International Journal of Biological Macromolecules 64 (2014) 1–5
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Polysaccharides from Enteromorpha prolifera enhance the immunity of normal mice Jianteng Wei a,b , Shuxian Wang c , Ge Liu d , Dong Pei a,b , Yongfeng Liu a,b , Yi Liu a,b , Duolong Di a,b,∗ a Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, PR China b Center of Resource Chemical & New Material, Qingdao, Qingdao 266100, PR China c Mariculture Institute of Shandong Province, Qingdao 266002, PR China d Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, PR China
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Article history: Received 2 September 2013 Received in revised form 29 October 2013 Accepted 22 November 2013 Available online 1 December 2013 Keywords: Enteromorpha prolifera Polysaccharide Immune response Immune-related enzyme NF-B
a b s t r a c t The effects of polysaccharides from Enteromorpha prolifera (PEP) on cell-mediated immunity, humoral immunity and mononuclear phagocytic system function were evaluated to assess the immunomodulatory potential of these macromolecules. Relevant immunological mechanisms were verified by biochemical assays and western blot analysis. Results showed that PEP could induce splenocyte proliferation. In vivo experiments on Kunming mice confirmed that PEP could improve cell-mediated immunity, humoral immunity and mononuclear phagocytic system function. To illustrate the mechanism, we determined several immune-related enzymes in the thymus and spleen. The results indicated that PEP could enhance the activities of alkaline phosphatase, superoxide dismutase and lactate dehydrogenase. PEP could also increase the level of NF-B. These results suggested that PEP exhibited potent immunomodulatory properties and could be used as a novel potential immunostimulant in food and pharmaceutical industries. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Enteromorpha is a type of edible marine algae used as a traditional Chinese medicine. This alga exhibits a strong propagation capability and is widely distributed along the coast of China, particularly in the eastern regions. In ancient China, Enteromorpha was used as a natural health product to treat many diseases, including epistaxis and inflammation. Enteromorpha is also considered as a rich source of carbohydrates, protein, crude fibre, minerals, fats and vitamins [1,2]. Polysaccharides from Enteromorpha possess a wide range of pharmacological properties, such as antitumor [3], biosorption [4], antioxidant [5] and anticoagulant [6] properties. In recent years, much attention has been directed towards the natural immunoregulatory properties of polysaccharides from Enteromorpha [7].
∗ Corresponding author at: Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, 18 Tianshui Middle Road, Lanzhou 730000, PR China. Tel.: +86 931 4968248; fax: +86 931 4968248. E-mail address: [email protected] (D. Di). 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.11.013
Enteromorpha prolifera is the dominant species in the Yellow Sea [8]. Previous studies investigated the immunomodulatory effects of polysaccharides from E. prolifera on the cellular level [9,10]. Watersoluble polysaccharides from E. prolifera can stimulate Raw 264.7 cells (mouse leukaemic monocyte macrophage cell line) by inducing considerable nitric oxide (NO) production [9]. The sulphated polysaccharides from E. prolifera stimulated Raw 264.7 to increase the production of various cytokines by upregulating mRNA expression. The results also showed that the sulphated polysaccharides significantly increase concanavalin A (Con A)-induced splenocyte proliferation, revealing the potential comitogenic activity of these polysaccharides [10]. These results confirmed the important function of polysaccharides from E. prolifera in triggering immune responses. In addition, polysaccharides from E. prolifera activated T cells by considerably increasing IFN-␥ and IL-2 secretions [10]. Despite extensive studies on the biological activities of polysaccharides from E. prolifera, few studies have been conducted on the immunoregulatory properties of polysaccharides from E. prolifera in normal mice. This study aimed to investigate the immunomodulatory effects of PEP on cell-mediated immunity, humoral immunity and mononuclear phagocytic system function in mice. Moreover, we investigated the effects of PEP on immunerelated enzymatic activities and NF-B levels.
