Figure 1.
HPLC profile, methylation analysis result and 1D NMR spectrums of GSP-2.
A: HPLC profile of GSP-2. Samples (2 mg/mL, 10 µL) were analyzed on an Agilent 1100 system equipped with an ELSD detector and TSK GMPWXL gel filtration columns (7.8×300 mm×2), with 20 mM CH3COONH4 as mobile phase at 0.6 mL/min and column temperature at 40°C. Commercially available T-series dextrans (MW 2000, 670, 410, 270, 150, 80, 50, 12, 5 and 1 kD). B: Methylation analysis result of GSP-2. GC-MS tests for methylation analysis were measured with a DB-5 column (30 m×0.25 mm×0.25 µm), and at temperatures programmed from 170–225 °C at 2 °C/min and then hold on 10 min, in the figure, a: T-Glcp, b: 1,3-linked Glcp, c: 1,4-linked Glcp, d: 1,6-linked Glcp, e: 1,3,6-linked Glcp. C-E: 1D NMR (1H, 13C, DEPT) spectra of GSP-2 in D2O with TMS as external standard, obtained on a Bruker AM 500 spectrometer with a dual probe in the FT mode at room temperature.
Table 1.
Amino acid composition of the polysaccharide (GSP-2) isolated from the fruiting bodies of Ganoderma sinense a.
Table 2.
GC–MS test result for the methylated sugar moieties of the polysaccharide GSP-2 a, b.
Figure 2.
2D NMR spectrums and proposed structure of the polysaccharides from the fruiting bodies of G. sinense.
(A: HSQC spectrum, B: HMBC spectrum, C: NOESY spectrum, D proposed strcuture).
Table 3.
1H NMR and 13C NMR-chemical shifts of GSP-2 a, b.
Figure 3.
Stimulating effect of GSP-2 on the proliferation of the mouse splenocytic B and T cells.
Freshly fractionated splenocyte, splenic B (solid bars) and T (open bars) cells were stimulated with, or without (Medium), dextran or GSP (30 µg/ml). LPS (2 µg/ml) and ConA (2 µg/ml) were included as controls. In a parallel experiment, mouse splenocytes were stimulated with, or without (Medium), GSP (30 µg/ml), dextran (30 µg/ml) or LPS (10 µg/ml) in triplicate wells in the presence, or absence, of PMB. 3H-TdR was added to the cultures for the last 8 hrs of incubation and then 3H-TdR incorporation (CPM) of each well counted. A: Parallel experiment to exclude the influence of the endotoxin contamination; B: Stimulating effect of GSP-2 to the mouse splenocytic B and T cells. All results are presented as mean ± SEM, *, P<0.05; **, P<0.01; ***, P<0.001 for difference from culture without treatment. (n = 9, repeated 3 times).
Figure 4.
Nitric oxide production and phagocytosis of GSP-2-treated RAW 264.7 cells.
A: The levels of NO production of RAW264.7 cells were assessed after incubation with GSP-2 or LPS for 24 h using Griess assay. All data are expressed as mean ± SEM of three individual experiments (n = 12). Significant difference: *, P<0.05; **, P<0.01; ***, P<0.001 for difference from culture without treatment. B: The viable RAW 264.7 cell populations were gated (left) and the FITC-positive population was shown in fluorescent-GSP-2-labeled cells (lower right histogram). C: RAW 264.7 cells were incubated with 10 µg GSP-2 (fluro-labeled) for 24 h. The cells were imaged at the 1st and 24th hour (lower histograms). After 24 h incubation, the cells were collected, washed and resuspended in PBS and the fluorescence of the samples was detected by flow cytometry. (Upper histograms: viable RAW 264.7 cells without fluorescent labeled GSP-2, lower histograms, 1st hour: viable RAW 264.7 cells with fluorescent labeled GSP-2, 24th hour).
Figure 5.
Cytokine productions of GSP-2-treated PBMCs.
Culture supernatants were collected 24-2 and the cytokines concentrations were specifically determined by ELISA. All data are expressed as mean ± SEM of three individual experiments (n = 12). Differences between the treated and untreated control groups were compared using one-way ANOVA. * P<0.05, ** P<0.01, *** P<0.001 for difference from culture without treatment.
Figure 6.
Cytokine productions of GSP-2-treated moDC.
Culture supernatants were collected 48-2 and the cytokines concentrations were specifically determined by ELISA. Lines represented mean percentage ± S.E.M. of duplicates (n = 8). LPS and Beta-glucan from E. gracilis were used as positive control. Significant difference: *, P<0.05; **, P<0.01; ***, P<0.001 for difference from culture without treatment.