Heterogeneous sulfated polysaccharides, which are ubiquitous in nature and occur in numerous organisms, possess various effective biological and pharmacological activities [1
]. Recently, green algae, with the unique structural features containing major repeating disaccharide units of α-L-Rhap
, have gained considerable attention for their use in functional foods and drugs due to their availability, functionalities, and low cost of production [4
]. Enteromorpha prolifera
, a type of green algae belonging to the phylum Chlorophyta
and class Chlorophyceae
, is found on seashores worldwide [6
]. Previous studies revealed that polysaccharides from E. prolifera
exert anti-oxidative, anti-microbial, and anti-hyperlipidemic effects, as well as possess the ability to improve glucose metabolism [7
]. Kim et al. (2011) found that water-soluble E. prolifera
polysaccharides produced by pyrohydrolysis and fractionation using ion-exchange chromatography could stimulate a macrophage cell line to induce the production of NO and various cytokines, but the associated mechanism was not elucidated [9
]. Furthermore, no information is currently available on the effects of acidolysis-degraded polysaccharides from E. prolifera
on macrophages. Considering that polysaccharides with a complex structure and large molecular weight were not conducive for this study, a low-molecular weight sulfated polysaccharide (EP2) was prepared and the potential immunomodulatory effects were investigated.
Macrophages derived from blood monocytes play a crucial role in host defense against infection by processing and presentation of antigens to the lymphocytes, killing pathogenic microorganisms, removing cell debris, as well as secreting pro-inflammatory mediators such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and nitric oxide (NO) [10
]. Hence, macrophages are thought to be the important target cells of some immunomodulatory drugs. In this study, we aimed to investigate the immunomodulatory effects of EP2 on RAW 264.7 cells. Cyclophosphamide (CYP) is one of the famous anticancer agents which remains extensively used in the treatment of hematological malignancies and various epithelial tumors. However, CTX can damage the DNA of normal cells and cause immunosuppression after mainly hydrolyzed by the hepatic cytochrome P450 enzymes [12
]. In our research, CYP was used to build an immunodeficiency model in mice, and the influence of EP2 on the immunodeficiency mice was evaluated.
As one of the main algal genera that cause green tide, the over-growth of E. prolifera
has a noticeable negative impact on the environment and aquaculture [9
]. Researchers have been making great efforts to assess the value of E. prolifera
in bio-oil extraction, marine aquaculture, food processing, and drug development. It is reported that Enteromorpha
contains a variety of active components, including polysaccharides which are the most important components [23
]. In this study, we obtained a low-molecular weight polysaccharide by degradation and anion exchange chromatography separation from E
. The polysaccharide mainly contained GlcA, Xyl, and Rha, and the molecular weight was 17.3 kDa with 18.99% sulfate content. To detect the immunomodulatory effects of EP2, we measured cell viability and NO production after RAW 264.7 cells were treated with EP2. As shown in Figure 1
, EP2 significantly increased the production of NO without being harmful to RAW 264.7 cells. The presence of NO in the culture medium of macrophage cells is considered one of the most reliable factors that indicate the classical activation of macrophages [24
NLRP3 inflammasome plays a crucial role in host immune responses to various pathogen-derived factors as well as danger-associated molecules, which involves the recruitment of apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1 [25
]. NLRP3 binds to pro-caspase-1 through ASC, subsequently activating caspase-1. The activation of capase-1 leads to the maturation of IL-1β [27
]. In this study, the expression of NLRP3 inflammasome and cleaved caspase-1 was increased by EP2 treatment, which was consistent with the increased expression of TNF-α, IL-1β, and IL-6. This suggests that EP2 can enhance immunoreaction. The pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 may have multiple functions during immunomodulatory process. Especially, the cytokines released from immune cells can stimulate the innate immune responses, which are essential for immunomodulation [29
The activation of NLRP3 inflammasome indicates the activation of NF-κB [30
]. Upon stimulation with pro-inflammatory factors, IκBα was phosphorylated, selectively ubiquitinated, and then quickly degraded, which in turn leads to the liberation of NF-κB [26
]. The liberated NF-κB dimers were transferred into the nucleus where it bound to the promoter site of the target gene to induce the transcription of pro-IL-1β and the self-assembly of NLRP3 [32
]. The presence of EP2 promoted the activation of NF-κB through the enhancement of the phosphorylation of IκB α and NF-κB p65 subunit. AP-1 is another transcription factor regulating inflammatory responsive genes. It consists of the members of Jun, Fos, or ATF families. The activation of AP-1 involved the heterogeneous dimerization of Jun and Fos, and the recognition of the transcription site [33
]. Treatment with EP2 induced the expression of c-Jun and c-Fos after 1 h. The activation of transcription factors promotes the synthesis and release of inflammatory factors. These results further demonstrate that EP2 stimulated the immune response of macrophages.
