Immune-Enhancing Effects of Crude Polysaccharides from Korean Ginseng Berries on Spleens of Mice with Cyclophosphamide-Induced Immunosuppression

Panax ginseng C. A. Meyer is well known as traditional herbal medicine, and ginseng berries are known to exhibit potential immune-enhancing functions. However, little is known about the in vivo immunomodulatory activity of Korean ginseng berries. In this study, crude Korean ginseng berries polysaccharides (GBP) were isolated and their immunomodulatory activities were investigated using cyclophosphamide (CY)-induced immunosuppressive BALB/c mice. In CY-treated mice, oral administration of GBP (50-500 mg/kg BW) remarkably increased their spleen sizes and spleen indices and activated NK cell activities. GBP also resulted in the proliferation of splenic lymphocytes (coordinating with ConA: plant mitogen which is known to stimulate T-cell or LPS: endotoxin which binds receptor complex in B cells to promote the secretion of pro-inflammatory cytokines) in a dose-dependent manner. In addition, GBP significantly stimulated mRNA expression levels of immune-associated genes including interleukin-1β (IL-1β), IL-2, IL-4, IL-6, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), toll-like receptor 4 (TLR-4), and cyclooxygenase-2 (COX-2) in CY-treated mice. These results indicate that GBP is involved in immune effects against CY-induced immunosuppression. Thus, GBP could be developed as an immunomodulation agent for medicinal or functional food application.

February 2022 ⎪ Vol. 32 ⎪ No. 2 from P. ginseng [25,26]. It has been reported that polysaccharides of P. ginseng can exert immunomodulatory activities in both in vitro and in vivo systems [27][28][29]. Crude polysaccharides from P. quinquefolius can increase macrophage phagocytosis and nitric oxide (NO) production as well as splenic lymphocyte proliferation [17]. Polysaccharides from Korean ginseng after cellulase-and α-amylase-based extraction can enhance spleen and thymus indices, lymphocyte proliferation, leukocyte count, NK cell activity, and serum levels of IL-2, IL-6, and IFN-γ in CY-induced immunosuppressed mice [30]. Moreover, we recently reported that Korean ginseng berries show immune-enhancing effects in RAW 264.7 cells [31]. However, still little is known about the immunomodulatory effect of polysaccharides from Korean ginseng berry in CY-induced immunosuppressive mice model. Therefore, our objective in the current study was to investigate the in vivo immune-enhancing properties of crude Korean ginseng berry polysaccharides (GBP).

Animals
Six-week-old male BALB/c mice (21-23 g) were obtained from the Central Lab, Animal Inc. (Korea). Animals were housed under standard conditions with temperature maintained at 22 ± 2 o C and a 12-h/12-h dark/light cycle for at least one week before the experiments. A standard laboratory diet and water were provided to all mice. All experimental processes were approved by the Gangneung-Wonju National University Committee.

Immunosuppressive Treatments
In this study, 40 mice were randomly divided into 8 groups (n = 5 per group) for various treatments (Table 1). CY was used to induce immunosuppressive models by intraperitoneal (IP) injection at a dose of 80 mg/kg body weight (BW). Levamisole (LEV) and commercial red ginseng syrup were used as positive controls at doses of 40 mg/kg BW and 100 mg/kg BW, respectively. LEV is an immunomodulatory agent that can modulate cellmediated, i.e., T cell-directed immunity [3,32]. Ginseng is a commercially available product that can modulate the immune system [33]. Mice were sacrificed at 24 h after the last treatment.

Measurement of Spleen Index
The spleen was dissected and weighed. The spleen index was calculated according to the formula as follows: Spleen index (mg/g) = the spleen weight/the body weight of mouse.

Preparation of Mouse Splenocytes
Splenocytes were isolated from spleens of BALB/c mice. After weighing, spleens of mice were placed in ice-cold PBS for splenocyte isolation. These splenocytes were extracted using RBC Lysis Buffer (eBioscience, USA) according to the manufacturer's instructions. Cells were adjusted to a concentration of 2 × 10 6 cells/ml in RPMI-1640 medium (Gibco Laboratories, USA) supplemented with 1% fetal bovine serum and 1% streptomycin (100 μg/ml) penicillin (100 IU/ml) (Welgene, Korea).

