Effects of Polysaccharides from Platycodon grandiflorum on Immunity-Enhancing Activity In Vitro

The study is aimed at investigating the immunoenhancement activity of polysaccharides from Platycodon grandiflorum polysaccharides (PGPSs) in vitro. In this study, some research on lymphocyte proliferation, cell cycle, and the levels of CD4+ and CD8+ T cells were performed. Four different concentrations of PGPSs (PGPStc, PGPS60c, PGPS80c, and PGPStp) were harvested and added to peripheral blood T lymphocytes. We observed significant increases in T lymphocyte proliferation at PGPStc groups individually or synergistically with phytohemagglutinin (PHA) at most concentrations, and their lymphocyte proliferation rates were the highest. The active sites of PGPStc and PGPS60c were subsequently chosen. Then, we utilized flow cytometry to determine lymphocyte cell cycle distribution and levels of CD4+ and CD8+ T cells. At most time points, PGPStc could facilitate lymphocyte cell cycle progression from the G0/G1 phase to the S and G2/M phases and, simultaneously, increase the levels of CD4+ and CD8+ T cells. These results indicate that PGPStc enhances the immune functions, suggesting that PGPStc could be a potential immunopotentiator for further in vivo and clinical trial experiments.


Introduction
Bioactive compounds from marine organisms with antimicrobial activity have been ascribed to a variety of metabolites, such as polysaccharides [1,2]. Polysaccharides with a low risk of side effects and toxicity, which can activate immune cells, improve immune function and have been found in normal cells. Interest in polysaccharides as new immunopotentiators for the development of veterinary vaccines has recently increased. Some studies have demonstrated that Astragalus and Echinacea polysaccharides have potential for use as immunopotentiators/adjuvants in inactivated rabies vaccines for veterinary use [3]. Taishan Robinia pseudoacacia polysaccharide can improve immunologic function and be used as a vaccine immunopotentiator for immune responses [4]. Astragalus polysaccharide (APS) and sulfated APS (SAPS) show dose-dependent growth-facilitating and immunomodulating function, and could be a potential trigger for immunomodulator in early LPS stimulation condition [5].
Platycodon grandiflorum (PG) has been used as either a food material as well as in traditional Chinese medicine to prevent or treat a variety of diseases, such as for treating cough, phlegm, hyperlipidemia, hypertension, diabetes and immunoregulation [6]. Polysaccharides extracted from PG had been shown to activate various cell types of the innate and adaptive immune systems. It is evident that PG polysaccharides induce DC maturation by activating MAPK and NF-κB signaling downstream of TLR4, and they might be used as adjuvants in DC-based cancer immunotherapy [7]. Moreover, another study has shown that MAPK/AP-1 and TLR4/NF-κB signaling pathways were involved in the macrophage activation by PG polysaccharides (PGPSs) [8].
In the present study, four types of polysaccharides, PGPS tc , PGPS 60c , PGPS 80c , and PGPS tp , were extracted from PG and purified. We investigated peripheral T lymphocyte proliferation activity at the cellular level in immune system, and further studies were examined on distributions of cell cycles and percentages of CD4 + and CD8 + T cells. Our research shows that PGPS tc had a significant impact on immunoenhancement activity and could be used as a novel immunopotentiator to develop.
The use and care of Hy-line adult Cocks (male, 60-day old) were from the Experimental Animal Center of the Shan Dong Agricultural University and were approved by the College Committee for animal experiments (Permit number: SDAUA-2014-012).

Preparation of PGPSs
The dried rhizome of PG was acquired from Jin Tai Lian Co. Ltd., Tai'an, China. Adopting alcohol sedimentation, three crude polysaccharides, crude total PG (PGPS tc ), and fractional PG polysaccharides (PGPS 60c and PGPS 80c ) were prepared in our laboratory. Briefly, PGPSs were obtained via water extraction and alcohol precipitation methods, and the final concentration was 1 g·mL −1 . PGPS tc was extracted by one-step ethanol precipitation, in which ethanol was added to the decoction to obtain ethanol concentration of 80% (v/v). Two fractional polysaccharides (PGPS 60c and PGPS 80c ) were extracted via stepwise ethanol precipitation with ethanol concentration at 60% and 80%, respectively.
The purified PGPS tp was obtained using Sevag's method to eliminate protein [9], through a Sephadex G-75 column and eluting with distilled water. The eluent in a dialysis sack was dialyzed against flowing distilled water for 12 h. Then, the dialysate was lyophilized, and the powder of purified PGPS tp was collected. The carbohydrate contents of PGPS tc , PGPS 60c , PGPS 80c and PGPS tp were 64%, 53.7%, 63.5% and 73%, respectively, as measured by a phenol hydrate-sulfuric acid method using analytical grade glucose as a standard sample [10]. The structure identification of PGPSs was assessed in our previous study [11].

