Enhanced Intestinal Immune Response in Mice after Oral Administration of Korea Red Ginseng-Derived Polysaccharide

(1) Background: The immunostimulatory role of the polysaccharide fraction (KRG-P) of Korea red ginseng (KRG) was studied in cells. However, its immunomodulatory activity is unknown. Therefore, we investigated the chemical properties of KRG-P and its intestinal immune responses in vitro and in vivo. (2) Methods: KRG-P monosaccharide composition and molecular weight were determined using high-performance liquid and size-exclusion chromatography systems. Immunoglobulin A (IgA) and α-defensin-1 transcript levels were measured using a SYBR Green qRT-PCR; defensin-1, Granulocyte-macrophage colony-stimulating factor (GM-CSF), and IgA protein levels were determined using Western blotting and ELISA kits. (3) Results: The molecular weight of KRG-P was estimated to be 106 kDa, and it contained neutral sugar (74.3%), uronic acid (24.6%), and proteins (1%). In vitro studies of intestinal immunomodulatory activity of KRG-P indicated that GM-CSF and IgA levels increased in Peyer’s patch cells to higher levels than those obtained with KRG and induced bone marrow cell proliferation. In in vivo study, oral KRG-P administration to mice upregulated the expression of α-defensin-1 and IgA in the small intestinal tissue and that of secreted IgA in the feces. (4) Conclusions: KRG-P contributed to the modulation of intestinal immunity and maintenance of intestinal homeostasis against intestinal infection.


Introduction
The human body is exposed to foreign materials, such as pathogenic bacteria and viruses, but it maintains homeostasis through the immune system. The immune system is classified into primary (the thymus and bone marrow) and secondary lymphoid systems (the spleen, lymph nodes, and mucosal-associated lymphoid tissue (MALT)) [1,2]. MALT acts as a physical and immunological barrier against harmful substances and pathogenic bacteria [3]. Gut-associated lymphoid tissue has the largest MALT area and consists of Peyer's patches, the lamina propria, and mesenteric lymph nodes, and Peyer's patches are known as inductive sites that secrete IgA onto the mucosal surface [4]. B, T, and dendritic cells of Peyer's patches are activated by antigens entering from the intestinal lumen, thereby secreting IgA that neutralizes the pathogens, and by producing cytokines, such as GM-CSF and Interleukine-6 (IL-6), which ultimately contribute to systemic immune activation [5]. In addition, Paneth cells present in the intestinal epithelium secrete an antimicrobial peptide called α-defensin-1 (Crytidin) after exposure to bacteria, lipopolysaccharide (LPS), lipid A, and muramyl dipeptide [6][7][8].
HPLC analytical conditions to determine polysaccharide fraction (KRG-P) monosaccharide composition.

Animals
Female BALB/c mice (6-8 weeks old) were purchased from Orientbio (Seongnam, Korea) and housed at 23 ± 2 • C with 55% ± 10% humidity under a 12/12 h light/dark cycle, with free access to a standard laboratory diet and water. For in vivo assays, the mice were treated for 10 days with KRG or KRG-P at 5 or 50 mg/kg by using oral gavage feeding needles. Mice in the normal group were treated with sterilized distilled water. All animal experiments were performed in accordance with the instruction of the Ethics Comminttee for Use of Experimental Animals at Gachon University (2020-010).

GM-CSF and IgA Production Analyses in Peyer's Patch Cells
BALB/c mice (7 weeks old, female) were sacrificed by cervical dislocation, and the small intestines were excised and placed on a sterilized paper. Peyer's patches were dissected out using fine scissors from the wall of the small intestine and transferred to a Petri dish containing RPMI 1640 medium with penicillin/streptomycin. The tissues were minced by using a sterilized metal mesh (100 µm) to release immune cells. The cell suspension was filtered with a sterilized cell strainer and cultured on a cell culture plate at a concentration of 2.0 × 10 6 cells/well (96-well plate), with RPMI 1640 medium containing 10% FBS, penicillin/streptomycin, and various concentrations of KRG or KRG-P at 37 • C in a humidified atmosphere (5% CO 2 , 95% air). After 5 days, the plate was centrifuged, and GM-CSF and IgA levels in the supernatants were analyzed using ELISA kits.