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2. Methods and materials
2.5. Assessment of cell-mediated immunity by delayed type hypersensitivity test
2.1. Materials and chemicals E. prolifera was collected from Qingdao, China in July 2012. The collected sample was washed, dried and stored until use. RPMI1640 and foetal bovine serum (FBS) were purchased from HyClone Co., USA. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 2,4-dinitro-1-fluorobenzene (DNFB) were purchased from Sigma Co., USA. Concanavalin A (Con A) and antimouse/rabbit antibodies were purchased from Beijing Solarbio Science & Technology Co., Ltd., China. Alkaline phosphatase (AKP), acid phosphatase (ACP), superoxide dismutase (SOD), and lactate dehydrogenase (LDH) kits were purchased from Nanjing Jiancheng Bioengineering Institute, China. Antibody against NF-B was purchased from Santa Cruz Biotechnology, Inc. All of the other reagents used in this study were analytical grade chemicals. 2.2. Preparation of polysaccharides Polysaccharides were prepared as described previously [11]. In brief, the polysaccharides were extracted with boiled water. Proteins were removed by adding 15% (m/m) trichloroacetic acid. The filtrate was then neutralized with 1 mol L−1 NaOH and centrifuged to remove any insoluble particles. Subsequently, 95% ethanol was added to allow the polysaccharides to precipitate overnight. The precipitate was collected by centrifugation, washed with acetone thrice, dissolved in distilled water, applied to a Sephadex G-100 column (5.0 cm × 100 cm) and eluted with distilled water. The main fraction termed PEP was collected and lyophilized. 2.3. Experimental animals Kunming mice (six weeks old to eight weeks old) weighing 18–22 g were purchased from Qingdao Food and Drug Administration (Qingdao, China). The study was conducted in accordance with the standard guidelines for laboratory animal use and care by the Welfare Committee of the Centre of Experimental Animals, Qingdao, China. Upon arrival, the mice were acclimated for 3 d before the treatment. The mice were randomly divided into four groups and each group was housed in individual cages (twelve mice per cage). Pellets and tap water were provided ad libitum. The animals were intragastrically fed with 200, 400, and 800 mg kg−1 PEP for 30 d. The control group was fed with double-distilled water. 2.4. In vitro splenocyte proliferation assay The immunomodulating activity of PEP was evaluated in vitro by measuring the effect on spleen cell proliferation as described by Wang et al. [14]. The mouse spleens were aseptically removed from the sacrificed mice by using scissors and forceps in 0.1 mol l−1 cold PBS, gently homogenized and passed through a 40 m nylon cell strainer to obtain single-cell suspensions. The erythrocytes were removed; the splenocytes were washed and resuspended in RPMI1640 medium (2% FBS) at a cell concentration of 5 × 106 ml−1 . The cells (100 l/well) were seeded in a 96-well plate and stimulated with 50 l of Con A (5 g ml−1 ). PEP solution (100 l) was added to each well, and the final concentration was adjusted to 12.5, 25, 50, and 100 g ml−1 accordingly. After incubation for 48 h at 37 ◦ C in a 5% CO2 incubator, 20 l of 5% MTT solution was added to each well. After incubation for another 4 h, the formazan precipitate was dissolved in 150 l of DMSO, and the optical density at 570 nm was then measured using SpectraMax M5 Absorbance Microplate Reader (Molecular Devices, USA). The experiment was performed in triplicate.