Peripheral Lymphocyte Proliferation Assay
The MTT assay was performed to measure peripheral lymphocyte proliferation as described in our previous study [12]. The absorbance of cells in each well was read at 570 nm using a microplate reader (DG-3022, Nanjing Huadong Electronics, Information & Technology Co., Ltd., Nanjing China).
The highest proliferation rate was evaluated: the highest proliferation rate = (the highest OD 570 value of the experimental group-the OD 570 value of the control group or the PHA group)/(the OD 570 value of the control group or the PHA group) × 100%. Then, PGPS tc and PGPS 60c with better activity were subsequently selected.

Detection of CD4 + and CD8 + T Lymphocytes
The expressions of CD4 + and CD8 + on lymphocytes were determined with a flow cytometer (BD Biosciences FACSCanto II Flow Cytometer, BioLegend, San Diego, CA, USA). After 24, 48, and 72 h interference by PGPSs, the cells were collected and washed twice with cold PBS. Then, 100 µL of PBS were added to resuspended. Then, the cells were stained with 10 µL of anti-CD4-FITC and 10 µL of anti-CD8-PE, and the mixture was incubated at 37 • C without light for 30 min. Finally, the stained cells were washed with PBS and separated at 1000 rpm for 10 min [15].

Statistical Analysis
SPSS 16.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis with one-way ANOVA. Multiple comparisons among the control and polysaccharide groups were conducted. All experiments were repeated at least thrice. Data were presented as means ± standard errors (SE). p-Values of differences less than 0.05 were considered significant.

Characterization of Platycodon grandiflorum
The structure identification of PGPSs was assessed in our previous research [11]. FT-IR analysis showed that PGPSs possess typical absorption peak of polysaccharides. Molecular weight distribution showed that all PGPSs had two peaks, at two different retention times. PGPSs were primarily composed of glucose, mannose, arabinose, and galactose, and they were linked mainly by (1→3,6)-β-D-Galp residues.

Cytotoxicity of PGPSs to Peripheral Lymphocytes
The OD 570 values of each group are presented in Table 1. The OD 570 values of PGPS 60c at 250 µg·mL −1 , PGPS 80c at 1000 µg·mL −1 , PGPS tc at 500 µg·mL −1 , and PGPS tp at 62.5 µg·mL −1 were just not lower than those of the control group significantly (p > 0.05). Therefore, the corresponding concentration could be reckoned as the safety concentration, respectively. For the convenience of comparison of PGPSs at the same level, the safety concentrations of PGPSs were unified as 62.5 µg·mL −1 .
Data within a column without the same superscripts differ significantly (p < 0.05). Table 2 indicates the changes of OD 570 values of each group. The OD 570 values of PGPS 60c , PGPS 80c , and PGPS tc at 3.907~62.5 µg·mL −1 and of PGPS tp at 15.625~62.5 µg·mL −1 were significantly higher than those of the control group (p < 0.05).   Table 2 indicates the changes of OD570 values of each group. The OD570 values of PGPS60c, PGPS80c, and PGPStc at 3.907~62.5 μg·mL −1 and of PGPStp at 15.625~62.5 μg·mL −1 were significantly higher than those of the control group (p < 0.05). The values are presented as means ± SE (n = 4). a-c Bars without the same superscripts differ significantly (p < 0.05). Figure 1 shows the highest lymphocyte proliferation rate. In a single stimulation with polysaccharides, the proliferation rate in PGPStc at 31.25 μg·mL −1 was the highest (80.61%), followed by PGPS80c at 62.5 μg·mL −1 (75%). The proliferation rates in the PGPStc and PGPS80c groups were likewise significantly higher than those in the PGPS60c and PGPStp groups (p < 0.05). The values are presented as means ± SE (n = 4). a-c Bars without the same superscripts differ significantly (p < 0.05). Figure 1 shows the highest lymphocyte proliferation rate. In a single stimulation with polysaccharides, the proliferation rate in PGPS tc at 31.25 µg·mL −1 was the highest (80.61%), followed by PGPS 80c at 62.5 µg·mL −1 (75%). The proliferation rates in the PGPS tc and PGPS 80c groups were likewise significantly higher than those in the PGPS 60c and PGPS tp groups (p < 0.05).         Figure 2 illustrates the highest lymphocyte proliferation rate of four groups. The proliferation rate in PGPS tc at 31.25 µg·mL −1 was the highest (75%), followed by PGPS tp at 7.813 µg·mL −1 (40%). The proliferation rate of cells in PGPS tc was considerably higher than those in PGPS 60c , PGPS 80c , and PGPS tp (p < 0.05).