Bone Marrow Cell Proliferation Assay
The proliferating activity of Peyer's patch-derived bone marrow cells was measured using the procedure reported previously [30][31][32]. In brief, mouse bone marrow cells were cultured with the supernatants collected from Peyer's patch cells, and with KRG or KRG-P at a concentration of 125, 250, or 500 µg/mL. After 4 days, bone marrow cell proliferation was measured using 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based EZ-Cytox reagent. EZ-Cytox was added to each well, and the plates were incubated for an additional 2 h. The absorbance was then measured at 450 nm using the FilterMax F5 microplate reader (Molecular Devices, San Jose, CA, USA).

Quantitative RT-PCR
Mouse small intestinal tissues were minced with the TaKaRa BioMasher (Tokyo, Japan) and passed through a QIAshredder (Qiagen, Hilden, Germany) to reduce the viscosity of the solution. Total RNA from small intestinal tissues was isolated and purified using the RNeasy Mini kit (Qiagen, Hilden, Germany) and reverse-transcribed into cDNA by using the RevertAid First Strand cDNA Synthesis kit (Fermentas, Waltham, MA, USA), according to the manufacturers' protocols. Quantitative RT-PCR was performed using a SYBR Green assay (Applied Biosystems, Foster City, CA, USA) and the indicated primers ( Table 2). β-Tubulin was used as an internal gene control. cDNA amplification and analysis were performed by using a QuantStudio-3 real-time PCR system (Applied Biosystems). Table 2. Primer sequences used for quantitative RT-PCR.

Preparation of Tissue Lysate and Immuno-Blotting
To analyze protein expression in the mouse small intestine, the tissue was minced with cold radioimmunoprecipitation assay buffer (RIPA) buffer (Rockland, Limerick, PA, USA), supplemented with 1 mM dithiothteitol (DTT) (Merk, Darmstadt, Germany), and diluted with a phosphatase inhibitor cocktail 2 solution (Merk). After centrifugation, the amount of protein in each supernatant was quantified, mixed with an SDS sample buffer, and denatured for 5 min at 95 • C. Protein electrophoresis and transfer as well as membrane development were all performed as described previously [23,24]. Briefly, electrophoresis was performed using 12% Tris-glycine SDS-polyacrylamide gel, and the protein bands were transferred onto a polyvinylidene difluoride membrane, which was blocked for unspecific binding of proteins by incubation at room temperature with 5% skim milk. After three washes with a Tris-Buffered Saline buffer containing 0.1% Tween ® 20, the membrane was incubated with anti-α-defensin-1 or β-Actin antibodies for 3 h, followed by washes and incubation with a secondary antibody. The proteins were detected by using the FUSION Solo Vilber Lourmat system (Collégien, France) with an ECL solution. The intensities of the protein bands were quantified using the ImageJ program and captured images.

Statistical Analysis
The results were expressed as the mean ± standard deviation (SD) of duplicate or triplicate experiments. The results were statistically analyzed with Mann-Whitney using Prism 8 (GraphPad Software, San Diego, CA, USA).

Effects of Intestinal Immunomodulatory Activity Mediated by Peyer's Patch Cells
Peyer's patch-mediated intestinal immunity-enhancing activities of polysaccharides have been previously reported [21,[30][31][32]. Thus, first we investigated the in vitro stimulatory activities of KRG and KRG-P on Peyer's patch cells, which play a central role in the intestinal immune system. As shown in Figure 2A, KRG-P-treated Peyer's patch cells strongly secreted GM-CSF in a concentrationdependent manner. However, KRG treatment did not affect GM-CSF production by Peyer's patch cells. LPS was used as a positive control for GM-CSF production. IgA production by Peyer's patch cells increased after treatment with 500 µg/mL KRG or KRG-P; nevertheless, only the last increase was statistically significant. Groups treated with lower concentrations of these compounds did not experience a change in IgA production ( Figure 2B).
Next, we investigated bone marrow cell proliferation mediated by Peyer's patch cells after treatment with KRG or KRG-P. The supernatants from Peyer's patch cells incubated with KRG-P for 4 days significantly promoted ex vivo bone marrow cell proliferation ( Figure 2C). The augmented bone marrow cell proliferation correlated with GM-CSF production by KRG-P, suggesting that KRG-P stimulated Peyer's patch cells to produce cytokines, and consequently induced bone marrow cell proliferation. Collectively, these results indicated that KRG-P may stimulate intestinal immunomodulation.