Measurement of Splenocyte Proliferation
EZ-Cytox Cell Viability Assay Kit (Daeillab Service, Korea) was used to evaluate splenic lymphocyte proliferation. Splenocytes were seeded into a 96-well plate and cultured at 37°C with 5% CO 2 for 1 h. These cells were then stimulated with 5 μg/ml concanavalin A (Con A) as T cell mitogen or 10 μg/ml lipopolysaccharide (LPS) as B cell mitogen. After incubation at 37°C with 5% CO 2 for 48 h, 25 μl EZ-Cytox reagent was added to each well and plates were incubated at 37°C with 5% CO 2 for another 1 h. The absorbance at 450 nm was then measured using a microplate reader (EL800; BioTek, USA).

Analysis of NK Cell-Mediated Cytotoxicity Assay
Splenocytes were co-cultured with or without YAC-1 cells (Korean Cell Line Bank) to obtain a 50:1 ratio of

Quantitative RT-PCR (qRT-PCR) Analysis
Tri reagent (Molecular Research Center, Inc., USA) was employed to extract total RNAs from splenic lymphocytes. Total RNAs were then reverse-transcribed to cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA). To amplify the cDNA, the cDNA was subjected to qRT-PCR using TB Green Premix Ex Taq II (Takara Bio Inc., Japan) on a QuantStudio 3 Flex Real-Time PCR System (Thermo Fisher Scientific, USA). Amplification conditions were as follows: initial denaturation at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 34 s. Primer sequences are listed in Table 2.

Statistical Analysis
The significance of differences was evaluated using one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test using Statistix 8.1 Statistics software (Statistix, USA). Significant difference between treatment groups was considered when the p-value was less than 0.05.

Effect of GBP on Spleen Index in CY-Treated Mice
The effects of ginseng berry polysaccharides (GBP) on spleen size and spleen indices of BALB/c mice are shown in Fig. 1. Compared with the normal group, the CY-treated group showed significant decreases of both spleen size and spleen index. Compared with the CY group, the LEV treatment group and the red ginseng syrup treatment group showed increased spleen index. GBP groups also showed significantly increased spleen indices in a dosedependent manner (50, 100, 250, and 500 mg/kg BW).

Effects of GBP on Splenic Lymphocyte Proliferation
The effects of GBP on mitogen-stimulated spleen lymphocyte proliferation are shown in Fig. 2. The CY group showed significantly reduced Con A-or LPS-stimulated cell proliferation (48.1% and 57.1%, respectively) compared to the normal group. GBP at doses of 250 and 500 mg/kg BW significantly stimulated the proliferation of splenocytes induced by Con A or LPS compared to the CY group. The proliferation of lymphocytes of the group treated with GBP at 500 mg/kg BW was higher than those of positive control groups (red ginseng syrup and LEV).

Effect of GBP on Splenic NK Cell Activity
As shown in Fig. 3, CY strongly inhibited the activity of NK cells to 42.9%. NK activity of the GBP group at 50 mg/kg BW showed little difference from that of the CY group. However, the NK cell activities of GBP treatment groups at doses of 100, 250, and 500 mg/kg BW were significantly higher than those of the CY group in a dosedependent manner. In addition, GBP at a dose of 500 mg/kg BW recovered the splenic NK cell activity up to a level similar to that of the normal group.

Effect of GBP on Gene Expression in CY-Treated Mice
To assess whether GBP could stimulate immune responses in splenic lymphocytes stimulated by T-cell mitogen (Con A) and B-cell mitogen (LPS), real-time PCR was performed to examine mRNA expression levels of immunerelated genes such as IL-1β, IL-2, IL-4, IL-6, TNF-α, IFN-γ, TLR-4, and COX-2. As shown in Fig. 4, mRNA expression levels of these genes in CY group were lower than those of the normal group. All GBP groups (50-500 mg/kg BW) showed markedly increased expression levels of immune-related genes than the CY group. GBP

Fig. 2. Effects of GBP on Con A-and LPS-induced splenocyte proliferation.
Results are expressed as means ± SD.
Different letters (a-h) indicate significant (p < 0.05) differences between treatment groups.

Fig. 1. Effects of GBP at various concentrations on spleen size and spleen index. (A) Effects on spleen size, (B)
Effects on spleen index. Results are expressed as means ± SD. Different letters (a-e) indicate significant (p < 0.05) differences between treatment groups.