Recently, several lines of evidence have indicated that MAPKs, a group of downstream Ser/Thr kinases, can modulate the activities of NF-κB and AP-1 [34
]. In response to various stimuli, the activation of JNK, ERK1/2, and p38 MAPK by phosphorylation is a key step leading to the expression of inflammatory mediators [36
]. JNK and p38 strengthen the translation of TNF-α mRNA and maintain the stability of the transcriptional process. Moreover, ERK1/2 promotes the transport of TNF-α mRNA from the nucleus to the cytoplasm [37
]. EP2 treatment promoted the phosphorylation of JNK, ERK1/2, and p38, which was supported by the increased level of TNF-α. EP2 promoted the expression of TLR4 which specifically mediates the innate immune response involved in various inflammatory disorders [39
]. The activation of TLR4 upon EP2 stimulation sent downstream signals by secreting a variety of pro-inflammatory mediators, NO, TNF-α, and IL-1β in a time-dependent manner. The addition of TLR4 inhibitors significantly inhibited the activation of EP2 in RAW 264.7 cells, but the inhibitor did not completely block the effect of EP2. This indicates that TLR4 played an important role in the activation of EP2 in macrophages, but the activity of EP2 was not completely dependent on TLR4. After all, the mechanism of action of polysaccharides in cells is complicated.
Furthermore, we characterized the immunomodulatory effects of EP2 on immunosuppressed mice and found that these effects occurred through the regulation of immune organs, inflammatory cell counts, and cytokines. CYP as an effective immunosuppressive and chemotherapeutic agent can damage the structure of DNA, interfere with the proliferation and differentiation of macrophages, kill immune cells, and weaken the immune system of the organism [40
]. It induces an imbalance in the immune function homeostasis. A previous study showed that CYP causes overall immunological dysfunction regardless of cell phenotypes by markedly repressing the production of cytokines [42
]. Because of the broad toxicity of CYP, the CYP-induced immunosuppression model is the most commonly used in immunostimulatory experiments [43
]. The administration of CYP reduced the body weight, spleen index, and thymus index. The weights are recognized as critical and intuitive indices for non-specific immunity. Thymus and spleen indices are assessed to evaluate the whole immune state of the organism [18
]. In our study, EP2 significantly increased the body weight, spleen index, and thymus index, indicating that EP2 could improve the immune function of developing immune organs. Moreover, peripheral WBC, NEUT, LYMPH, and platelet counts showed that treatment with EP2 inhibited CYP-induced immunosuppression, which is an important limiting factor in the outcome and recovery of tumor patients receiving chemotherapy. This suggests that EP2 can enhance the host’s specific and non-specific immunity, including cellular and humoral immune systems.
4. Materials and Methods
High-glucose Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Hyclone (Logan, UT, USA). Lipopolysaccharide (LPS) (Escherichia coli 0111: B4) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The cyclophosphamide (CYP) were obtained by Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China).