Effects of Intestinal Immunomodulatory Activity Mediated by Peyer's Patch Cells
Peyer's patch-mediated intestinal immunity-enhancing activities of polysaccharides have been previously reported [21,[30][31][32]. Thus, first we investigated the in vitro stimulatory activities of KRG and KRG-P on Peyer's patch cells, which play a central role in the intestinal immune system. As shown in Figure 2A, KRG-P-treated Peyer's patch cells strongly secreted GM-CSF in a concentration-dependent manner. However, KRG treatment did not affect GM-CSF production by Peyer's patch cells. LPS was used as a positive control for GM-CSF production. IgA production by Peyer's patch cells increased after treatment with 500 µg/mL KRG or KRG-P; nevertheless, only the last increase was statistically significant. Groups treated with lower concentrations of these compounds did not experience a change in IgA production ( Figure 2B).
Next, we investigated bone marrow cell proliferation mediated by Peyer's patch cells after treatment with KRG or KRG-P. The supernatants from Peyer's patch cells incubated with KRG-P for 4 days significantly promoted ex vivo bone marrow cell proliferation ( Figure 2C). The augmented bone marrow cell proliferation correlated with GM-CSF production by KRG-P, suggesting that KRG-P stimulated Peyer's patch cells to produce cytokines, and consequently induced bone marrow cell proliferation. Collectively, these results indicated that KRG-P may stimulate intestinal immunomodulation.

Effects of KRG and KRG-P Oral Administration on Mouse IgA Production
Based on the in vitro results, we investigated the in vivo intestinal immunomodulation activity of KRG and KRG-P. According to our previous report [26], activation of innate immune cells, such as natural killer (NK) cells and macrophages, was confirmed after oral administration of natural polysaccharides at 5-50 mg/kg to mice. Therefore, in this experiment, KRG or KRG-P was orally and daily administered to BALB/c mice for 10 days at 5 and 50 mg/kg. During the oral administration of KRG or KRP-P, we measured the bodyweight of each mouse every 2-3 days. As shown in Figure 3A, the bodyweight of the normal group slightly increased, and that of the KRG or KRG-P group showed similar patterns. These results indicated that KRG or KRG-P treatment did not affect the bodyweight of mice ( Figure 3A).
Next, to verify the modulating activity of KRG or KRG-P on the intestinal immune system, we measured IgA production in mouse feces on days 6 and 11 after oral administration. On the 6th day, IgA production increased in all treatment groups, but not significantly, compared with that in the normal group ( Figure 3B). However, on the 11th day, secreted IgA levels significantly increased in the KRG-P-treated group in a dose-dependent manner, and in the KRG-treated group only at the concentration of 50 mg/kg ( Figure 3C). In addition, to confirm IgA secretion in feces, we measured IgA mRNA expression levels in the jejunum region of the intestinal tissue. As shown in Figure 3D, the KRG-P-treated group (5 and 50 mg/kg) exhibited a significant increase in IgA mRNA levels. Collectively, oral administration of KRG-P augmented IgA production in feces and intestinal tissue;

