Bioactive compounds from marine organisms with antimicrobial activity have been ascribed to a variety of metabolites, such as polysaccharides [1
]. 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
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
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
(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, PGPStc, PGPS60c, PGPS80c, and PGPStp, 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 PGPStc had a significant impact on immunoenhancement activity and could be used as a novel immunopotentiator to develop.
2. Materials and Methods
RPMI-1640 media (Gibco, Grand Island, NY, USA) filtered (0.22 μm) and added with benzylpenicillin (100 IU·mL−1), streptomycin (100 IU·mL−1), and fetal bovine serum (10%) was utilized to culture cells. Phytohemagglutinin (PHA-P, Sigma, St. Louis, MO, USA) was dissolved into 0.5 mg·mL−1 with RPMI-1640 media. 3-(4,5)-Dimethylthiazol-2,5-diphenyltetrazolium bromide (MTT) was dissolved with PBS, and the final concentration was 5 mg·mL−1. Dimethyl sulfoxide (purchased from Solarbio Technology Co., Ltd., Beijing, China). Lymphocyte separation medium (purchased from Tianjin Haoyang Biological Manufacturing Co., Ltd., Tianjin, China). A PHA solution was placed at −20 °C, and an MTT solution was stored at 4 °C for 2 weeks without light.
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).
2.2. 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 (PGPStc), and fractional PG polysaccharides (PGPS60c and PGPS80c) 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. PGPStc 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 (PGPS60c and PGPS80c) were extracted via stepwise ethanol precipitation with ethanol concentration at 60% and 80%, respectively.
The purified PGPStp
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 PGPStp
was collected. The carbohydrate contents of PGPStc
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
2.3. 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 OD570 value of the experimental group—the OD570 value of the control group or the PHA group)/(the OD570 value of the control group or the PHA group) × 100%. Then, PGPStc and PGPS60c with better activity were subsequently selected.
2.4. Cell Cycle Distribution Analysis
Cell cycle was analyzed by flow cytometer (BD Biosciences FACSCanto II Flow Cytometer, BioLegend, San Diego, CA, USA) [13
]. The lymphocytes as described above were challenged with PGPS60c
, selected according to the preliminary experimental results) at 37 °C, 5% CO2
. After 24, 48, and 72 h treatments, the lymphocyte cells were collected, fixed with 70% cold ethanol, and left overnight. After washing twice with cold PBS, the lymphocyte cells were labeled with propidium iodide (PI) solution (50 μg·mL−1
) in the presence of RNase A (500 μg·mL−1
) and incubated at 37 °C without light for 30 min. The distribution of cells in G0/G1, S, and G2/M phases were analyzed via ModFit LT software (FACS Calibur™, Becton-Dickinson, Franklin Lakes, NJ, USA). The proliferation index was evaluated as SPF and PI values according to the following formula [14
]. SPF = S/(G0/G1 + S + G2/M) × 100%. PI = (S + G2/M)/(G0/G1 + S + G2/M) × 100%.
2.5. Detection of CD4+ and CD8+ T Lymphocytes
The expressions of CD4+
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
2.6. 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.
T lymphocyte, which can be induced by PHA, is responsible for cell-mediated immunity [16
]. 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 PGPS60c
), and PGPStp
) alone increased the OD570
values, the highest proliferation rate of cells was presented in PGPStc
, followed by PGPS80c
, suggesting that they promoted cell proliferation of the lymphocytes. Besides, PGPS60c
), and PGPStp
) also enhanced PHA-induced OD570
values, and the highest proliferation rate of cells was presented in PGPStc
, followed by PGPStp
, which implied that they synergized with PHA in promoting proliferation of the lymphocytes. Thus, the active sites of PGPStc
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
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+
cells and the ratio of CD4+
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+
]. These results were consistent with our data, which proved the cellular and humoral immune functions. In this study, PGPStc
also improved the proportion of CD4+
T cells effectively, and PGPStc
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
) 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 PGPStc
could accelerate the cell cycle and promote lymphocytes proliferation, which underlines the importance of PGPStc
in these processes. This is supported by other related research [25
]. These results showed that the immune-enhancing activity of PGPStc
may be related to its ability which can stimulate the proliferation of lymphocytes.