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Cite this: DOI: 10.1039/c4fo01192a
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Lentinula edodes-derived polysaccharide enhances systemic and mucosal immunity by spatial modulation of intestinal gene expression in mice Xiaofei Xu,a,b Jiguo Yang,a Zhen Luoa and Xuewu Zhang*a Mushroom polysaccharides have been reported to possess significant biological activities. However, their molecular mechanisms are not fully elucidated. In this study, the immunostimulating activity of a newly purified heteropolysaccharide L2 from Lentinula edodes is evaluated in Caco-2 cells and a Caco-2/ RAW264.7 co-culture system, as well as in mice. Subsequently, the customized RT-PCR array containing 112 genes is employed to investigate the effects of L2 on gene expressions in the small intestine, cecum and colon. The results show that L2 significantly enhances immune responses by differentially affecting
Received 25th December 2014, Accepted 18th May 2015 DOI: 10.1039/c4fo01192a www.rsc.org/foodfunction
the gene expressions of small intestine, cecum and colon, in which 55, 26 and 25 genes are markedly changed, respectively. In particular, 3 core regulation networks are identified for various parts of gut. These data demonstrate the potential of L2 as a potent immune stimulator and for the first time provide a detailed landscape of tissue-specific gene expressions and core regulation networks in response to L. edodes-derived heteropolysaccharide treatment.
1 Introduction Natural ingredients used in the prevention or treatment of disease have become a more prominent element in functional food marketing. β-glucan are well-known biological response modifiers that have been intensively investigated for several decades.1 β-glucan are derived from different sources (e.g. yeast, mushrooms, oat, barley, seaweeds and bacteria) and have been reported to possess significant biological activities such as antibacterial activity,2 antifungal activity,3 antiviral activity,4 immunomodulation,5 anticancer activity,6 blood cholesterol- and glucose-lowering activities.7,8 β-glucans in mushrooms are important biologically active substances for their widespread availability on Earth and long history of use in medicine and food of East Asia.9 L. edodes is the first medical mushroom and the second most popular edible mushroom in the world.10 Lentinan is a typical β-glucan from L. edodes with β-(1,3) glycosidic linkage in the main chain,11 which have been widely studied since 1970.12 However, its oral bioavailability is reportedly limited due to its large molecular weight.13 Except for lentinan, heteropolysaccharides in the L. edodes are also obtained with different extraction methods,
a College of Light Industry and Food Sciences, South China University of Technology, Guangzhou, China. E-mail: [email protected] b Treerly Women’s Nutrition and Health Institute, Guangzhou, China
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to date.14–16 However, the immunomodulating properties of these heteropolysaccharides have seldom been studied in vivo. In our previous study,16 a new heteropolysaccharide, L2 (26 kDa), was isolated from the fruiting body of L. edodes. Chemical characterization indicated that L2 consists of glucose (87.5%), galactose (9.6%), and arabinose (2.8%). Notably, L2 does not possess a triple-helix structure, while such a structure is considered to be important to lentinan for bioactivity.17 In addition, L2 demonstrates the ability to alter the composition of gut microbiota along the intestinal tract after oral administration.18 Interestingly, gut microbiota are found to modulate the expression of a large set of genes along the intestinal tracts of mice.19 Thus, for facilitating the application of L2 in functional food, it is necessary to determine the immunomodulating properties of an L2 in vitro and in vivo gut models. Furthermore, the responses of host intestinal gene expression induced by the oral administration of L2 are also needed to better understand the immunomodulating molecular mechanism of L2. In this investigation, the cytokine responses of intestinal epithelial-like Caco-2 cells to L. edodesderived L2 as an in vitro model for analyzing direct interactions between L2 and intestinal mucosa of the body were first examined. Then, animal models were applied to assess local and systematic effects in response to L2. In particular, the cellular and humoral immunoresponses were assessed. Subsequently, the customized RT-PCR array involved in 112 genes was employed to analyze the variations of gene expressions in the
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small intestine, cecum and colon of mice, and the intestinal tissue-specific regulation networks were also identified.
later, L2 (500 μg ml−1) or Zymonsan A (400 μg ml−1) was added. After co-culturing for additional 24 hours, cytokines in the basal medium were assayed by ELISA method.
2
Experimental
2.4
2.1
Reagents
L. edodes-derived polysaccharide L2 was prepared as previously described.16 RPMI 1640 medium, Dulbecco’s modified Eagle’s medium (DMEM, glutamine, high glucose) and fetal calf serum were purchased from Gibco (Invitrogen Corporation, USA). YAC-1 cells were purchased from Sinovac Biotech Co., Ltd. (Shanghai, China). Trypan blue (T6146), 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, M2128) were purchased from Sigma-Aldrich (St. Louis, Mo., USA). Human TNF-α, IFN-γ, IL-6, IL-8, IL-10, IL-12, and IL-17; mouse TNF-α, IFN-γ, IL-1α, IL-2, IL-6, and IL-12; and secretary IgA (SIgA) enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D Systems (USA). All chemical reagents used in this study were of analytical grade. 2.2 Establishment of simulated in vitro small-intestine model (Caco-2 model) Human intestinal epithelial cells (Caco-2) were cultured on Transwell insert plates (4.67 cm2, 0.4 μm pore size, Corning CoStar Corp., Cambridge, MA). The medium was MEM (Eagle’s Minimum Essential Medium), supplemented with 1% MEM nonessential amino acids (NEAA), 100 μg ml−1 streptomycin, 10% fetal bovine serum (FBS), 2 mmol L−1 L-glutamine, and 10 mmol L−1 Hepes. Cell cultures were incubated in a humidified 5% CO2 incubator at 37 °C. On day 1, 5, 10 and 15, morphology was observed by optical microscopy, and sodium fluorescein and alkaline phosphatase activities were assayed. 