Korean J Physiol Pharmacol.  2010 Jun;14(3):133-137. 10.4196/kjpp.2010.14.3.133.

Stimulatory Effects of Ginsan on the Proliferation and Viability of Mouse Spleen Cells

• Ko EJ
• Joo HG

• KMID: 2285393
• DOI: http://doi.org/10.4196/kjpp.2010.14.3.133

Abstract

Ginsan is an acidic polysaccharide purified from Panax ginseng, a famous oriental herb. Although a variety of biological activities of ginsan have been studied, the effects of ginsan on spleen cells are not fully elucidated. We investigated the effect of ginsan on the viability and proliferation of spleen cells. Using Cell Counting Kit-8(R) solution and trypan blue solution, we found that ginsan significantly enhanced viability and proliferation. Multiple clusters, indicating proliferation, were observed in ginsan-treated spleen cells and, carboxyfluorescein succinimidyl ester and surface marker staining assay revealed that ginsan promoted proliferation from CD19+ B cells rather than CD4+ or CD8+ T cells. In addition, ginsan decreased the percentage of late apoptotic cells. Ginsan increased the surface expression of CD25 and CD69 as well as production of interleukin-2 from spleen cells, suggesting increased activation. Taken together, these results demonstrate that ginsan increases the viability and proliferation of spleen cells via multiple mechanisms, valuable information for broadening the use of ginsan in clinical and research settings.

Keyword

B lymphocytes; Ginsan; Stimulation; Proliferation; Spleen cells

MeSH Terms

Animals
B-Lymphocytes
Cell Count
Diminazene
Fluoresceins
Interleukin-2
Mice
Panax
Polysaccharides
Spleen
T-Lymphocytes
Trypan Blue
Diminazene
Fluoresceins
Interleukin-2
Polysaccharides
Trypan Blue

Figure

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  Fig. 1. Effect of ginsan on the proliferation/viability of spleen cells. The spleen cells were cultured at a concentration of 2×105 cells/200 μl/ well in 96-well culture plates. (A) Proliferation assay using Cell Counting Kit-8® solution, in which spleen cells were treated with 0∼200 μg/ml ginsan for 4 days. (B) Trypan blue exclusion test, in which spleen cells were treated with 0∼100 μg/ml ginsan for 3 days. Data are mean±SD from three or four individual wells.

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  Fig. 2. Proliferating clusters in ginsan-treated spleen cells. Spleen cells were cultured as described in Fig. 1, and treated with medium alone, 10 μg/ml or 50 μg/ml ginsan for 3 days. (A) Cell morphology was observed using an inverted microscope and the image was obtained by a digital camera. (B) Cell size of the treated spleen cells was analyzed by flow cytometry.

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  Fig. 3. Selective proliferation of CD19+ B lymphocytes by ginsan treatment. Spleen cells were stained with CFSE and cultured in 96-well culture plates with medium alone or 10 μg/ml or 50 μg/ml ginsan for 2 days. After harvesting, the cells were labeled with surface marker-specific antibodies. The numbers indicate the percentage of proliferating cells of a specific subset.

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  Fig. 4. Ginsan protects spleen cells against spontaneous cell death. Spleen cells were cultured at a concentration of 5×106 cells/5 ml/well in 6-well culture plate. Following ginsan treatment, spleen cells were harvested and stained with annexin V-FITC/PI or rhodamine 123. The numbers indicate cell percentage and mean fluorescence intensity, respectively.

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  Fig. 5. Enhanced expression of activation markers on spleen cells treated with ginsan. Spleen cells were cultured and treated as described in Fig. 4. The number of histograms indicates the percentage of highly expressed cells.

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  Fig. 6. IL-2 responsiveness of ginsan-activated spleen cells. (A, B) Spleen cells were cultured as described Fig. 4 and treated with ginsan for 2 days. The harvested cells were stained with CFSE and incubated in 24-well culture plates in the presence of IL-2 for 5 days. The percentage of low-intensity CFSE signals indicates proliferating cells. (C) Spleen cells were cultured as described in Fig. 1 and treated with the indicated concentrations of ginsan. After 3 days of treatment, the supernatants were collected and IL-2 and IL-4 were quantified. Data are mean±SD from four individual wells.

Reference

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