

Heparin- and heparan sulfate-like glycosaminoglycans (HLGAGs) represent an important class of molecules that interact with and modulate the activity of growth factors, enzymes, and morphogens. Of the many biological functions for this class of molecules, one of its most important functions is its interaction with antithrombin III (AT-III). AT-III binding to a specific heparin pentasaccharide sequence, containing an unusual 3-O sulfate on a N-sulfated, 6-O sulfated glucosamine, increases 1,000-fold AT-III's ability to inhibit specific proteases in the coagulation cascade. In this manner, HLGAGs play an important biological and pharmacological role in the modulation of blood clotting. Recently, a sequencing methodology was developed to further structure-function relationships of this important class of molecules. This methodology combines a property-encoded nomenclature scheme to handle the large information content (properties) of HLGAGs, with matrix-assisted laser desorption ionization MS and enzymatic and chemical degradation as experimental constraints to rapidly sequence picomole quantities of HLGAG oligosaccharides. Using the above property-encoded nomenclature-matrix-assisted laser desorption ionization approach, we found that the sequence of the decasaccharide used in this study is DeltaU(2S)H(NS,6S)I(2S)H(NS, 6S)I(2S)H(NS,6S)IH(NAc,6S)GH(NS,3S,6S) (+/-DDD4-7). We confirmed our results by using integral glycan sequencing and one-dimensional proton NMR. Furthermore, we show that this approach is flexible and is able to derive sequence information on an oligosaccharide mixture. Thus, this methodology will make possible both the analysis of other unusual sequences in HLGAGs with important biological activity as well as provide the basis for the structural analysis of these pharamacologically important group of heparin/heparan sulfates.

One- and two-dimensional 13C-n.m.r. characterization of two series of oligosaccharides derived from porcine intestinal mucosal heparin by degradation with heparinase

Two tetrasaccharides, two hexasaccharides, and a disaccharide have been purified from heparinase digests of porcine intestinal mucosal heparin in sufficient quantities to permit 13C-n.m.r. characterization of the species. The two tetrasacchrides are the sulfated iduronic acid-containing 4en-Hex pA2SO 3-(1→4)-α- d-Glc pNSO 3;6SO 3-(1→4)-α- l-Ido pA2SO 3-(1→4)- d-Glc pNSO 3;6SO 3 and the non-sulfated glucuronic acid-containing 4en-Hex pA2SO 3-(1→4)-α- d-Glc pNSO 3;6SO 3-(1→4)-β- d-Glc pA-(1→4)- d-Glc pNSO 3;6SO 3. The two hexasaccharides are related to the two tetrasaccharides by the insertion of α-linked l-Ido pA2SO 3-(1→4)- d-Glc pNSO 3;6SO 3 after the non-reducing end sulfated glucosamine residue. The disaccharide is 4en-Hex pA2SO 3-(1→4)-α- d-Glc pNSO 3;6SO 3. The disaccharide, together with each of the iduronate-containing oligosaccharides, form one series of related di-, tetra-, and hexa-saccharides, while the disaccharide together with the glucuronate-containing oligosaccharides form a second series. Using inverse detection as a means of increasing sensitivity, two-dimensional n.m.r. 13C 1H heterocorrelation spectra have been obtained for all five oligosaccharides. The use of two-dimensional heterocorrelation n.m.r. spectroscopy offers a much less ambiguous means of making 13C resonance assignments than do traditional one-dimensional methods, while the use of inverse detection gives both greater sensitivity than direct detection, as well as values for the one-bond 13 1H coupling constants. From a knowledge of the assignments of resonances in the 1H spectra of these species, it has been possible to assign almost all of the 13C resonances of these five oligosaccharides. Some corrections to previously published assignments for the tetrasaccharides have been made. In addition, one-bond 13C 1H coupling constant data have been obtained for all of the anomeric protons.

Molecular basis of symbiotic host specificity in rhizobium meliloti: nodH and nodPQ genes encode the sulfation of lipo-oligosaccharide signals

The symbiosis between Rhizobium and legumes is highly specific. For example, R. meliloti elicits the formation of root nodules on alfalfa and not on vetch. We recently reported that R. meliloti nodulation ( nod) genes determine the production of acylated and sulfated glucosamine ollgosaccharide signals. We now show that the biochemical function of the major host-range genes, nodH and nodPQ, is to specify the 6-O-sulfation of the reducing terminal glucosamine. Purified Nod factors (sulfated or not) from nodH + or nodH − strains exhibited the same plant specificity in a variety of bioassays (root hair deformations, nodulation, changes in root morphology) as the bacterial cells from which they were purified. These results provide strong evidence that the molecular mechanism by which the nodH and nodPQ genes mediate host specificity is by determining the sulfation of the extracellular Nod signals.

A novel 3-0 sulfatase from human urine acting on methyl-2-deoxy-2-sulfamino-α-D-glucopyranoside 3-sulfate

Human urine contains a novel sulfatase which is specific for the 3-0 sulfate ester of sulfaminoglucopyranoside 3-sulfate. Of the three isomeric sulfamate derivatives, 3,4 and 6-0 sulfate esters, only the 3-0 ester is hydrolyzed. Enzymatic activity requires that the amino group be sulfated; sulfate is not released if the amino group is free or acetylated. The enzyme has been purified 70-fold. It has a pH optimum of 6.3 and is inhibited by inorganic sulfate and phosphate. The specificity of this enzyme suggests that a 3-0 sulfated glucosamine moiety may have a role in the physiological activity of heparin or heparan sulfate.