Delayed-type hypersensitivity test (DTH) was carried out according to a previously described protocol [12]. Mice were smeared with 50 l of freshly prepared DNFB in acetone-sesame oil solution (1:1, v/v) on the belly 5 days prior to the assay. On Day 5 following immunization, the right ears of the mice were smeared with 10 l DNFB solution homogeneously. The mice were euthanized after DNFB treatment for 12 h. Ear punches (8 mm in diameter) were obtained and weighed. The difference between left and right ear weight indicates the extent of the delayed type hypersensitivity. 2.6. Assessment of humoral immunity by serolysin test Humoral immunity was evaluated by serolysin test as previously described [13]. In brief, 0.2 ml of 5% chicken red blood cells prepared in normal saline was intragastrically injected in mice 5 days prior to the assay. On Day 5 following immunization, 1.0 ml of 0.2% chicken red blood cells and 1.0 ml of mice serum were mixed with 1.0 ml of cell suspension and incubated for 1 h at 37 ◦ C. The optical density of the supernatant was measured at 415 nm. 2.7. Assessment of mononuclear phagocytic system function by carbon clearance test Mononuclear phagocytic system function assay was carried out as previously described [14]. Thirty days after PEP treatment, mice were injected with India ink (0.1 ml/10 g body weight) intravenously through a lateral tail vein. Blood samples (20 l) were taken from the retroorbital venous plexus at 2 min (t1 ) and 10 min (t2 ) intervals after India ink staining injection and mixed with 2 ml 0.1% Na2 CO3 . The optical densities of the samples were then determined at 600 nm (OD1 for 2 min and OD2 for 10 min). The phagocytic index (˛) was calculated according to the following formula: a=
body weight × K 1/3 liver weight + spleen weight
where K was calculated according to the following formula: K=
(lgOD1 − lgOD2 ) . t2 − t1
2.8. Biochemical assay The organ homogenates (including the thymus and spleen) were prepared in 0.1 g ml−1 wet weight of ice-cold physiological saline. The samples were centrifuged for 10 min at 5000 × g at 4 ◦ C, and the supernatants were used to measure AKP, ACP, SOD, and LDH levels. Enzyme activities were determined with the assay kits (Nanjing Jiancheng, China). The optical density of LDH was measured at 440 nm, the optical density of ACP and AKP was measured at 520 nm, and the optical density of SOD was measured at 550 nm [14–16]. 2.9. Western blot assay To determine the effect of PEP on NF-B, western blot was performed as previously described [17]. Briefly, the organs were washed twice with ice-cold PBS and then broken with homogenizer in RIPA buffer containing fresh protease inhibitor mixture (50 g ml−1 aprotinin, 0.5 mM phenylmethanesulfonyl fluoride, 1 mM sodium orthovanadate, 10 mM sodium fluoride, and 10 mM -glycerolphosphate). Total protein concentration was quantified using the BCA protein assay (Biocolor BioScience &
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Table 2 Effect of PEP on serum serolysin of mice. Group
Dose (mg kg−1 )
Absorbance of serum serolysin (A540 nm )
Control 1 2 3
– 200 400 800
0.56 0.83 0.82 0.91
± ± ± ±
0.31 0.18* 0.20* 0.15*
Note: Data are presented as mean ± SD (n = 12) and evaluated by one-way ANOVA using SPSS version 11.5. Differences were considered to be statistically significant when p < 0.05. * p < 0.05 compared with control group.
Fig. 1. Effect of polysaccharides from Enteromorpha prolifera on splenocyte proliferation. Splenocyte proliferation assay was carried out as described in Section 2. Data is presented as mean ± SD. The absorbance was obtained in more than three independent experiments. *p < 0.05 vs. control group with Con A group; #p < 0.05 vs. control group without Con A group.
Technology, China). Protein samples were resolved on 10–15% SDSpolyacrylamide gels. The separated protein samples were then transferred to nitrocellulose membranes and probed with protein specific antibodies followed by HRP-conjugated secondary antibody. 2.10. Statistical analysis The data were analyzed using SPSS version 11.5 and the difference was considered significant if p < 0.05. The data was presented as mean ± SD.
