Korean J Physiol Pharmacol.  2012 Dec;16(6):431-436. 10.4196/kjpp.2012.16.6.431.

Synergistic Induction of iNOS by IFN-gamma and Glycoprotein Isolated from Dioscorea batatas

• Huong PT
• Lee MY
• Lee KY
• Chang IY
• Lee SK
• Yoon SP
• Lee DC
• Jeon YJ

• KMID: 2285452
• DOI: http://doi.org/10.4196/kjpp.2012.16.6.431

Abstract

Dioscorea species continue to be used in traditional Chinese medicine, and represent a major source of steroid precursors for conventional medicine. In the previous study, We isolated glycoprotein (GDB) from Dioscorea batatas, characterized, and demonstrated immunostimulating activity in C57BL/6 mice. The aim of this study was to investigate the mechanism whereby GDB activates macrophages. Macrophages activation by GDB was investigated by analyzing the effects of GDB on nitric oxide (NO) production, iNOS expression, mitogen activated protein kinase (MAPK) phosphorylation, and transcription factor activation. In the presence of IFN-gamma, GDB strongly stimulated macrophages to express iNOS and produce NO. Furthermore, the activation of p38 was synergistically induced by GDB plus IFN-gamma , but SB203580 (a p38 inhibitor) inhibited GDB plus IFN-gamma-induced p38 activation. This study indicates that GDB is an important activator of macrophages. Furthermore, due to the critical role that macrophage activation plays in innate immune response, the activation effects of GDB on macrophages suggest that GDB may be a useful immunopotentiating agent.

Keyword

Glycoprotein; iNOS; Macrophages; NO

MeSH Terms

Animals
Dioscorea
Glycoproteins
Imidazoles
Immunity, Innate
Macrophage Activation
Macrophages
Medicine, Chinese Traditional
Mice
Nitric Oxide
Phosphorylation
Protein Kinases
Pyridines
Transcription Factors
Glycoproteins
Imidazoles
Nitric Oxide
Protein Kinases
Pyridines
Transcription Factors

Figure

• 

  Fig. 1 Induction of iNOS expression by GDB plus IFN-γ. (A) GDB was isolated as described in "Methods". Isolated GDB was diluted 2- and 4- folds and analyzed by SDS-PAGE. (B) RAW264.7 cells were treated with GDB (25 µg/ml) and IFN-γ (10 ng/ml) for 24 h. Morphological change and iNOS expression were analyzed using immunofluorescence staining (upper column; with staining, lower column; without staining) and inverted microscope. (C) RAW264.7 cells were treated with GDB (1, 10, and 25 µg/ml) in the presence or absence of IFN-γ (10 ng/ml) for 8 h. Total RNA was then isolated and analyzed for iNOS using RT-PCR. (D) RAW264.7 cells were treated with GDB (1, 10, and 25 µg/ml) in the presence or absence of IFN-γ for 24 h. Cell lysates were then prepared and analyzed for the iNOS using Western blot analysis. One representative of three experiments is shown.

• 

  Fig. 2 Induction of NO production by GDB plus IFN-γ. (A) RAW264.7 cells were treated with IFN-γ (1, 2.5, 5, and 10 ng/ml) in the presence or absence of GDB (25 µg/ml) for 24 h. Supernatants were then isolated and analyzed for nitrite generation. LPS (200 ng/ml) was used as a positive control. (B) RAW264.7 cells were treated with GDB (1, 10, 25, and 50 µg/ml) in the presence or absence of IFN-γ (10 ng/ml) for 24 h. The results are presented as the mean±S.D. *Denotes significant differences (p<0.05) from the control group. One representative of three experiments is shown.

• 

  Fig. 3 Activation of p38 by GDB plus IFN-γ. (A) RAW264.7 cells were treated with GDB (1, 10, and 25 µg/ml) in the presence or absence of IFN-γ (10 ng/ml) for 30 min. Cell lysates were prepared and subjected to immunoblot assay using antibodies against phospho- and nonphospho-p38, ERK1/2 and SAPK/JNK proteins. (B) The density of p38 was analyzed by Image J program. β-actin was used as a control. One representative of three experiments is shown. *Denotes significant differences (p<0.05) from the control group.

• 

  Fig. 4 Effect of SB203580 on iNOS expression in GDB plus IFN-γ-stimulated RAW 264.7 cells. (A) RAW 264.7 cells were pretreated with SB 203580 (30 µM) or PD98059 (50 µM) for 30 min before incubation with GDB (25 µg/ml) and IFN-γ (10 ng/ml) or LPS (200 ng/ml) for 24 h. (A) Supernatants were then isolated and analyzed for nitrite generation. (B) RAW264.7 cells were treated with Polymyxin B in the presence of GDB plus IFN-γ or LPS for 24 h. Supernatants were then isolated and analyzed for nitrite generation. One of two representative experiments is shown. *Denotes significant differences (p<0.05) from the control group.

• 

  Fig. 5 Effects of GDB on the activation of NF-κB in RAW264.7 cells. RAW264.7 cells were treated with GDB (1, 10, and 25 µg/ml) in the presence or absence of IFN-γ (10 ng/ml) for 2 h. Nuclear extracts were isolated and analyzed for the determination of (A) NF-κB and (B) AP1 and Oct1. (C) using EMSA. One representative of three experiments is shown.

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