Characterization and Immunopotentiating Effects of the Glycoprotein Isolated from Dioscorea Batatas

Pham Thi Thu Huong; Chan-Ho Lee; Mei Hong Li; Min Young Lee; Joon-Ki Kim; Sun-Mee Lee; Jong-Heum Seon; Dong-Cheol Lee; Young Jin Jeon

doi:10.4196/kjpp.2011.15.2.101

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

Original Article

Korean J Physiol Pharmacol 2011;15(2):101-106.

Published online: 18 February 2011

DOI: https://doi.org/10.4196/kjpp.2011.15.2.101

Characterization and Immunopotentiating Effects of the Glycoprotein Isolated from Dioscorea Batatas

Pham Thi Thu Huong^1,^∗, Chan-Ho Lee^2,^∗, Mei Hong Li¹, Min Young Lee¹, Joon-Ki Kim², Sun-Mee Lee², Jong-Heum Seon³, Dong-Cheol Lee³, Young Jin Jeon^1,

¹Department of Pharmacology, School of Medicine, Chosun University, Gwangju 501-759, Korea

²School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Korea

³Department of Biotechnology and Biomedicine, Chungbuk Provincial College, Chungbuk 373-806, Korea

Corresponding to: Young Jin Jeon, Department of Pharmacology, School of Medicine, Chosun University, 375, Susuk-dong, Dong-gu, Gwangju 501-759, Korea. (Tel) 82-62-230-6338, (Fax) 82-62-232-9213, (E-mail) yjjeon@chosun.ac.kr

^∗ P.T.T. Huong and C.H. Lee contributed equally to this work. Pham Thi Thu Huong, Chemical Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 685-1, Yangcheon-ri, Ochang-eup, Cheongwon 363–883, Korea.

Jong-Heum Seon, Greencross Co., 320-1, Songdae-ri, Ochang-eup, Cheongwon 363–886, Korea.

Received 25 March 2011 Revised 16 April 2011 Accepted 18 April 2011

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

Keywords: Glycoprotein, Macrophages, TNF-α, IL-1β

References

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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.

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.

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.

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.

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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