Edible mushrooms are known to be a highly nutritious foodstuff and to exhibit tonic and medicinal attributes in folk medicines [1, 2]. The pharmacological effects of mushrooms vary greatly,ranging from antihypertensive actions,immunity enhancement,cholesterol lowering actions,to anti-tumor activities [3, 4],and various mushrooms have long been used in traditional Chinese medicine and other folk medicines [5, 6]. Polysaccharides isolated from botanical sources,such as fungi,algae,lichens,and higher plants,have also attracted a great deal of attention in biomedicine because of their broad spectrum of therapeutic properties and relatively low toxicity [7, 8, 9]. Pleurotus nebrodensis is native to China,Southern Europe,and Central Asia . However,few literatures focus on polysaccharides from P. nebrodensis and their bioactivities.In our previous study,a novel polysaccharide (PN-S) has been purified from P. nebrodensis and verified to be nontoxic .
The aim of the present study was to determine the capacity of the water-soluble PN-S to induce proliferation and exert immune-stimulating effects in a murine monocytemacrophage cell line RAW264.7 and to identify the underlying biochemical mechanisms. To the best of our knowledge,this was the first report on the evaluation of the immune activity potential of the edible P. nebrodensis polysaccharides. Our results indicated that,in addition to its nutritional properties,PN-S might be a very promising candidate polysaccharide for the development of immunityenhancing medicines.Materials and Methods
The murine monocyte-macrophage RAW264.7 cell line was obtained from the Bioresource Collection and Research Center of the Food Industry Research and Development Institute (Hsinchu,Taiwan,ROC). Human myelomonocytic leukemia U937 cell line was obtained from the American Type Culture Collection (ATCC,Manassas,VA,USA). RPMI1640 medium was purchased from Thermo (Beijing,China). Fetal bovine serum (FBS) was obtained from Gibco GRL (Grand Island,NY,USA). Penicillin-streptomycin solution,EDTA- trypsin,phosphate buffered saline (PBS),dimethyl sulfoside side (DMSO),3-(4,5-dimethylthiazol- 2-yl)- 2,5-diphenyltertrazolium bromide (MTT),and cell lysis solution were purchased from Solarbio (Beijing,China). Propidium iodide (PI) was purchased from Sigma- Aldrich (St. Louis,MO,USA). RNeasy Mini Kit was purchased from Qiagen (Hilden,Germany). The PrimeScript 1st Strand cDNA Synthesis Kit was obtained from Takara Bio (Dalian,China). The SYBR Green PCR master mix was purchased from Invitrogen (Shanghai,China).Isolation and purification of the polysaccharide PN-S
The polysaccharide PN-S was extracted from fresh P. nebrodensis by decoction and alcohol sedimentation techniques. Briefly,the mushrooms were ground and mixed with distilled water at a grain : water ratio of 1 : 20 (W/V). The mixture was extracted three times in a water bath at 100 °C with stirring for 4 h. After extracted,the mixture was cooled to room temperature (25 ± 2 °C),filtered through a gauze and centrifuged at 4 000 r·min-1 for 15 min. The supernatant was collected,concentrated with a rotary evaporator,precipitated with four volumes of 50% ice-cold ethanol,and freeze-dried (Thermo Scientific,Rockford,IL,USA). The crude polysaccharide was further purified through a Sepharose 4B gel permeation column (60 cm × 2.6 cm) with ultrapure water as eluent using the Automated Fraction Collector. A main single fraction (PN-S) was obtained and was then collected and freeze-dried. The molecular mass of PN-S was determined to be 200 kDa by gel filtration. The percentage of total protein and carbohydrate constituent were determined to be 2.6% ± 0.9% and 92.4% ± 6.1% (W/W) by the phenol-sulfuric acid and Bradford’s method. The component sugars of PN-S were determined by gas chromatography (GC). The polysaccharide was hydrolyzed with acid,reduced and acetylated,indicating that the neutral sugar components were xylose,mannose,glucose,and galactose,at a molar ratio of 1 : 2.7 : 34.4 : 1.5,with a trace of arabinose. Using Fourier transform infrared spectroscopy (FTIR) (Bruker,Ettlingen,Germany),the structure of PN-S was determined to possess a backbone composed of α-Dglucopyranosyl (Glcp) residues. Ultraviolet spectrophotometry (Agilent Santa Clara,CA,USA) confirmed the absence of nucleic acid.Cell culture
RAW264.7 cells and U937 cells were grown and maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillin/streptomycin (100 U·mL-1 P + 0.1 mg·mL-1 S) at 37 °C in a 5% CO2 atmosphere. After grown to 90% confluence in a T25 tissue culture flask (TPP Biochrom AG,Trasadingen,Switzerland),the cells were plated at a density of 1 × 106 or 5 × 103 cells/mL in 24-well plates or 96-well plates to perform the following bio-assays. The plates were incubated at 37 °C for 2-4 h to allow cell attachment to the bottom. After the supernatant was removed,an aliquot with equal volume of test sample was added to the well.Cell viability and proliferation