2.3 Cytokines secretions in Caco-2 cells or Caco-2/RAW264.7 co-culture system Caco-2 cells were seeded at 1 × 106 cells per well onto Transwell insert plates, and the cell cultures were described as previously mentioned. At day 15, Transwell insert plates were added by various concentrations of L2 (500 μg ml−1, 250 μg ml−1, and 125 μg ml−1), Zymonsan A (Sigma-Aldrich; 400 μg ml−1, 200 μg ml−1, and 100 μg ml−1), and LPS (Sigma-Aldrich) (50 pg ml−1), in the apical side. After 24 h of co-culture, cytokines in the basal medium were assayed by the ELISA method. In the case of LPS stimulation, the cell cultures were the same as mentioned above. At day 15, LPS (50 pg ml−1) was added into the apical side of the Transwell insert plates, and 1 h later, L2 (500 μg ml−1, 250 μg ml−1, and 125 μg ml−1) or Zymonsan A (400 μg ml−1, 200 μg ml−1, and 100 μg ml−1) was added. After 24 h of co-culture, cytokines in the basal medium were assayed by the ELISA method. For the Caco-2/RAW264.7 co-culture system, the cell cultures were the same as mentioned above. At day 15, murine RAW264.7 cells were seeded at 1 × 105 cells per well onto the basal side of the Transwell insert plates. After 24 h of coculture, LPS (50 pg ml−1) was added in the apical side, and 1 h
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Animal experiments
All experiments were performed in compliance with the relevant laws and were approved by the Animal Care Welfare Committee of Guangzhou University of Chinese Medicine. Fourteen 8-week-old specific pathogen-free male C57BL/6 mice were divided into two groups (normal and L2 groups, n = 7). Gavage administration of L2 40 mg kg−1 (body weight) to L2 groups for 28 consecutive days was carried out. Detailed information of animal experiments is described in another study.18 On the last day, mice were weighed and peripheral blood samples were taken and stored at 4 °C for further analysis. Subsequently, mice were sacrificed by cervical dislocation, and spleen and thymus were immediately removed in a sterile environment and weighed. The small intestine, cecum and colon tissues were sterilely collected separately and immediately stored at −80 °C for further analysis. 2.5
Immune activity assays in mice
Splenocytes were obtained by a gentle disruption of the spleen and placed in cold phosphate-buffered saline (PBS) and filtered through a 200-mesh sieve to obtain single-cell suspension as previously described on the day of sacrifice.20 After treatment with erythrocyte lysis buffer, the cells were resuspended at a final density of 3 × 106 cells per ml in a RPMI-1640 medium supplemented with 10% fetal calf serum, 100 U per ml streptomycin and 100 U per ml penicillin. The co-mitogenic activities were assayed using a slight modification of a previously described method.21 Splenocytes were prepared according to a method mentioned above. The cell suspension (1 ml) was placed in each well of a 24-well flatbottomed microplate with or without 75 μl ConA (7.5 μg ml−1). It was then cultured for 72 h at 37 °C in a humidified 5% CO2 atmosphere and then further incubated for 4 h with 50 μl of 5 mg ml−1 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT, Sigma, USA) per well. DMSO (150 μl) was added to the culture to dissolve the colored material, and the absorbance at 570 nm was measured with an ELISA reader (Bio-Rad Model 6800, USA). The splenic natural killer (NK) cells were those of effector and YAC-1 and were used as the target cells. The assay was carried out according to the previously described method.22 The NK activity of effector cells was calculated as cytotoxicity by the following formula: cytotoxicity (%) = (A + B − C)/A × 100%, where A is the absorbance of the well of target cells, B is the absorbance of the well of effector cells, C is the absorbance of the experimental well. The blood samples were centrifuged at 1000g and 4 °C for 20 min, and the upper layer contained the serum. Small-intestinal contents were suspended with 1 ml of sterile PBS and then centrifuged at 1000g and 4 °C for 20 min. The amount of TNF-α, IFN-γ, IL-1α, IL-2, IL-6, IL-12 in serum and SIgA in the supernatant of small-intestinal contents were analyzed by the
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mouse TNF-α, IFN-γ, IL-1α, IL-2, IL-6, IL-12, and SIgA ELISA kits, respectively, according to the manufacturer’s instructions.
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2.6
gene or significant expressions ( p < 0.05) in various expressed genes based on ANOVA analysis were considered as the up- or down-regulation of a specific gene expression.
Total RNA extraction and RT-PCR array analysis
Small intestine, cecum, and colon tissues of three mice from each group were randomly selected. The TRIZOL (Invitrogen, Carlsbad, Calif., USA) method was used to isolate total RNAs from small intestine, cecum and colon tissues. The quality of isolated RNA was evaluated using RT2 RNA QC PCR arrays (Qiagen) according to the manufacturer’s instructions. Reverse transcription of RNA to cDNA was performed using an RT-PCR kit (catalog#CTB101; Chutian Biosciences, China) on the ABI 9700 thermocycler (ABI, Foster City, CA) according to manufacturer’s instructions, and additional removal of genomic DNA from the RNA sample and a specific control of reverse transcription were also included. RT-PCR arrays were performed in 384-well plates with a LightCycler 480 (Roche Diagnostics, Mannheim, Germany) using SYBR MasterMix (catalog#CTB103; Chutian Biosciences, China). Each PCR contained 10 ng of synthesized cDNA. Self-selected 112 genes (Table 1) were simultaneously amplified in the sample. Amplification was carried out at an initial denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s and extension at 72 °C for 20 s. Relative changes in gene expressions were calculated using a ΔΔCt (threshold cycle) method. Housekeeping genes, such as B2M, ACTB, GAPDH, RPL27, HPRT1 and OAZ1, were used to normalize the amount of RNA. Fold-change values were calculated using the formula of 2-ΔΔCt. More than twofold changes in
Table 1
2.7
Network analysis of differentially expressed genes
R spider is employed to implement a network-based analysis of differentially expressed genes by the union of signaling and metabolic pathways from Reactome and KEGG knowledge databases.23 2.8
Statistical analysis
The Student’s t-test was performed using SPSS. P-values < 0.05 were considered significant unless otherwise stated.