Discussion
In this study, we investigated the potential of Korean GBPs to activate immune systems in a CY-induced immunosuppressed mice model. Modulation of splenic lymphocytes was determined by cell proliferation, NK cell activity, and immune-associated gene expression. CY is an inducer of immunosuppression. It is a widely used alkylating agent for cancer therapy [8]. In mice models, the effect of CY could be shown as reduction of immune organ index. The capacity of the immune system decreases after CY treatment [5,12]. In addition, immunestimulating agents such as LEV and red ginseng syrup were applied as positive controls to recover the immunosuppressive condition induced by CY.
The spleen is one of the most critical immune organs. It plays an important role in the body's immune function by regulating immune responses. Their weights and organ indices indicate the function of innate immunity to some extent [10,34]. In this study, spleen indices of mice treated with LEV, red ginseng syrup, and GBP were higher than that of the CY group. Treatments with GBP (50-500 mg/kg BW) remarkably increased spleen weights of CY-treated mice, indicating that GBP could regulate immune organs to improve immune damage caused by CY (Fig. 1). Similar to our study results, Song et al. have reported that polysaccharides extracted from Korean ginseng with enzyme-assisted extraction can increase spleen and thymus indices, enhance the proliferation of lymphocytes and NK cell activity, and increase serum levels of IL-2, IL-6, and IFN-γ in CY-induced BALB/c mice [30].
Lymphocytes are major cellular components of the adaptive immune response. They can be divided mainly into T cells and B cells depending on their developmental site, surface, antigen, receptor, and function [3]. They are involved in activating the processes of antigen presentation and mitogen stimulation [35]. Splenic lymphocyte proliferation is an important biomarker for the activation of cellular and humoral immune responses [36]. This activity is induced by mitogen Con A or LPS, which is commonly used to evaluate T or B lymphocyte activity [34]. In the present study, proliferation assay results showed that GBP could significantly promote Con A-and LPSstimulated splenocyte proliferation and restore its decrease induced by CY (Fig. 2). Especially, high doses of GBP (250-500 mg/kg BW) showed strong immune-enhancing effects on mitogen-induced proliferation. Similar to our results, American ginseng polysaccharides can also promote the proliferation of B cells or T cells in these dose ranges [17].
Wang et al. [24] have reported that ginseng fruit polysaccharides can inhibit tumor growth and lung metastasis, promote spleen lymphocyte proliferation, increase NK cell activities, and increase serum concentrations of IL-2 and IFN-γ in a Lewis lung carcinoma (LLC)-bearing mouse model. Our results showed that NK cell activity was attenuated by CY administration, whereas GBP treatment significantly enhanced the activity of NK cells from splenocytes, similar to positive controls (red ginseng syrup and LEV).
After activation, splenic lymphocytes can secrete several cytokines, chemokines, and other inflammatory mediators to regulate functions of other innate and adaptive immune cells [16,37]. Cytokines play an important role in immune responses by modulating immune-related cells (B cells, T cells, and NK cells) and non-immune cells (endothelial cells, epidermal cells, and fibroblasts) [3,9]. Helper T cells (Th cells) are essential immunoregulatory cells of the body [38]. Th 1 cells can produce type 1 cytokines (IL-2, TNF-α, IFN-γ), while Th 2 cells can produce type 2 cytokines (IL-4, IL-5, IL-6, IL-10, IL-13) [39]. Many studies have shown that plantderived polysaccharides can modulate the secretion of cytokines [4,12,16]. The present results showed that GPB treatment increased expression levels of cytokines (IL-1β, IL-2, IL-4, IL-6, TNF-α, and IFN-γ) in a dose-dependent manner and that high doses (250 and 500 mg/kg BW) of GBP could improve these cytokines more so than the normal control (Fig. 4). GBP treatment also upregulated TLR-4 and COX-2 expression depending on its concentration (Fig. 4). COX-2 is an inflammatory mediator. COX-2-mediated PGE 2 plays an important role in IFNγ and IL-12 production of BALB/c spleen cells [40]. TLR-4 has a fundamental role in the initiation of adaptive immunity, activation of cells, and pathogen recognition. It can also induce the expression of TNF-α, IL-1β, and IL-6 [4]. These results suggest that GBP could improve immune activity by upregulating the expression of cytokines induced by CY.
Korean GBPs exhibited potent immunomodulatory properties in a CY-induced immunosuppressed mice model. GBP treatment enhanced immune responses by improving the spleen index. It markedly restored splenocyte proliferation and NK cell activity. It also upregulated gene expression of immune-regulated cytokines. This is the first study to report that GBP possesses immune-enhancing activity in an in vivo mice model, suggesting that GBP could be developed as an effective immunomodulatory agent.