The cell viabilities of RAW264.7 macrophages and U937 cells treated with PN-S at different concentrations were determined by MTT  assay. RAW264.7 macrophages in the absence or presence of samples at different concentrations (100 μL/well) were cultured in 96-well plates for 24,48,and 72 h. Aliquot of 20 μL of 5 mg·L-1 MTT in PBS was added to each well. The plates were incubated for another 4 h and centrifuged. The culture medium was then discarded. The plates were washed carefully twice with PBS buffer. Aliquot of 150 μL of DMSO was added to each well and oscillated for 30 min to extract the insoluble formazan formed. The absorbance was measured at 570 nm on a plate reader (ELISA reader,ASYS Hitech,GmbH,Austria). Cell viability was calculated as follows:
Index of proliferation =(A - B)/(C - B)
Non-cytotoxic concentrations of PN-S were selected to conduct immune-stimulating assessments.Neutral red uptake assay
After a 24-h incubation at 37 °C in 5% CO2,the medium was removed,and 100 μL of 0.1% neutral red in PBS was added to each well and incubated for additional 4 h. The cells were washed with PBS thrice,and then 100 μL of 1% acetic acid solution (V/V) in 50% ethanol (V/V) was added to each well to extract the dye phagocytized by macrophages. After rapid agitation on a microtiter plate shaker,the absorbance was read at 540nm by using an ELISA reader.
RAW264.7 cells (5 × 106 cells/mL) were grown on cover slips in 6-well plates and treated with PN-S at different concentrations. The cells were prepared for scanning electron microscopic (SEM) by treatment with 4% (V/V) glutaraldehyde at 25 °C and 1% osmium tetroxide for 30 min. The osmium-fixed cells were then dehydrated with a graded ethanol series from 10% to absolute ethanol before air drying at room temperature and coated with gold. The morphological changes were observed under a SEM (Hitachi,Tokyo,Japan).Cell cycle analysis
RAW264.7 cells (1 × 106 cells/mL) were seeded in 6-well plates and exposed to PN-S (0,10,20 and 40 μg·mL-1) for 48h. The cells were washed in PBS and collected by trypsinization,fixed in 70% glacial ethanol,washed in PBS,resuspended in 1 mL of PBS containing 50 U·mL-1 RNase and 50 μg·mL-1 PI,and then incubated for 40 min in the dark at 4 °C. Cell cycle analysis was performed by flow cytometry (BD,Franklin Lakes,NJ,USA),and the population of cells in various phases were calculated using the Modifit LT software program. Each experiment was conducted thrice.Measurement of cytokine levels in RAW264.7 macrophage treated by PN-S using ELISA
The RAW264.7 macrophage culture supernatant in each individual treatment was collected to measure IL-6,NO,INF-γ,and TNF-α levels. The IL-6,NO,INF-γ,and TNF-α concentrations were assayed according to the cytokine ELISA protocol following the instructions of the manufacturer (mouse DuoSet ELISA Development system,R&D Systems,Minneapolis,MN). The sensitivity of these cytokine assays was<15.6 pg·mL-1.Measurement of mRNA expressions of IL-6,iNOS,INF-γ,and TNF-α by qRT-PCR
The RAW264.7 cells were treated with PN-S for 48 h. The cells were collected Total RNA was extracted using the RNeasy Mini-Kit according to the manufacturer’s protocol. First-strand cDNA was synthesized using the PrimeScript 1st Strand cDNA Synthesis Kit,with the Oligo dT-Adaptor Primer. Gene expression was monitored by Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR),carried out using the SYBR Green PCR master mix. Primers for IL-6,iNOS,INF-γ,TNF-α,and actin genes are listed in Table1. qRT-PCR was performed using the ABI step one plus system (Mx3000P,Agilent,USA)followed by melting curve analysis with the following cycling program: initial activation at 95 °C for 10 min,followed by 40 cycles of denaturation at 95 °C for 30 s,annealing at 55 °C for 1 min,and extension at 72 °C for 1 min.
Each experiment was repeated at least three times. Numerical data are presented as mean ± SEM. The differences among different treatments were analyzed using one-way ANOVA. All statistical analyses were performed by using SPSS 17.0 software (Chicago,IL,USA). P<0.05,P<0.01 were considered statistically significant.Results Cell viability
The effects of PN-S at various concentrations on the viability of RAW264.7 macrophages and U973 cells are shown in Fig.1A and Fig.1B,respectively. In a dose-dependent manner (5-40 μg·mL-1),PN-S promoted cell proliferation in both RAW264.7 and U973 cells. However,RAW264.7 cells treated with 80 μg·mL-1 PN-S showed no statistical difference in cell viability. Compared with the control cells PN-S In addition,PN-S showed cytotoxicity at 160 μg·mL-1 (Fig.1A) Similar results were obtained in U973 cells treated with 80 and 160 μg·mL-1 of PN-S. Therefore,PN-S treatments at 10,20,and 40 μg·mL-1 for 48 h were selected in subsequent cytokine secretion assays to avoid its cytotoxicity.