3
Results
3.1 Effects of L. edodes-derived polysaccharide L2 on secretion of cytokines in vitro The morphologies of Caco-2 cells cultured on Transwell insert plates were observed by optical microscopy at days 1, 5, and 15. The results displayed that Caco-2 cells were uniformly distributed and densely arranged with a monolayer and clear borders after 15 days of culture (Fig. 1). In fact, a sodium fluorescein assay indicated that the permeability coefficient was smaller than 0.5 × 10−6 cm s−1 after 15 days, revealing dense cell layers. The activity of alkaline phosphatase increased with time, i.e. 0.093, 0.255, 0.812 and 0.527 U g−1 at days 1, 5, 10 and 15, respectively, which meant that the polarization of cells
Self-selected and housekeeping genes for PCR array
Gene category (numbers)
Gene names
Chemokines (7) Barrier function (7) Antibacterial genes (6) Interferons (4) Interleukins (12) TNF Superfamily (6) Innate immunity Pattern Recognition Receptors (16)
cxcl1, cxcl11, Ccl5, Cxcl9, Csf1, Ccl12, Csf2 (GM-CSF) Sprr1a, Sprr1b, Muc2, Muc4, Muc13, Lamb1, Lama1 Nox1, Nos2, Duox2, Duoxa2, Reg3γ, Reg3β Ifna2, Ifna4, Ifnb1, Ifng Il10, Il12b, Il17a, Il17b, Il17c, Il1a, Il1b, Il2, Il23a, Il4, Il6, Il22 Tnfrsf1a, Tnfrsf13c, Lta (Tnfb), Tnfa, Tnfsf15, Tnfsf4
Cellular Immunity Th1 Markers/Immune Response (3) Th2 Markers/Immune Response (3) Th17 Markers (1) Treg Markers (1) Regulators of T-Cell Activation (5) T-Cell Proliferation (6) Antigen Dependent B-Cell Activation (2) B-Cell Proliferation (3) Macrophage Activation (1) Cell Function Regulators (3) Natural Killer Cell Activation (2) TLR Signaling TICAM1 (TRIF) – Dependent (MYD88 – Independent) (5) MYD88-Dependent (5) NF-κB Pathway (11) JNK/p38 Pathway (5) Reference genes (6)
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Muc13, Clec7a (Dectin-1), Mct-1, Tlr1, Tlr2, Tlr3, Tlr4, Tlr5, Tlr6, Tlr7, Tlr8, Tlr9, Gpr40, Gpr41, Gpr42, Gpr43 Ccr5,Cd80, Stat4 Bcl6, Cd86, Stat6 Stat3 Foxp3 cb1b, Dpp4, Foxp3, Icam1, Icosl, Lag3, Prkcq, Thy1 Casp3, Cd3e, Ctla4, Tnfrsf13c, Tnfsf13b, Tnfsf14 Cd28, Cd4 Bcl2, Cd38, Cd81,Tnsf5 Csf1 (MCSF) Bcl3, Malt1, Bcl10 Cd2, H60a Irf3, Tbk1, PeIi1, Traf6, Ticam1 (Trif) Irak1, Irak2, Tak1, Myd88, Tirap Casp8, Chuk (Ikka), Ikbkb, Irak1, Irak2, Map3k1, Nfkb1, Nfkb2, Nfkbia, Nfkbib, Nfrkb, Rela Fos, Jun, Map2k4, Mapk8, Mapk9 B2 m, Actb, Gapdh, Oazl, Rpl27, Hprt1
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Fig. 1
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Morphological observations of Caco-2 cells at days 1, 5, and 15.
occurred. This demonstrated the successful establishment of a simulated in vitro small-intestinal model. Subsequently, the effects of L2 under various concentrations (125, 250 and 500 μg ml−1) on the secretions of cytokines were determined in the Caco-2 cell model. Fig. 2A showed that L2 significantly facilitated the secretions of proinflammatory cytokines (INF-γ, TNF-α, IL-8 and IL-12) and anti-inflammatory cytokine IL-10 ( p < 0.01), compared with the control. L2 also significantly enhanced the production of IL-17 at high concentrations (250 and 500 μg ml−1) ( p < 0.01), and pro- or anti-inflammatory cytokine IL-6 at a concentration of 500 μg ml−1 ( p < 0.05). As a positive control, the effects of Zymosan A under various concentrations (100, 200 and 400 μg ml−1) on the secretions of cytokines in a Caco-2 cell model were determined too. Fig. 2B indicated that Zymosan A also significantly enhanced the productions of pro-inflammatory cytokines (INF-γ, TNF-α, IL-8 and IL-12; p < 0.05 or 0.01); however, the secretion of IL-12 was decreased with the increased concentration. Moreover, at low concentrations, Zymosan A significantly facilitated the secretions of IL-10 (100 and 200 μg ml−1) and IL-17 (100 μg ml−1) ( p < 0.01), but the production of IL-6 was significantly increased at high concentrations (200 and 400 μg ml−1; p < 0.05). Thus, the responsive variations of cytokines to L2 and Zymosan A were primarily in IL-10 and IL-17, respectively. In LPS-stimulated Caco-2 cells, Fig. 2C showed that LPS (50 pg ml−1) significantly enhanced the secretion of 6 cytokines (TNF-α, IL-6, IL-8, IL-10, IL-12, and INF-γ; p < 0.01) except for IL-17, compared with the control. Under the stimulation of LPS, L2 (500 μg ml−1) significantly stimulated the productions of IL-17 and INF-γ ( p < 0.01), but significantly inhibited the secretions of IL-6 and IL-12 ( p < 0.05), relative to LPS. Similarly, under the stimulation of LPS, Zymosan A (400 μg ml−1) significantly stimulated the secretion of INF-γ and IL-17 but inhibited the production of IL-6 ( p < 0.01), compared with LPS. The differential effects of L2 and Zymosan A lied in the inhibiting degree of secretion of IL-12 in the case of LPS stimulation. For the LPS-stimulated Caco-2/RAW264.7 co-culture system, Fig. 2D displayed that LPS (50 pg ml−1) significantly stimulated the secretion of 4 cytokines (INF-γ, IL-8, IL-10, and IL-12) ( p < 0.05 or 0.01), compared with the control. L2 (500 μg ml−1) significantly enhanced the productions of 4 cytokines (IL-6, IL-12,
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IL-17, and INF-γ; p < 0.05 or 0.01), relative to LPS. Zymosan A (400 μg ml−1) significantly stimulated the productions of 3 cytokines (IL-6, IL-12, and INF-γ; p < 0.05 or 0.01), but inhibited the secretion of IL-10 ( p < 0.01) and IL-17 ( p < 0.05), compared with LPS. At this time, the differential effects of L2 and Zymosan A lied in IL-10 and IL-17. 3.2 Effects of L. edodes-derived polysaccharide L2 on the activity of immune cells and the secretion of cytokines in serum in vivo Basically, all mice displayed no significant variations in food intake, body weight gain and activities of daily living. The average food intake per mouse was 5.09 g per day and 5.99 g per day for normal and L2-treated groups, respectively. Compared with normal mice, the spleen and thymus indices were significantly ( p < 0.01) increased in L2-treated mice, and the natural killer cell (NK cell) activities and the proliferation of T cell of splenocytes in the L2-treated groups were also significantly elevated (P < 0.05; Table 2). Fig. 3 showed that L2 significantly increased the productions of six cytokines (TNF-α, IFN-γ, IL-1α, IL-2, IL-6, and IL-12) in serum, although SIgAs in small-intestinal contents were similar, compared with normal groups. These results revealed that L. edodes-derived polysaccharide L2 indeed enhanced the cellular and humoral immunities of mice. 3.3 Effects of L. edodes-derived polysaccharide L2 on gene expression in mucosa of mice Among 112 immune-related genes examined herein, 78 genes exhibited significant changes (>2-fold or p < 0.05). The results showed various impact patterns of L2 treatment in the mucosa of mice. In particular, L2 exhibited the greatest effects on the genes of the small intestine, a total of 55 (49.1% of 112 genes), including 41 up-regulated and 14 down-regulated genes. There were 26 genes (23.2% of 112 genes) significantly changed in cecum, including 9 up-regulated and 17 down-regulated genes. In the colon, 25 genes (22.3% of 112 genes) were significantly altered, including 8 up-regulated and 17 down-regulated genes. It means that the major acting site of L2 was the small intestine. In addition, L2 primarily stimulated overexpression in the small intestine and under-expression in the cecum and colon. For chemokine-related genes (Table 3), all of the varied genes were up-regulated, 4 genes (Cxcl9, Cxcl11, Csf1 and