Effects of KRG and KRG-P Oral Administration on Mouse IgA Production
Based on the in vitro results, we investigated the in vivo intestinal immunomodulation activity of KRG and KRG-P. According to our previous report [26], activation of innate immune cells, such as natural killer (NK) cells and macrophages, was confirmed after oral administration of natural polysaccharides at 5-50 mg/kg to mice. Therefore, in this experiment, KRG or KRG-P was orally and daily administered to BALB/c mice for 10 days at 5 and 50 mg/kg. During the oral administration of KRG or KRP-P, we measured the bodyweight of each mouse every 2-3 days. As shown in Figure 3A, the bodyweight of the normal group slightly increased, and that of the KRG or KRG-P group showed similar patterns. These results indicated that KRG or KRG-P treatment did not affect the bodyweight of mice ( Figure 3A).
Next, to verify the modulating activity of KRG or KRG-P on the intestinal immune system, we measured IgA production in mouse feces on days 6 and 11 after oral administration. On the 6th day, IgA production increased in all treatment groups, but not significantly, compared with that in the normal group ( Figure 3B). However, on the 11th day, secreted IgA levels significantly increased in the KRG-P-treated group in a dose-dependent manner, and in the KRG-treated group only at the concentration of 50 mg/kg ( Figure 3C). In addition, to confirm IgA secretion in feces, we measured IgA mRNA expression levels in the jejunum region of the intestinal tissue. As shown in Figure 3D, the KRG-P-treated group (5 and 50 mg/kg) exhibited a significant increase in IgA mRNA levels. Collectively, oral administration of KRG-P augmented IgA production in feces and intestinal tissue; meanwhile, KRG administration produced a slight increase in IgA production. These results suggested that polysaccharides, rather than ginsenosides, of red ginseng may affect IgA production in mice.
Polymers 2020, 12, x FOR PEER REVIEW 7 of 10 meanwhile, KRG administration produced a slight increase in IgA production. These results suggested that polysaccharides, rather than ginsenosides, of red ginseng may affect IgA production in mice.

Effects of KRG and KRG-P Oral Administration on Intestinal Mouse α-Defensin-1 Expression
Paneth cells, present in the intestinal epithelium, produce and secrete α-defensin, lysozyme, and proinflammatory mediators that play a role in maintaining intestinal homeostasis [6,7]. Therefore, we determined the protein levels of α-defensin-1, an antimicrobial and antiviral peptide, in the intestinal tissues of mice orally administered KRG or KRG-P. As shown in Figure 4A, α-defensin-1 protein expression in the jejunum markedly increased after oral administration of 5 and 50 mg/kg KRG-P compared with that in the control group, and after 50 mg/kg KRG administration, which showed a slight increase. KRG-P and KRG administration increased the mRNA expression of αdefensin-1 compared with the control group ( Figure 4B). In most of the 50 mg/kg KRG-P-treated mice, the mRNA and protein levels of α-defensin-1 significantly increased ( Figure 4B). Meanwhile, the observed increase in α-defensin-1 mRNA levels was not statistically significant in the group receiving 5 mg/kg compared with that in the normal group. These results suggested that oral administration of KRG-P may have better protective activity against intestinal infections than KRG. The mice were sacrificed at 11 days after oral treatment, and tissues from the jejunum region of the small intestine were then collected. Intestinal IgA mRNA levels were measured using qRT-PCR. Data are presented as the means ± SD of three independent experiments. * p < 0.05 vs. the normal group. ** p < 0.01 vs. the normal group.

Effects of KRG and KRG-P Oral Administration on Intestinal Mouse α-Defensin-1 Expression
Paneth cells, present in the intestinal epithelium, produce and secrete α-defensin, lysozyme, and proinflammatory mediators that play a role in maintaining intestinal homeostasis [6,7]. Therefore, we determined the protein levels of α-defensin-1, an antimicrobial and antiviral peptide, in the intestinal tissues of mice orally administered KRG or KRG-P. As shown in Figure 4A, α-defensin-1 protein expression in the jejunum markedly increased after oral administration of 5 and 50 mg/kg KRG-P compared with that in the control group, and after 50 mg/kg KRG administration, which showed a slight increase. KRG-P and KRG administration increased the mRNA expression of α-defensin-1 compared with the control group ( Figure 4B). In most of the 50 mg/kg KRG-P-treated mice, the mRNA and protein levels of α-defensin-1 significantly increased ( Figure 4B). Meanwhile, the observed increase in α-defensin-1 mRNA levels was not statistically significant in the group receiving 5 mg/kg compared with that in the normal group. These results suggested that oral administration of KRG-P may have better protective activity against intestinal infections than KRG.