Cell Cycle Analysis
As shown in Figure 3, compared to cell control group, PHA induced a notable decrease in the percent of cells in the G0/G1 phase (p < 0.05) but a significant increase of cells in the S and G2/M phases at all the time points (p < 0.05). The ratio of cells decreased in the G0/G1 phase and increased in the S phase in the PGPS tc group over 24 h after incubation (p < 0.05), and the percentage of cells in the G2/M phase had no significant change after treatment for 24 h. However, after treatment with PGPS tc for 48 h, there was a significant reduction in the amount of cells in the G0/G1 phase but an increase in the S and G2/M phases (p < 0.05) which did not significantly change compared with the PHA group. The same in the G2/M phase as the PGPS 60c group. Treated with PGPS tc for 72 h, a significant reduction occurred in the percentage of cells in the G0/G1 phase, whereas there was an increase in the G2/M phases (p < 0.05); however, there were no notable differences when compared with the PHA group. At most time points, SPF and PI in PGPS tc were the largest among all groups (Tables 4 and 5).

Cell Cycle Analysis
As shown in Figure 3, compared to cell control group, PHA induced a notable decrease in the percent of cells in the G0/G1 phase (p < 0.05) but a significant increase of cells in the S and G2/M phases at all the time points (p < 0.05). The ratio of cells decreased in the G0/G1 phase and increased in the S phase in the PGPStc group over 24 h after incubation (p < 0.05), and the percentage of cells in the G2/M phase had no significant change after treatment for 24 h. However, after treatment with PGPStc for 48 h, there was a significant reduction in the amount of cells in the G0/G1 phase but an increase in the S and G2/M phases (p < 0.05) which did not significantly change compared with the PHA group. The same in the G2/M phase as the PGPS60c group. Treated with PGPStc for 72 h, a significant reduction occurred in the percentage of cells in the G0/G1 phase, whereas there was an increase in the G2/M phases (p < 0.05); however, there were no notable differences when compared with the PHA group. At most time points, SPF and PI in PGPStc were the largest among all groups (Tables 4 and 5).

Changes in Lymphocytes CD4 + and CD8 + T Cells
The changes of CD4 + T cells are presented in Figure 4. The percentages of CD4 + T cells in PGPS tc at 24, 48, and 72 h, and in PGPS 60c at 48 h and 72 h, were markedly higher than those in the PHA group (p < 0.05). At the time points of 48 h and 72 h, the proportion of CD4 + T cells in the PGPS tc group were markedly higher than those in the PGPS 60c group (p < 0.05).

Changes in Lymphocytes CD4 + and CD8 + T Cells
The changes of CD4 + T cells are presented in Figure 4. The percentages of CD4 + T cells in PGPStc at 24, 48, and 72 h, and in PGPS60c at 48 h and 72 h, were markedly higher than those in the PHA group (p < 0.05). At the time points of 48 h and 72 h, the proportion of CD4 + T cells in the PGPStc group were markedly higher than those in the PGPS60c group (p < 0.05). The changes in CD8 + T cells are presented in Figure 5. At 48 h and 72 h, the proportion of CD8 + T cells in the PGPStc group was markedly higher than those in the PHA group (p < 0.05). At 72 h, the proportion of CD8 + T cells in the PGPStc group was markedly higher than those in the PGPS60c group (p < 0.05).
At 72 h, the proportion of CD8 + T cells in the PGPStc group was observably higher than those in the PGPS60c group (p < 0.05).
The changes in CD4 + /CD8 + ratios are presented in Figure 6. At three time points, the CD4 + /CD8 + ratios in the PGPStc and PGPS60c groups were significantly higher than that in the cell control group (p < 0.05), which indicated that the polysaccharides increased the ratio of CD4 + /CD8 + . The changes in CD8 + T cells are presented in Figure 5. At 48 h and 72 h, the proportion of CD8 + T cells in the PGPS tc group was markedly higher than those in the PHA group (p < 0.05). At 72 h, the proportion of CD8 + T cells in the PGPS tc group was markedly higher than those in the PGPS 60c group (p < 0.05).
At 72 h, the proportion of CD8 + T cells in the PGPS tc group was observably higher than those in the PGPS 60c group (p < 0.05).
The changes in CD4 + /CD8 + ratios are presented in Figure 6. At three time points, the CD4 + /CD8 + ratios in the PGPS tc and PGPS 60c groups were significantly higher than that in the cell control group (p < 0.05), which indicated that the polysaccharides increased the ratio of CD4 + /CD8 + .