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Fig. 2 Secretion of cytokines: (A) In Caco-2 cells under different concentrations of L2 (125, 250 and 500 μg ml−1); *p < 0.05, **p < 0.01 vs. control; (B) in Caco-2 cells under various concentrations of Zymosan A (100, 200 and 400 μg ml−1); *p < 0.05, **p < 0.01 vs. control; (C) in Caco-2 cells prostimulated by LPS (50 pg ml−1) under concentrations of L2 (500 μg ml−1) and Zymosan (400 μg ml−1); *p < 0.05, **p < 0.01 vs. LPS. bp < 0.01 vs. control. (D) In co-cultured Caco-2/RAW264.7 cells pro-stimulated by LPS (50 pg ml−1) under concentrations of L2 (500 μg ml−1) and Zymosan (400 μg ml−1); *p < 0.05, **p6)-glucans, Mycol. Res., 2007, 111(Pt 6), 635–652. 5 E. A. Murphy, J. M. Davis and M. D. Carmichael, Immune modulating effects of β-glucan, Curr. Opin. Clin. Nutr. Metab. Care, 2010, 13(6), 656–661. 6 S. H. Albeituni and J. Yan, The effects of β-glucans on dendritic cells and implications for cancer therapy, Anticancer Agents Med. Chem., 2013, 13(5), 689–698. 7 R. Nicolosi, S. J. Bell, B. R. Bistrian, I. Greenberg, et al., Plasma lipid changes after supplementation with betaglucan fiber from yeast, Am. J. Clin. Nutr., 1999, 70(2), 208– 212. 8 H. C. Lo, F. A. Tsai, S. P. Wasser, J. G. Yang, et al., Effects of ingested fruiting bodies, submerged culture biomass, and acidic polysaccharide glucuronoxylomannan of Tremella mesenterica Retz.:Fr. on glycemic responses in normal and diabetic rats, Life Sci., 2006, 78(17), 1957–1966. 9 S. P. Wasser, Medicinal mushrooms as a source of antitumor and immunomodulating polysaccharides, Appl. Microbiol. Biotechnol., 2002, 60, 258–274. 10 P. S. Bisen, R. K. Baghel, B. S. Sanodiya, G. S. Thakur, et al., Lentinus edodes: A Macrofungus with Pharmacological Activities, Curr. Med. Chem., 2010, 22, 2419–2430. 11 G. Chihara, J. Hamuro, Y. Y. Maeda, Y. Arai, et al., Fractionation and purification of the polysaccharides with marked antitumor activity, especially lentinan, from Lentinus edodes (Berk.) Sing. (an edible mushroom), Cancer Res., 1970, 30, 2776–2781. 12 Y. Y. Zhang, S. Li, X. H. Wang, L. N. Zhang, et al., Advances in Lentinan: Isolation, structure, chain conformation and bioactivities, Food Hydrocolloids, 2011, 25(2), 196–206. 13 P. M. Kidd, The use of mushroom glucans and proteoglycans in cancer treatment, Altern. Med. Rev., 2000, 5(1), 4–27. 14 E. R. Carbonero, A. H. P. Gracher, D. L. Komura, R. Marcon, et al., Lentinus edodes heterogalactan: Antinociceptive and anti-inflammatory effects, Food Chem., 2008, 111, 531–537. 15 H. Fu, W. Y. Guo, H. Yin, Z. X. Wang, et al., Inhibition of Lentinus edodes polysaccharides against liver tumour growth, Int. J. Phys. Sci., 2011, 6(1), 116–120. 16 X. F. Xu, H. D. Yan and X. W. Zhang, Structure and Immuno-Stimulating Activities of a New Heteropoly-
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experimental drugs in oncology based on patient survival information, Cell. Death. Dis., 2014, 5, e1051. J. Hou, J. Aerts, B. den Hamer, W. van Ijcken, et al., Gene expression-based classification of non-small cell lung carcinomas and survival prediction, PLoS One, 2010, 5(4), e10312. Y. Pawitan, J. Bjöhle, L. Amler, A. L. Borg, et al., Gene expression profiling spares early breast cancer patients from adjuvant therapy: derived and validated in two population-based cohorts, Breast Cancer Res. Treat., 2005, 7(6), R953–R964. Z. Hu, C. Fan, C. Livasy, X. He, et al., A compact VEGF signature associated with distant metastases and poor outcomes, BMC Med., 2009, 7, 9. S. Pradervand, H. Yasukawa, O. G. Muller, H. Kjekshus, et al., Small proline-rich protein 1A is a gp130 pathwayand stress-inducible cardioprotective protein, EMBO J., 2004, 23(22), 4517–4525. Y. Kim, S. H. Kim, K. Y. Whang, Y. J. Kim, et al., Inhibition of Escherichia coli O157:H7 attachment by interactions between lactic acid bacteria and intestinal epithelial cells, J. Microbiol. Biotechnol., 2008, 18(7), 1278–1285. R. Prakash, S. Bharathi Raja, H. Devaraj and S. N. Devaraj, Up-regulation of MUC2 and IL-1β expression in human colonic epithelial cells by Shigella and its interaction with mucins, PLoS One, 2011, 6(11), e27046. R. C. Rickert, J. Jellusova and A. V. Miletic, Signaling by the tumor necrosis factor receptor superfamily in B-cell biology and disease, Immunol. Rev., 2011, 244(1), 115–133. K. Warnatz, U. Salzer, M. Rizzi, B. Fischer, et al., B-cell activating factor receptor deficiency is associated with an adult-onset antibody deficiency syndrome in humans, Proc. Natl. Acad. Sci. U. S. A., 2009, 106(33), 13945–13950. S. S. Kim, D. P. Richman, S. S. Zamvil and M. A. Agius, Accelerated central nervous system autoimmunity in BAFFreceptor-deficient mice, J. Neurol. Sci., 2011, 306(1–2), 9–15. I. Moisini and A. Davidson, BAFF: a local and systemic target in autoimmune diseases, Clin. Exp. Immunol., 2009, 158(2), 155–163. S. T. Jeon, W. J. Kim, S. M. Lee, M. Y. Lee, et al., Reverse signaling through BAFF differentially regulates the expression of inflammatory mediators and cytoskeletal movements in THP-1 cells, Immunol. Cell. Biol., 2010, 88(2), 148–156. S. M. Lee, E. J. Kim, K. Suk and W. H. Lee, BAFF and APRIL induce inflammatory activation of THP-1 cells through interaction with their conventional receptors and activation of MAPK and NF-κB, Inflammation Res., 2011, 60(9), 807– 815. A. P. Sage, D. Tsiantoulas, L. Baker, J. Harrison, et al., BAFF receptor deficiency reduces the development of atherosclerosis in mice–brief report, Arterioscler., Thromb., Vasc. Biol., 2012, 32(7), 1573–1576. A. Gewirtz, Gut microbiota, low-grade inflammation, and metabolic syndrome, FASEB J., 2015, 29(1 suppl.), 368.2.