The pinocytic activity of macrophages triggered by PN-S was examined by measuring the uptake of neutral red (0.1%). Compared with the control group,the pinocytic activity of the macrophages treated with PN-S at 10 μg·mL-1 for 48 h was enhanced significantly (P<0.05). The macrophages treated with 20 and 40 μg·mL-1 of PN-S exhibited greater phagocytic activity (P<0.01,Fig.2).
Representative micrographs of the macrophages observed through SEM are shown in Fig.3. The typical morphology of a normal macrophage is shown in Fig.3A,which shows smooth surfaces. However,the cells treated with 20 μg·mL-1 of PN-S visibly increased in size and became irregular in shape,compared with the normal macrophages. Moreover,the PN-S-treated macrophages became more adherent to the plastic culture dishes with several microvilli-like structures (Fig.3C) In the 10 and 40 μg·mL-1 PN-S treated groups (Figs. 3B and 3D,respectively),although the cells were larger than normal cells,the surface folds and microvilli-like structures were fewer than that in 20 μg·mL-1 PN-S-treated cells.
In order to quantitatively evaluate the PN-S effects on cell proliferation,flow cytometry analysis was carried out with PI staining. Table2 illustrates the percentages of cells in G0/G1,S,and G2/M phase,respectively,as calculated using Multi-cycle software. In a concentrationdependent manner,PN-S increased the proportion of cells at G2/M phases (from 11.89% to 25.04%) and cells in the S phase (from 15.77% to 24.92 decreased of the percentage of cells at G0/G1 phase (from 72.34% to 43.07%). The percentages of apoptotic cells in PN-S treated cells were not significantly different from that of the control cells (Table2).
The RAW264.7 macrophages were treated with PN-S at the indicated non-cytotoxic concentrations for 48 h and the cytokine secretions,including IL-6,NO,INF-γ,and TNF-α,were determined. The results indicated that cytokines increased dramatically in the cells treated with 20 and 40 μg·mL-1 PN-S (Table3). Equal change of cytokine secretion in cell culture supernatant was displayed in the 20 and 40 μg·mL-1 PN-S. Cytokine changes were also observed at 10 μg·mL-1 PN-S,except for INF-γ,compared with normal control.
The effects of PN-S on the expressions of macrophage cytokines measured by qRT-PCR are shown in Fig.4 As shown in Fig.4A,the levels of IL-6 mRNA in RAW264.7 treated with a different concentrations of PN-S were significantly increases,compared with that of the normal control (P<0.05,P<0.01,and P<0.05). Significant increases in mRNA levels of iNOS were observed in RAW264.7 macrophage treated with PN-S,compared with normal control (P<0.05,P<0.01,and P<0.01; Fig.4B). The increases in the mRNA levels of INF-γ were also concentration-dependent (Fig.4C). As shown in Fig.4D,PN-S treatment increased the mRNA level of TFN-α significantly,compared with that of controls (P<0.05,P<0.01,and P < 0.01).
As phagocytic cells,macrophages are widely distributed throughout the body and are important in host defense. The phagocytotic function of phagocytes is an important indicator of the body’s immune competence . In the present study,The morphology of the PN-S-treated cells displayed characteristics of activated macrophages,indicating that macrophages were activated by PN-S. We also found increased expression of IL-6,cytokine immune system signaling molecule . As one of the most important endogenous mediators of immunity,iNOS plays a critical role in normal immune functions,including macrophage activation,and host defense against intracellular pathogens,among others [15, 16]. IFN-γ,originally called macrophageactivating factor,is secreted by Th1 cells,dendritic cells and NK cells. Also known as immune interferon,IFN-γ is the only Type II interferon that is serologically distinct from Type I interferons,and it is acid-labile,while the type I variants are acid-stable. IFN-γ has antiviral,immunoregulatory,and anti-tumor properties [17, 18, 19]. A growing body of evidence suggests that locally produced cytokines,such as TNF-α,have a critical role in various physiological and pathological processes,including immune and inflammatory responses [20, 21]. In the present study,we found that PN-S induced proliferation was accompanied by cell cycle dominated in S and G2/M phase in macrophage cells treated with PN-S. This coincided with the observation that PN-S might acted as an accelerator that controlled entry into the G2/M phase and promoted the replication of DNA and the synthesis of RNA and protein. Our experimental data also illustrated the effects of PN-S on the mRNA and protein levels of cytokines in RAW264.7 cells.
Taken together,our study documented that PN-S effectively modulated phagocytosis levels and enhanced immune activity of murine peritoneal macrophages. We anticipate that PN-S treatment will be beneficial to immunity-related diseases via regulation of cytokine production.
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