4.2. Extraction and Purification of the Polysaccharide
(1000 g) was extracted with 0.1 M HCl (30 L) at room temperature for 4 h. Then, the supernatant was filtered through celite, neutralized, concentrated and precipitated by ethanol. The sediment (8 g, the yield was 20.1%) underwent anion exchange chromatography on a DEAE-Bio Gel Agarose FF gel (6 cm D × 40 cm H) with elution by water (5 L), 0.3 M NaCl (5 L), 1 M NaCl and 2 M NaCl, respectively. After being ultrafiltrated, concentrated, and precipitated by ethanol, the polysaccharides in 1 M NaCl (the yield was 53.2%) underwent autohydrolysis and were precipitated by ethanol according to the modified method [44
]. The precipitate was fractionated on a Bio-Gel P-10 Gel column (2.6 cm D × 100 cm H) eluted with 0.5 M NH4
into two fractions (EP1 and EP2). The degraded polysaccharides (EP1 and EP2) was dialyzed against distilled water and then lyophilized.
4.3. Composition Analysis
Chemical compositions of polysaccharides were elucidated from the yields, total sugar content, sulfated content, uronic acid (UA) content, molar ratio of monosaccharides and the molecular weight. The content of total sugar was determined according to the modified phenol-sulfuric acid method, using rhamnose as a standard [45
]. An ion chromatography with a Shodex IC SI-52 4E column (4.0 mm D × 250 mm H) was applied to perform the sulfated content with 3.6 mM Na2
at a flow rate of 0.8 mL/min at 45 °C referred to the previous study [46
]. UA was determined using a modified carbazole method [47
], using glucuronic acid as a reference. Finally, the molar ratio of monosaccharide composition was determined according to the method described by Geng et al. [17
]. Prior to HPLC, the polysaccharide was first hydrolyzed and derivatized with PMP. Briefly, polysaccharides (10 mg/mL) were hydrolysed by trifluoroacetic acid (2 M) under a nitrogen atmosphere for 4 h at 110 °C. Then, 0.2 mL of hydrolysed mixture was neutralized to pH 7 with sodium hydroxide. Later, the mixture was converted into its PMP derivatives and separated by HPLC chromatography on a YMC Pack ODS AQ column (4.6 mm D × 250 mm L). The molecular weight was analyzed by HPGPC on a TSK G3000 PWxl column (7 μm, 8.0 mm D × 300 mm H) (TOSOH, Tokyo, Japan) eluted with 0.05M Na2
at a flow rate of 0.5 mL/min at 30 °C with refractive index detection. Ten different molecular weight dextrans purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China) were used as weight standards.
4.4. Endotoxin Test
The concentration of endotoxin was determined by using the chromogenic end-point TAL assay kit (Solarbio, Beijing, China). The experiment was performed according to the manufacturer’s instructions. The endotoxin in the sample activates a cascade of enzymes in TAL, the activated enzyme splits the synthetic substrate, releasing a yellow colored product with maximum absorbance at 405 nm. The yellow product can further react with diazo reagents forming purple product with maximum absorbance at 545 nm. The absorbance of both the yellow product and purple product are proportional to endotoxin levels.
4.5. Cell Culture and Cell Viability Assay
The RAW 264.7 murine macrophages were obtained from the National Infrastructure of Cell Line Resource (Beijing, China) and cultured in DMEM high glucose medium supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin and 10% heat-inactivated FBS. Cultures were maintained at 37 °C in a humidified 5% CO2 incubator. RAW 264.7 cells were seeded at a density of 1 × 105 cells/mL in a 96-well plate overnight and then treated with various concentrations of polysaccharides or 1 μg/mL of LPS for 24 h. After 24-h incubation, CCK-8 reagent (10 µL/mL) was added to each well and the absorbance was measured at 450 nm using a microplate reader.
4.6. Nitric Oxide Production
RAW 264.7 cells at a density of 1 × 105 cells/mL were incubated with different concentrations of polysaccharides or LPS (1 μg/mL) for 24 h. After incubation, supernatants were collected and reacted with Griess reagent according to the manufacturer’s instructions (Beyotime Biotechnology, Shanghai, China). A NaNO2 standard curve was used to calculate nitrite concentration.