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Cite this: DOI: 10.1039/c4fo01192a
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Lentinula edodes-derived polysaccharide enhances systemic and mucosal immunity by spatial modulation of intestinal gene expression in mice Xiaofei Xu,a,b Jiguo Yang,a Zhen Luoa and Xuewu Zhang*a Mushroom polysaccharides have been reported to possess significant biological activities. However, their molecular mechanisms are not fully elucidated. In this study, the immunostimulating activity of a newly purified heteropolysaccharide L2 from Lentinula edodes is evaluated in Caco-2 cells and a Caco-2/ RAW264.7 co-culture system, as well as in mice. Subsequently, the customized RT-PCR array containing 112 genes is employed to investigate the effects of L2 on gene expressions in the small intestine, cecum and colon. The results show that L2 significantly enhances immune responses by differentially affecting
Received 25th December 2014, Accepted 18th May 2015 DOI: 10.1039/c4fo01192a www.rsc.org/foodfunction
the gene expressions of small intestine, cecum and colon, in which 55, 26 and 25 genes are markedly changed, respectively. In particular, 3 core regulation networks are identified for various parts of gut. These data demonstrate the potential of L2 as a potent immune stimulator and for the first time provide a detailed landscape of tissue-specific gene expressions and core regulation networks in response to L. edodes-derived heteropolysaccharide treatment.
1 Introduction Natural ingredients used in the prevention or treatment of disease have become a more prominent element in functional food marketing. β-glucan are well-known biological response modifiers that have been intensively investigated for several decades.1 β-glucan are derived from different sources (e.g. yeast, mushrooms, oat, barley, seaweeds and bacteria) and have been reported to possess significant biological activities such as antibacterial activity,2 antifungal activity,3 antiviral activity,4 immunomodulation,5 anticancer activity,6 blood cholesterol- and glucose-lowering activities.7,8 β-glucans in mushrooms are important biologically active substances for their widespread availability on Earth and long history of use in medicine and food of East Asia.9 L. edodes is the first medical mushroom and the second most popular edible mushroom in the world.10 Lentinan is a typical β-glucan from L. edodes with β-(1,3) glycosidic linkage in the main chain,11 which have been widely studied since 1970.12 However, its oral bioavailability is reportedly limited due to its large molecular weight.13 Except for lentinan, heteropolysaccharides in the L. edodes are also obtained with different extraction methods,
a College of Light Industry and Food Sciences, South China University of Technology, Guangzhou, China. E-mail: [email protected] b Treerly Women’s Nutrition and Health Institute, Guangzhou, China
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to date.14–16 However, the immunomodulating properties of these heteropolysaccharides have seldom been studied in vivo. In our previous study,16 a new heteropolysaccharide, L2 (26 kDa), was isolated from the fruiting body of L. edodes. Chemical characterization indicated that L2 consists of glucose (87.5%), galactose (9.6%), and arabinose (2.8%). Notably, L2 does not possess a triple-helix structure, while such a structure is considered to be important to lentinan for bioactivity.17 In addition, L2 demonstrates the ability to alter the composition of gut microbiota along the intestinal tract after oral administration.18 Interestingly, gut microbiota are found to modulate the expression of a large set of genes along the intestinal tracts of mice.19 Thus, for facilitating the application of L2 in functional food, it is necessary to determine the immunomodulating properties of an L2 in vitro and in vivo gut models. Furthermore, the responses of host intestinal gene expression induced by the oral administration of L2 are also needed to better understand the immunomodulating molecular mechanism of L2. In this investigation, the cytokine responses of intestinal epithelial-like Caco-2 cells to L. edodesderived L2 as an in vitro model for analyzing direct interactions between L2 and intestinal mucosa of the body were first examined. Then, animal models were applied to assess local and systematic effects in response to L2. In particular, the cellular and humoral immunoresponses were assessed. Subsequently, the customized RT-PCR array involved in 112 genes was employed to analyze the variations of gene expressions in the
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small intestine, cecum and colon of mice, and the intestinal tissue-specific regulation networks were also identified.
later, L2 (500 μg ml−1) or Zymonsan A (400 μg ml−1) was added. After co-culturing for additional 24 hours, cytokines in the basal medium were assayed by ELISA method.