Discussion
T lymphocyte, which can be induced by PHA, is responsible for cell-mediated immunity [16,17]. By means of accelerating the clearance of pathogens and producing immunomodulatory cytokines, cellular immunity responses play a critical role in the host defense system against infections [18]. In this study, it was observed that PGPS 60c , PGPS 80c , PGPS tc (3.907~62.5 µg·mL −1 ), and PGPS tp (15.625~62.5 µg·mL −1 ) alone increased the OD 570 values, the highest proliferation rate of cells was presented in PGPS tc , followed by PGPS 80c , suggesting that they promoted cell proliferation of the lymphocytes. Besides, PGPS 60c (15.625~31.25 µg·mL −1 ), PGPS tc (3.907~31.25 µg·mL −1 ), and PGPS tp (3.907~15.625 µg·mL −1 ) also enhanced PHA-induced OD 570 values, and the highest proliferation rate of cells was presented in PGPS tc , followed by PGPS tp , which implied that they synergized with PHA in promoting proliferation of the lymphocytes. Thus, the active sites of PGPS tc and PGPS 60c were selected and subjected to further experiments by synthetic analysis. Zhang et al. have demonstrated that in vitro Ganoderma lucidum polysaccharide (GLP) could significantly enhance lymphocytes proliferation singly or synergistically with ConA [19]. Liu et al. have reported that Atractylodes macrocephala polysaccharide (AMP) and its selenium-modified products at certain concentrations could significantly promote lymphocytes proliferation and enhance cellular immunity [20].
CD4 + is a T helper (Th), while CD8 + is a Tcytotoxic (Tc) lymphocyte, and these are two common T lymphocytes vital for adaptive immunity [21]. Some researchers have reported that the percentage of CD4 + or CD8 + cells and the ratio of CD4 + /CD8 + increased when treated with ginseng fruit polysaccharides (CMPs) substantially compared with the negative control group, which is in line with the literature [22]. The effect of Jujube polysaccharide conjugates (JPCs) treatment was better than Ginsenoside treatment on the growth of counts of CD4 + T cell and the ratio of CD4 + /CD8 + [23]. These results were consistent with our data, which proved the cellular and humoral immune functions. In this study, PGPS tc and PGPS 60c also improved the proportion of CD4 + and CD8 + T cells effectively, and PGPS tc produced optimal effects. The cell cycle refers to a series of processes that take place in a cell to allow division and duplication period. Research showed that polysaccharide isolated from Yu-Ping-Feng (YPF-PS) could significantly increase lymphocyte cells entering into the S and G2/M phases at most time points by the in vitro and in vivo tests [24]. Previous reports have shown that Atractylodis macrocephalae Koidz polysaccharides (RAMPS 60c and RAMPS tp ) could promote cells into the S and G2/M phases [14]. This result was consistent with the present data, which indicated that they can regulate the cell cycle progression. In this study, the results clearly showed that PGPS tc could accelerate the cell cycle and promote lymphocytes proliferation, which underlines the importance of PGPS tc in these processes. This is supported by other related research [25]. These results showed that the immune-enhancing activity of PGPS tc may be related to its ability which can stimulate the proliferation of lymphocytes.

Conclusions
Polysaccharides from Platycodon grandiflorum (PGPS) exhibited the immunoenhancement activity in vitro. Moreover, PGPS tc could significantly promote the proliferation of lymphocyte individually or synergistically with PHA-P at most concentrations, facilitate lymphocyte cell cycle progression from the G0/G1 phase to the S and G2/M phases, and increase the levels of CD4 + and CD8 + T cells, which indicated that PGPS tc could improve immune functions. Therefore, PGPS tc might be helpful for developing a novel immunopotentiator. Further studies of PGPS are under investigation.