Conclusions
The intestinal tract is exposed to foreign substances, such as food and pathogenic bacteria. Therefore, maintaining homeostasis in the intestine is particularly important for the defense mechanism of the body. Peyer's patches, an important organ of the intestinal tract, contains specialized M cells in the epithelium that uptake antigens from the intestinal lumen. These antigens activate B-lymphocytes in the Peyer's patch, which secrete IgA to prevent mucosal infections. In this study, we first confirmed that KRG-P stimulated Peyer's patch cells to produce GM-CSF and IgA and induced bone marrow cell proliferation ex vivo ( Figure 2). Recently, Kim et al. reported that αamylase-and amyloglucosidase-treated polysaccharides (non-starch-like fraction) isolated from KRG induced the significant production of IL-6 and GM-CSF by Peyer's patches [21]. Yu et al. reported that hot water-extracted crude polysaccharide fraction isolated from Atractylodes lancea DC showed bone marrow cell proliferation activity mediated by Peyer's patches [31]. Furthermore, we showed that oral administration of KRG-P (5 and 50 mg/kg) increased IgA secretion in mouse feces on day 11 ( Figure 3C); mRNA expression of IgA also increased in the 50 mg/kg-treated group ( Figure 3D). These results suggested that polysaccharide fractions can stimulate Peyer's patch immune cells and produce cytokines and IgA. IgA is mainly produced by Peyer's patches and plays a central role in protection against pathogens and homeostatic regulation of the intestine. Thus, our results showed that KRG-P has intestinal immune stimulatory activity. Moreover, we showed that protein and mRNA expression levels of α-defensin-1 were significantly increased by oral administration of 50 mg/kg KRG-P (Figure 4). These results suggested that KRG-P possessed protection activity against intestinal infection and maintained homeostasis of the intestinal lumen through activation of intestinal immunity. Future studies should focus on the effects of KRG-P on the expression of intestinal tight junction proteins, such as E-cadherin, ZO-1, and occludin, that are related to the regulation of intestinal barrier permeability and changes of the microbiome. Effects of oral administration of KRG and KRG-P on α-defensin-1 expression in mouse intestinal tissues. BALB/c mice were orally administered the indicated doses of KRG or KRG-P daily for 10 days. (A) e α-defensin-1 protein levels were determined by immunoblotting with the specific antibody. Actin was used as a loading control. The bar chart displays the intensity of α-defensin-1 after normalizing with that of β-actin by using ImageJ software. (B) α-defensin-1 mRNA levels were determined by using qRT-PCR. An actin gene was used as a housekeeping gene for normalization. Data are presented as the means ± SD of three independent experiments. * p < 0.05 vs. the normal group.

Conclusions
The intestinal tract is exposed to foreign substances, such as food and pathogenic bacteria. Therefore, maintaining homeostasis in the intestine is particularly important for the defense mechanism of the body. Peyer's patches, an important organ of the intestinal tract, contains specialized M cells in the epithelium that uptake antigens from the intestinal lumen. These antigens activate B-lymphocytes in the Peyer's patch, which secrete IgA to prevent mucosal infections. In this study, we first confirmed that KRG-P stimulated Peyer's patch cells to produce GM-CSF and IgA and induced bone marrow cell proliferation ex vivo ( Figure 2). Recently, Kim et al. reported that α-amylaseand amyloglucosidase-treated polysaccharides (non-starch-like fraction) isolated from KRG induced the significant production of IL-6 and GM-CSF by Peyer's patches [21]. Yu et al. reported that hot water-extracted crude polysaccharide fraction isolated from Atractylodes lancea DC showed bone marrow cell proliferation activity mediated by Peyer's patches [31]. Furthermore, we showed that oral administration of KRG-P (5 and 50 mg/kg) increased IgA secretion in mouse feces on day 11 ( Figure 3C); mRNA expression of IgA also increased in the 50 mg/kg-treated group ( Figure 3D). These results suggested that polysaccharide fractions can stimulate Peyer's patch immune cells and produce cytokines and IgA. IgA is mainly produced by Peyer's patches and plays a central role in protection against pathogens and homeostatic regulation of the intestine. Thus, our results showed that KRG-P has intestinal immune stimulatory activity. Moreover, we showed that protein and mRNA expression levels of α-defensin-1 were significantly increased by oral administration of 50 mg/kg KRG-P (Figure 4). These results suggested that KRG-P possessed protection activity against intestinal infection and maintained homeostasis of the intestinal lumen through activation of intestinal immunity. Future studies should focus on the effects of KRG-P on the expression of intestinal tight junction proteins, such as E-cadherin, ZO-1, and occludin, that are related to the regulation of intestinal barrier permeability and changes of the microbiome.