2
Experimental
2.4
2.1
Reagents
L. edodes-derived polysaccharide L2 was prepared as previously described.16 RPMI 1640 medium, Dulbecco’s modified Eagle’s medium (DMEM, glutamine, high glucose) and fetal calf serum were purchased from Gibco (Invitrogen Corporation, USA). YAC-1 cells were purchased from Sinovac Biotech Co., Ltd. (Shanghai, China). Trypan blue (T6146), 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, M2128) were purchased from Sigma-Aldrich (St. Louis, Mo., USA). Human TNF-α, IFN-γ, IL-6, IL-8, IL-10, IL-12, and IL-17; mouse TNF-α, IFN-γ, IL-1α, IL-2, IL-6, and IL-12; and secretary IgA (SIgA) enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D Systems (USA). All chemical reagents used in this study were of analytical grade. 2.2 Establishment of simulated in vitro small-intestine model (Caco-2 model) Human intestinal epithelial cells (Caco-2) were cultured on Transwell insert plates (4.67 cm2, 0.4 μm pore size, Corning CoStar Corp., Cambridge, MA). The medium was MEM (Eagle’s Minimum Essential Medium), supplemented with 1% MEM nonessential amino acids (NEAA), 100 μg ml−1 streptomycin, 10% fetal bovine serum (FBS), 2 mmol L−1 L-glutamine, and 10 mmol L−1 Hepes. Cell cultures were incubated in a humidified 5% CO2 incubator at 37 °C. On day 1, 5, 10 and 15, morphology was observed by optical microscopy, and sodium fluorescein and alkaline phosphatase activities were assayed. 2.3 Cytokines secretions in Caco-2 cells or Caco-2/RAW264.7 co-culture system Caco-2 cells were seeded at 1 × 106 cells per well onto Transwell insert plates, and the cell cultures were described as previously mentioned. At day 15, Transwell insert plates were added by various concentrations of L2 (500 μg ml−1, 250 μg ml−1, and 125 μg ml−1), Zymonsan A (Sigma-Aldrich; 400 μg ml−1, 200 μg ml−1, and 100 μg ml−1), and LPS (Sigma-Aldrich) (50 pg ml−1), in the apical side. After 24 h of co-culture, cytokines in the basal medium were assayed by the ELISA method. In the case of LPS stimulation, the cell cultures were the same as mentioned above. At day 15, LPS (50 pg ml−1) was added into the apical side of the Transwell insert plates, and 1 h later, L2 (500 μg ml−1, 250 μg ml−1, and 125 μg ml−1) or Zymonsan A (400 μg ml−1, 200 μg ml−1, and 100 μg ml−1) was added. After 24 h of co-culture, cytokines in the basal medium were assayed by the ELISA method. For the Caco-2/RAW264.7 co-culture system, the cell cultures were the same as mentioned above. At day 15, murine RAW264.7 cells were seeded at 1 × 105 cells per well onto the basal side of the Transwell insert plates. After 24 h of coculture, LPS (50 pg ml−1) was added in the apical side, and 1 h
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Animal experiments
All experiments were performed in compliance with the relevant laws and were approved by the Animal Care Welfare Committee of Guangzhou University of Chinese Medicine. Fourteen 8-week-old specific pathogen-free male C57BL/6 mice were divided into two groups (normal and L2 groups, n = 7). Gavage administration of L2 40 mg kg−1 (body weight) to L2 groups for 28 consecutive days was carried out. Detailed information of animal experiments is described in another study.18 On the last day, mice were weighed and peripheral blood samples were taken and stored at 4 °C for further analysis. Subsequently, mice were sacrificed by cervical dislocation, and spleen and thymus were immediately removed in a sterile environment and weighed. The small intestine, cecum and colon tissues were sterilely collected separately and immediately stored at −80 °C for further analysis. 2.5
Immune activity assays in mice
Splenocytes were obtained by a gentle disruption of the spleen and placed in cold phosphate-buffered saline (PBS) and filtered through a 200-mesh sieve to obtain single-cell suspension as previously described on the day of sacrifice.20 After treatment with erythrocyte lysis buffer, the cells were resuspended at a final density of 3 × 106 cells per ml in a RPMI-1640 medium supplemented with 10% fetal calf serum, 100 U per ml streptomycin and 100 U per ml penicillin. The co-mitogenic activities were assayed using a slight modification of a previously described method.21 Splenocytes were prepared according to a method mentioned above. The cell suspension (1 ml) was placed in each well of a 24-well flatbottomed microplate with or without 75 μl ConA (7.5 μg ml−1). It was then cultured for 72 h at 37 °C in a humidified 5% CO2 atmosphere and then further incubated for 4 h with 50 μl of 5 mg ml−1 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT, Sigma, USA) per well. DMSO (150 μl) was added to the culture to dissolve the colored material, and the absorbance at 570 nm was measured with an ELISA reader (Bio-Rad Model 6800, USA). The splenic natural killer (NK) cells were those of effector and YAC-1 and were used as the target cells. The assay was carried out according to the previously described method.22 The NK activity of effector cells was calculated as cytotoxicity by the following formula: cytotoxicity (%) = (A + B − C)/A × 100%, where A is the absorbance of the well of target cells, B is the absorbance of the well of effector cells, C is the absorbance of the experimental well. The blood samples were centrifuged at 1000g and 4 °C for 20 min, and the upper layer contained the serum. Small-intestinal contents were suspended with 1 ml of sterile PBS and then centrifuged at 1000g and 4 °C for 20 min. The amount of TNF-α, IFN-γ, IL-1α, IL-2, IL-6, IL-12 in serum and SIgA in the supernatant of small-intestinal contents were analyzed by the
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mouse TNF-α, IFN-γ, IL-1α, IL-2, IL-6, IL-12, and SIgA ELISA kits, respectively, according to the manufacturer’s instructions.
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2.6
gene or significant expressions ( p < 0.05) in various expressed genes based on ANOVA analysis were considered as the up- or down-regulation of a specific gene expression.
Total RNA extraction and RT-PCR array analysis
Small intestine, cecum, and colon tissues of three mice from each group were randomly selected. The TRIZOL (Invitrogen, Carlsbad, Calif., USA) method was used to isolate total RNAs from small intestine, cecum and colon tissues. The quality of isolated RNA was evaluated using RT2 RNA QC PCR arrays (Qiagen) according to the manufacturer’s instructions. Reverse transcription of RNA to cDNA was performed using an RT-PCR kit (catalog#CTB101; Chutian Biosciences, China) on the ABI 9700 thermocycler (ABI, Foster City, CA) according to manufacturer’s instructions, and additional removal of genomic DNA from the RNA sample and a specific control of reverse transcription were also included. RT-PCR arrays were performed in 384-well plates with a LightCycler 480 (Roche Diagnostics, Mannheim, Germany) using SYBR MasterMix (catalog#CTB103; Chutian Biosciences, China). Each PCR contained 10 ng of synthesized cDNA. Self-selected 112 genes (Table 1) were simultaneously amplified in the sample. Amplification was carried out at an initial denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s and extension at 72 °C for 20 s. Relative changes in gene expressions were calculated using a ΔΔCt (threshold cycle) method. Housekeeping genes, such as B2M, ACTB, GAPDH, RPL27, HPRT1 and OAZ1, were used to normalize the amount of RNA. Fold-change values were calculated using the formula of 2-ΔΔCt. More than twofold changes in
Table 1
2.7
Network analysis of differentially expressed genes
R spider is employed to implement a network-based analysis of differentially expressed genes by the union of signaling and metabolic pathways from Reactome and KEGG knowledge databases.23 2.8
Statistical analysis
The Student’s t-test was performed using SPSS. P-values < 0.05 were considered significant unless otherwise stated.
