Fig. 1. Effect of sulfated glucosamine and glucosamine on membrane lipid peroxidation and cytotoxicity. (A) Diphenyl-1-pyrenylphosphine (DPPP) fluorescence probe labeled mouse macrophages, (RAW264.7) were treated with predetermined sample concentrations and incubated for 1 h. 3 mM AAPH in PBS was then added to each well and lipid oxidation was measured as the emission of DPPP oxide fluorescence after 6 h of at λ_excitation = 361 nm and λ_emission = 380 nm after 6 h. The results shown are representative of three independent experiments. Error bars represent the S.E. (B) Effects of glucosamine and sulfated glucosamine on the viability RAW264.7 cells. Cells (4 × 10⁴ cells) were cultured in DMEM medium with or without sulfated glucosamine or glucosamine and viability was measured by MTT assay at the indicated time as described under experimental procedure. The viability of cells is shown as means of ± S.E. Statistically significant differences (P < 0.05) could not be observed in sulfated glucosamine or glucosamine treated groups. Statistical comparisons, P < 0.05 and P < 0.01.

Fig. 2. Sulfated glucosamine and glucosamine mediated suppression of radical mediated oxidation in chondrocyte membrane proteins. Lysates of SW1353 cells were treated with indicated concentrations of sulfated glucosamine and glucosamine. Amount of carbonyl group was measured using 2,4-dinitrophenyl hydrazine reagent as described under experimental procedure. Amount of carbonyl groups was expressed in nmol/mg of protein, using a molar absorption co-efficient of 22,000 M^− 1 cm^− 1. The results shown are representative of separate experiments performed in triplicate. Error bars represent the S.E. Statistical comparisons, P < 0.05 and P < 0.01.

Fig. 3. DNA oxidative protection by sulfated glucosamine. Genomic DNA from human chondrocytes (SW1353) was pre-treated with glucosamine or sulfated glucosamine and exposed to OH using Fenton chemistry. After 30 min, reaction mixture containing about 1 μg of DNA was electrophorased on a 1% agarose gel for 30 min at 100 V and visualized by UV light after stained with 1 mg/ml ethidium bromide.

Fig. 4. Effect of sulfated glucosamine and glucosamine on inhibition of intracellular generation of reactive oxygen species. Intracellular formation of reactive oxygen species was assessed as described previously using oxidation sensitive dye, DCFH-DA. SW1353 cells were loaded with 20 μmol/l DCFH-DA in HBSS and then treated with different concentrations of sulfated glucosamine and glucosamine and incubated for 1 h. Cells were then treated with 500 μmol/l H₂O₂ dissolved in HBSS and formation of DCF due to oxidation of DCFH was read after every 30 min at the excitation wavelength of 485 nm and the emission wavelength of 528 nm. Dose-dependant and time-dependant effects of treatment groups were plotted and compared with fluorescence intensity of control. Pre-treatment with sulfated glucosamine decreased the DCF fluorescence dose and time-dependently. The results shown are the means of ± S.E. of independent experiments. Statistical comparisons, P < 0.05 and P < 0.01.

Fig. 5. (A) Suppression of NF-κB gene promoter activity in sulfated glucosamine and glucosamine treated SW1353 cells. Cells were co-transfected with NF-κB-luciferase reporter vector and β-galactosidase expression vector by lipofectamine. After 24 h of transfection, cells were subcultured and treated with different concentrations of test compounds followed by stimulation with 500 μM H₂O₂. Luciferase enzyme activity that represents NF-κB promoter activity was determined using luciferin as the substrate. The level of reporter gene expression was determined as a ratio, compared with non treated cells and represented as relative luciferase activity. Statistical comparisons, P < 0.05 and P < 0.01. (B and C) Western blot analysis of p65 and p50 protein expressions in SW1353 cells stimulated with H₂O₂. After treatment of test compounds, proteins in nucleus and cytosol were extracted and resolved by denaturing SDS-PAGE. Proteins (20–40 μg) were diluted in sample buffer and resolved in 4–20% Novex gradient gel. Proteins were transferred onto nitrocellulose membranes and incubated with primary and secondary antibodies respectively. Western blot bands were developed with enhanced chemiluminescence reagents and visualized using LAS3000® Luminescent image analyzer. Detection of β-actin was used as control for equal loading of protein.

Fig. 6. Effect of glucosamine and sulfated glucosamine on regulation of GSH level in SW1353 cells. SW1353 cells were treated with predetermined concentrations of sulfated glucosamine and glucosamine and incubated for 30 min. Cellular GSH level was determined using mBBr as a thiol-staining reagent according to the method described in the text measuring mBBr-GSH fluorescence intensity at λ_excitation = 360 nm and λ_emission = 465 nm. The average fluorescence values of cell populations were plotted and compared with blank group in which cells were grown without treatment of test compounds. Data are presented as means ± S.E. from three independent determinations. Statistical comparisons, *P < 0.05 and **P < 0.01.

Fig. 7. Determination of MPO released in stimulated neutrophils and cell viability. (A) HL-60, human neutrophil cell line was preincubated with various concentrations of sulfated glucosamine and glucosamine for 30 min followed by stimulation with TNF-α (0.05 μg/ml) for 30 min. Assay mixture containing 0.05 ml of 2 mM H₂O₂ in 0.1 M phosphate buffer (pH 6.0) and 0.05 ml of 0.02 M O-dianisidine was added and amount of MPO released was measured spectrophotometrically at 460 nm. The total MPO activity was plotted as an absorbance value and it was compared with the control group in which cells were grown in absence of inhibitors. Data are representative of three independent experiments. Error bars represent the S.E. Effects of glucosamine and sulfated glucosamine on the viability HL-60 cells. (B) Cells (4 × 10⁴) were cultured in DMEM medium with or without sulfated glucosamine or glucosamine and viability was measured by MTT assay at the indicated time as described under experimental procedure. The viability of cells is shown as means of ± S.E. Statistically significant differences (P < 0.05) could not be observed in sulfated glucosamine or glucosamine treated groups.

Scavenging activity of Glc and SGlc on OH, O₂^‾ and carbon-centered radicals at different concentrations

Radical scavenging capacities were determined as a percentage of signal intensities compared to control under specified conditions. Values are means of triplicate analyses ± S.E.M.