4.7. Immunofluorescence Staining Analysis
RAW 264.7 cells were grown on cover slips in six-well plates and treated with EP2 for 6 h. The cells were treated as mentioned, and then fixed and incubated with a blocking buffer for 1 h to suppress nonspecific binding. Next, cells were incubated with the primary antibody at 4 °C overnight, followed by incubation with a FITC-conjugated secondary antibody at room temperature for another 1 h. DAPI was used as nuclear staining after 3-min incubation with cells. Samples were visualized under a fluorescent inverted microscope (Olympus Fluoview FV1000, Tokyo, Japan).
4.8. Western Blot
RAW 264.7 cells at a density of 1 × 106 cells/mL were seeded in six-well plates, then treated with EP2 for 0, 0.5, 1, 3 and 6 h. After that, cells were washed twice with phosphate buffered saline and harvested in cold lysis buffer containing protease inhibitors or phosphatase inhibitors. The proteins were collected by centrifugation. The concentration of proteins was determined by a BCA protein assay (Beyotime Biotechnology, Shanghai, China). The proteins were separated by 12.5% SDS-PAGE gels and transferred to a PVDF membrane (Millipore, Carrigtwohill, Ireland). The membrane was sealed with 5% bovine serum albumin for 2 h, then hybridized with primary antibodies (TNF-α, IL-1β, iNOS, COX-2, MMP-9, IL-6, TLR4, NLRP3, caspase-1, P38, ERK1/2, JNK, IκB, P65, c-jun, c-fos) (Abcam, Cambridge, UK) overnight at 4 °C. After being rinsed with TBST three times, the horseradish peroxidase-conjugated IgG secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) (1:2000) was added. Enhanced chemiluminescence (Millipore, Carrigtwohill, Ireland) was applied to detect the bands of proteins.
The study was approved by the Animal Care and Ethics Committee of Qingdao University in compliance with the Principles of Laboratory Animal Care that were developed by the National society for Medical Research. The animals were kept with free access to food and tap water and housed under standard conditions, with controlled temperature (22 ± 2 °C) and a 12 h light–dark cycle. Before the study proceeded, fifty male ICR mice (22–25 g) were given seven days to acclimatize to the feeding environment.
The mice were randomly divided into five groups (n
= 10 for each group) (1) control; (2) CYP only; (3) EP2 (20 mg/kg/d) + CYP; (4) EP2 (40 mg/kg/d) + CYP; (5) EP2 (80 mg/kg/d) + CYP. The mice in groups (3–5) received different concentrations of EP2 intraperitoneally for consecutive 14 days. In addition to the control group, mice in other groups were injected intraperitoneally with CYP (50 mg/kg/d) at day 8, 10, 12 and 14. Mice were given gavage of normal saline (0.1 mL/20 g) in the control group. The experimental design was showed in Figure 7
. Mice in each group were weighed from the first day of feeding. The thymus index and spleen index of each group were calculated as thymus and spleen weight.
4.10. Inflammatory Cell Counts
Twenty-four hours after the last drug administration, whole blood samples were collected into heparin tubes. The total number of red blood cell (RBC), peripheral white blood cell (WBC), neutrophil (NEUT), lymphocyte (LYMPH) and platelet (PLT) were counted using a SYSMEX XS-500i Analyzer (Kobe, Japan).
4.11. Enzyme Linked Immunosorbent Assay (ELISA)
TNF-α and IL-1β were measured by ELISA kit. Briefly, the blood samples were collected by the heart punctures in mice, then centrifuged at 1200 g for 10 min to obtain serum. The levels of TNF-α, IL-6 and IL-1β in the serum were detected according to the instruction (Multi Sciences (Lianke) Biotech, Co., Ltd, Hangzhou, China).
4.12. Statistical Analysis
The data and statistical analyses comply with the recommendations on experimental design and analysis. Data analysis was performed using 19.0 SPSS software (SPSS Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) was used to compare the mean differences among the groups. The results are shown as the mean ± SEM and a level of p ≤ 0.05 or p ≤ 0.01 was considered to be statistically significant.