3
Results
3.1 Effects of L. edodes-derived polysaccharide L2 on secretion of cytokines in vitro The morphologies of Caco-2 cells cultured on Transwell insert plates were observed by optical microscopy at days 1, 5, and 15. The results displayed that Caco-2 cells were uniformly distributed and densely arranged with a monolayer and clear borders after 15 days of culture (Fig. 1). In fact, a sodium fluorescein assay indicated that the permeability coefficient was smaller than 0.5 × 10−6 cm s−1 after 15 days, revealing dense cell layers. The activity of alkaline phosphatase increased with time, i.e. 0.093, 0.255, 0.812 and 0.527 U g−1 at days 1, 5, 10 and 15, respectively, which meant that the polarization of cells
Self-selected and housekeeping genes for PCR array
Gene category (numbers)
Gene names
Chemokines (7) Barrier function (7) Antibacterial genes (6) Interferons (4) Interleukins (12) TNF Superfamily (6) Innate immunity Pattern Recognition Receptors (16)
cxcl1, cxcl11, Ccl5, Cxcl9, Csf1, Ccl12, Csf2 (GM-CSF) Sprr1a, Sprr1b, Muc2, Muc4, Muc13, Lamb1, Lama1 Nox1, Nos2, Duox2, Duoxa2, Reg3γ, Reg3β Ifna2, Ifna4, Ifnb1, Ifng Il10, Il12b, Il17a, Il17b, Il17c, Il1a, Il1b, Il2, Il23a, Il4, Il6, Il22 Tnfrsf1a, Tnfrsf13c, Lta (Tnfb), Tnfa, Tnfsf15, Tnfsf4
Cellular Immunity Th1 Markers/Immune Response (3) Th2 Markers/Immune Response (3) Th17 Markers (1) Treg Markers (1) Regulators of T-Cell Activation (5) T-Cell Proliferation (6) Antigen Dependent B-Cell Activation (2) B-Cell Proliferation (3) Macrophage Activation (1) Cell Function Regulators (3) Natural Killer Cell Activation (2) TLR Signaling TICAM1 (TRIF) – Dependent (MYD88 – Independent) (5) MYD88-Dependent (5) NF-κB Pathway (11) JNK/p38 Pathway (5) Reference genes (6)
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Muc13, Clec7a (Dectin-1), Mct-1, Tlr1, Tlr2, Tlr3, Tlr4, Tlr5, Tlr6, Tlr7, Tlr8, Tlr9, Gpr40, Gpr41, Gpr42, Gpr43 Ccr5,Cd80, Stat4 Bcl6, Cd86, Stat6 Stat3 Foxp3 cb1b, Dpp4, Foxp3, Icam1, Icosl, Lag3, Prkcq, Thy1 Casp3, Cd3e, Ctla4, Tnfrsf13c, Tnfsf13b, Tnfsf14 Cd28, Cd4 Bcl2, Cd38, Cd81,Tnsf5 Csf1 (MCSF) Bcl3, Malt1, Bcl10 Cd2, H60a Irf3, Tbk1, PeIi1, Traf6, Ticam1 (Trif) Irak1, Irak2, Tak1, Myd88, Tirap Casp8, Chuk (Ikka), Ikbkb, Irak1, Irak2, Map3k1, Nfkb1, Nfkb2, Nfkbia, Nfkbib, Nfrkb, Rela Fos, Jun, Map2k4, Mapk8, Mapk9 B2 m, Actb, Gapdh, Oazl, Rpl27, Hprt1
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Fig. 1
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Morphological observations of Caco-2 cells at days 1, 5, and 15.
occurred. This demonstrated the successful establishment of a simulated in vitro small-intestinal model. Subsequently, the effects of L2 under various concentrations (125, 250 and 500 μg ml−1) on the secretions of cytokines were determined in the Caco-2 cell model. Fig. 2A showed that L2 significantly facilitated the secretions of proinflammatory cytokines (INF-γ, TNF-α, IL-8 and IL-12) and anti-inflammatory cytokine IL-10 ( p < 0.01), compared with the control. L2 also significantly enhanced the production of IL-17 at high concentrations (250 and 500 μg ml−1) ( p < 0.01), and pro- or anti-inflammatory cytokine IL-6 at a concentration of 500 μg ml−1 ( p < 0.05). As a positive control, the effects of Zymosan A under various concentrations (100, 200 and 400 μg ml−1) on the secretions of cytokines in a Caco-2 cell model were determined too. Fig. 2B indicated that Zymosan A also significantly enhanced the productions of pro-inflammatory cytokines (INF-γ, TNF-α, IL-8 and IL-12; p < 0.05 or 0.01); however, the secretion of IL-12 was decreased with the increased concentration. Moreover, at low concentrations, Zymosan A significantly facilitated the secretions of IL-10 (100 and 200 μg ml−1) and IL-17 (100 μg ml−1) ( p < 0.01), but the production of IL-6 was significantly increased at high concentrations (200 and 400 μg ml−1; p < 0.05). Thus, the responsive variations of cytokines to L2 and Zymosan A were primarily in IL-10 and IL-17, respectively. In LPS-stimulated Caco-2 cells, Fig. 2C showed that LPS (50 pg ml−1) significantly enhanced the secretion of 6 cytokines (TNF-α, IL-6, IL-8, IL-10, IL-12, and INF-γ; p < 0.01) except for IL-17, compared with the control. Under the stimulation of LPS, L2 (500 μg ml−1) significantly stimulated the productions of IL-17 and INF-γ ( p < 0.01), but significantly inhibited the secretions of IL-6 and IL-12 ( p < 0.05), relative to LPS. Similarly, under the stimulation of LPS, Zymosan A (400 μg ml−1) significantly stimulated the secretion of INF-γ and IL-17 but inhibited the production of IL-6 ( p < 0.01), compared with LPS. The differential effects of L2 and Zymosan A lied in the inhibiting degree of secretion of IL-12 in the case of LPS stimulation. For the LPS-stimulated Caco-2/RAW264.7 co-culture system, Fig. 2D displayed that LPS (50 pg ml−1) significantly stimulated the secretion of 4 cytokines (INF-γ, IL-8, IL-10, and IL-12) ( p < 0.05 or 0.01), compared with the control. L2 (500 μg ml−1) significantly enhanced the productions of 4 cytokines (IL-6, IL-12,
