Journal List > Korean J Physiol Pharmacol > v.15(2) > 1025726

Huong, Lee, Li, Lee, Kim, Lee, Seon, Lee, and Jeon: Characterization and Immunopotentiating Effects of the Glycoprotein Isolated from Dioscorea Batatas

Abstract

We demonstrate that glycoprotein isolated from Dioscorea batatas (GDB) has immunostimulatory effects including macrophage activation. Analysis of infiltration of inflammatory cells into peritoneal cavity showed GDB treatment significantly increased the recruitment of macrophages, lymphocytes, neutrophils, and monocytes into the peritoneal cavity. Treatment of spleen cells isolated from C57BL/6 mice with GDB significantly increased the proliferation of B cells and T cells induced by LPS and ConA, respectively. Treatment with GDB significantly increased the cytolytic capacity of NK cells and macrophages against YAC-1 and B16 cells, respectively. In order to further confirm and investigate the mechanism of GDB on macrophage activation, we analyzed the effects of GDB on the cytokine expression including iNOS, IL-1β, and TNF-α in mouse macrophage cell line, RAW 264.7 cells. RT-PCR and ELISA showed that GDB increased the expression of IL-1β, and TNF-α, whereas iNOS was not induced by GDB. Collectively, this series of experiments indicates that GDB stimulates immune system including macrophage activation.

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Fig. 1.
Characterization of glycoprotein isolated from Dioscorea batatas. (A) The lyophilized proteins were dissolved in 50 mM Tris-HCl buffer (pH 8.0) and subjected to an anion-exchange chromatography. Fractionation of proteins was performed by a step-wise salt gradient (0.1, 0.2, 0.3, and 1 M) NaCl in equilibrium buffer. (B) Peak fractions were further analyzed by SDS-PAGE.
kjpp-15-101f1.tif
Fig. 2.
Effect of GDB on (A) LPS- and (B) ConA-stimulated splenic lymphocyte proliferation. Splenocytes were isolated from C57BL/6 mice. The results are presented as the mean±S.E.M. ∗∗Denote significant differences (p<0.05, p<0.01) vs. the control group. Experiments were repeated three times.
kjpp-15-101f2.tif
Fig. 3.
Effects of GDB on (A) NK cell- and (B) macrophage-mediated cytotoxicity. Cytotoxicity was measured as described in Methods and was expressed as the cytolytic percentage of target cells. IL-2 and LPS/IFN-γ were used as a positive control of NK cell- and macrophage-mediated cytotoxicity, respectively. The results are presented as the mean±S.E.M. ∗∗Denotes significant differences (p <0.01) vs. the control group. Experiments were repeated three times.
kjpp-15-101f3.tif
Fig. 4.
Effects of GDB on macrophage activation. (A) RAW264.7 cells were treated with GDB for 8 h. Total RNA was then isolated and analyzed for iNOS, IL-0, and TNF-α using RT-PCR. The production of (B) NO2, (C) TNF-α, and (D) IL-1β were analyzed from the cell lysates treated with GDB for 24 h. The results are presented as the mean± S.E.M. ∗∗Denotes significant differences (p<0.01) vs. the control group. One representative of three experiments is shown.
kjpp-15-101f4.tif
Table 1.
Effect of GDB on the recruitment of inflammatory cells
  Control Saline 25 mg/kg 50 mg/kg 100 mg/kg 200 mg/kg
Macrophage (×106) 0.27±0.01 0.84±0.04∗∗ 1.00±0.12 1.07±0.02∗∗,†† 1.20±0.11∗∗, 1.01±0.09
Lymphocyte (×106) 0.59±0.07 1.00±0.07 1.67±0.09∗∗++ 1.67±0.08∗∗†† 1.89±0 1.5∗∗,†† 1.70±0.1 ∗∗††
Neutrophil (×106) 0.26±0.02 0.82±0.08∗∗ 1.24±0.09∗∗ + 0.99±0.13∗∗ 1.26±0.1∗∗, 1.40±0.26∗∗
Eosinophil (×106) 0.01±0.01 0.28±0.03∗∗ 0.28±0.04 0.42±0.08 0.37±0.03 0.43±0.07∗∗
Monocyte (×106) 0.23±0.04 0.42±0.04 0.78±0.01∗∗, + 0.93±0.02∗∗†† 1.01±0.2 1 1 4±0 07∗∗, ††

Animals orally received vehicle/GDB administrations (25, 50, 100 or 200 mg/kg) for 2 weeks. Differential counting for macrophages, lymphocytes, neutrophils, eosinophils, and monocytes was performed.

∗∗ The results are presented as the mean±S.E.M. of 10 mice per group,

Denotes the significant differences (p<0.05, p<0.01) from the control group,

†† denotes the significant differences (p<0.05, p<0.01) from the vehicle group.

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