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IL-17, and INF-γ; p < 0.05 or 0.01), relative to LPS. Zymosan A (400 μg ml−1) significantly stimulated the productions of 3 cytokines (IL-6, IL-12, and INF-γ; p < 0.05 or 0.01), but inhibited the secretion of IL-10 ( p < 0.01) and IL-17 ( p < 0.05), compared with LPS. At this time, the differential effects of L2 and Zymosan A lied in IL-10 and IL-17. 3.2 Effects of L. edodes-derived polysaccharide L2 on the activity of immune cells and the secretion of cytokines in serum in vivo Basically, all mice displayed no significant variations in food intake, body weight gain and activities of daily living. The average food intake per mouse was 5.09 g per day and 5.99 g per day for normal and L2-treated groups, respectively. Compared with normal mice, the spleen and thymus indices were significantly ( p < 0.01) increased in L2-treated mice, and the natural killer cell (NK cell) activities and the proliferation of T cell of splenocytes in the L2-treated groups were also significantly elevated (P < 0.05; Table 2). Fig. 3 showed that L2 significantly increased the productions of six cytokines (TNF-α, IFN-γ, IL-1α, IL-2, IL-6, and IL-12) in serum, although SIgAs in small-intestinal contents were similar, compared with normal groups. These results revealed that L. edodes-derived polysaccharide L2 indeed enhanced the cellular and humoral immunities of mice. 3.3 Effects of L. edodes-derived polysaccharide L2 on gene expression in mucosa of mice Among 112 immune-related genes examined herein, 78 genes exhibited significant changes (>2-fold or p < 0.05). The results showed various impact patterns of L2 treatment in the mucosa of mice. In particular, L2 exhibited the greatest effects on the genes of the small intestine, a total of 55 (49.1% of 112 genes), including 41 up-regulated and 14 down-regulated genes. There were 26 genes (23.2% of 112 genes) significantly changed in cecum, including 9 up-regulated and 17 down-regulated genes. In the colon, 25 genes (22.3% of 112 genes) were significantly altered, including 8 up-regulated and 17 down-regulated genes. It means that the major acting site of L2 was the small intestine. In addition, L2 primarily stimulated overexpression in the small intestine and under-expression in the cecum and colon. For chemokine-related genes (Table 3), all of the varied genes were up-regulated, 4 genes (Cxcl9, Cxcl11, Csf1 and
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Fig. 2 Secretion of cytokines: (A) In Caco-2 cells under different concentrations of L2 (125, 250 and 500 μg ml−1); *p < 0.05, **p < 0.01 vs. control; (B) in Caco-2 cells under various concentrations of Zymosan A (100, 200 and 400 μg ml−1); *p < 0.05, **p < 0.01 vs. control; (C) in Caco-2 cells prostimulated by LPS (50 pg ml−1) under concentrations of L2 (500 μg ml−1) and Zymosan (400 μg ml−1); *p < 0.05, **p < 0.01 vs. LPS. bp < 0.01 vs. control. (D) In co-cultured Caco-2/RAW264.7 cells pro-stimulated by LPS (50 pg ml−1) under concentrations of L2 (500 μg ml−1) and Zymosan (400 μg ml−1); *p < 0.05, **p6)-glucans, Mycol. Res., 2007, 111(Pt 6), 635–652. 5 E. A. Murphy, J. M. Davis and M. D. Carmichael, Immune modulating effects of β-glucan, Curr. Opin. Clin. Nutr. Metab. Care, 2010, 13(6), 656–661. 6 S. H. Albeituni and J. Yan, The effects of β-glucans on dendritic cells and implications for cancer therapy, Anticancer Agents Med. Chem., 2013, 13(5), 689–698. 7 R. Nicolosi, S. J. Bell, B. R. Bistrian, I. Greenberg, et al., Plasma lipid changes after supplementation with betaglucan fiber from yeast, Am. J. Clin. Nutr., 1999, 70(2), 208– 212. 8 H. C. Lo, F. A. Tsai, S. P. Wasser, J. G. Yang, et al., Effects of ingested fruiting bodies, submerged culture biomass, and acidic polysaccharide glucuronoxylomannan of Tremella mesenterica Retz.:Fr. on glycemic responses in normal and diabetic rats, Life Sci., 2006, 78(17), 1957–1966. 9 S. P. Wasser, Medicinal mushrooms as a source of antitumor and immunomodulating polysaccharides, Appl. Microbiol. Biotechnol., 2002, 60, 258–274. 10 P. S. Bisen, R. K. Baghel, B. S. Sanodiya, G. S. Thakur, et al., Lentinus edodes: A Macrofungus with Pharmacological Activities, Curr. Med. Chem., 2010, 22, 2419–2430. 11 G. Chihara, J. Hamuro, Y. Y. Maeda, Y. Arai, et al., Fractionation and purification of the polysaccharides with marked antitumor activity, especially lentinan, from Lentinus edodes (Berk.) Sing. (an edible mushroom), Cancer Res., 1970, 30, 2776–2781. 12 Y. Y. Zhang, S. Li, X. H. Wang, L. N. Zhang, et al., Advances in Lentinan: Isolation, structure, chain conformation and bioactivities, Food Hydrocolloids, 2011, 25(2), 196–206. 13 P. M. Kidd, The use of mushroom glucans and proteoglycans in cancer treatment, Altern. Med. Rev., 2000, 5(1), 4–27. 14 E. R. Carbonero, A. H. P. Gracher, D. L. Komura, R. Marcon, et al., Lentinus edodes heterogalactan: Antinociceptive and anti-inflammatory effects, Food Chem., 2008, 111, 531–537. 15 H. Fu, W. Y. Guo, H. Yin, Z. X. Wang, et al., Inhibition of Lentinus edodes polysaccharides against liver tumour growth, Int. J. Phys. Sci., 2011, 6(1), 116–120. 16 X. F. Xu, H. D. Yan and X. W. Zhang, Structure and Immuno-Stimulating Activities of a New Heteropoly-
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