increases the difference between left and right ear weight, although it was not statistically significant compared with the control group. By contrast, significant differences were observed in mice treated with 400 and 800 mg kg−1 PEP (20.2 ± 2.8 mg and 22.0 ± 7.8 mg, respectively) compared with the control group. Therefore, the data suggest that PEP enhances cell-mediated immunity in mice. 3.3. PEP enhances humoral immunity Serolysin test was performed to evaluate whether PEP can enhance humoral immunity. The results show that PEP dramatically increases the level of serum hemolysin, but the increase was not in a dose-dependent manner (Table 2). The optical density of serum hemolysin was 0.56 ± 0.31 in the control group, whereas the optical densities of serum hemolysin were 0.83 ± 0.18, 0.82 ± 0.20, and 0.91 ± 0.15 in the experimental groups treated with 200, 400, and 800 mg kg−1 PEP, respectively. The data suggests that PEP enhances humoral immunity. 3.4. PEP improves carbon clearance ability
3. Results 3.1. PEP induces splenocyte proliferation Splenocyte proliferation plays an important role in the activation of both cellular and humoral immune responses. The effect of PEP on splenocyte proliferation was examined using MTT assay and the result is shown in Fig. 1. Con A triggers spleen splenocyte differentiation and the differentiated splenocytes of the Con A-treated group remarkably increased compared with those of the control group. However, the splenocytes in PEP-treated group displayed a marked increase at doses of 50 and 100 g ml−1 compared with the Con A-treated group. The data indicate that PEP has potential comitogenic activity. 3.2. PEP enhances cell-mediated immunity DTH was used as an in vivo assessment of cell-mediated immunity. We investigated whether DTH responsiveness was greater in the PEP-treated group compared with the control group. The results are shown in Table 1. Treatment of mice with 200 mg kg−1 PEP
The phagocytic indexes of mice treated with high doses of PEP (400 and 800 mg kg−1 ) were 5.32 ± 0.40 and 5.24 ± 0.54, respectively (Table 3). These indexes are significantly higher compared with the control group. These data indicate that PEP improves carbon clearance ability. 3.5. PEP affects spleen index of mice The spleen and thymus indexes of mice were quantified to examine the effect of PEP on these organs. The body weight and thymus index of mice were 46.9 ± 3.9 and 2.08 ± 0.51 in the control group, respectively, and there was no significant difference in PEP-treated group compared with the control group. Moreover, the intermediate dose of PEP (400 mg kg−1 ) increases the spleen index of mice, whereas no significant increase was observed in the other two groups (200 and 800 mg kg−1 ) compared with the control group (Table 4). 3.6. PEP affects enzyme activities of the thymus and the spleen The enzyme activities of AKP, ACP, SOD, and LDH of the thymus and spleen were investigated to assess the effect of
Table 1 Effect of PEP on delayed type hypersensitivity of mice. Group
Dose (mg kg−1 )
Difference between left and right ear weight (mg)
Control 1 2 3
– 200 400 800
14.3 18.4 20.2 22.0
± ± ± ±
5.6 5.8 2.8* 7.8*
Note: Data are presented as mean ± SD (n = 12) and evaluated by one-way ANOVA using SPSS version 11.5. Differences were considered to be statistically significant when p < 0.05. * p < 0.05 compared with control group.
Table 3 Effect of PEP on phagocytic index of mice. Group −1
Dose (mg kg ) Phagocytic index (˛)
Control
1
2
3
– 4.86 ± 0.42
200 5.05 ± 0.66
400 5.32 ± 0.40*
800 5.24 ± 0.54*
Note: Data are presented as mean ± SD (n = 12) and evaluated by one-way ANOVA using SPSS version 11.5. Differences were considered to be statistically significant when p < 0.05. * p < 0.05 compared with control group.
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Fig. 2. Effect of polysaccharides from Enteromorpha prolifera on activities of alkaline phosphatase (AKP) (a), acid phosphatase (ACP) (b), superoxide dismutase (SOD) (c) and lactate dehydrogenase (LDH) (d) in the murine thymus, spleen, and peritoneal macrophage. All data are presented as mean ± SD (n = 6). Differences were considered to be statistically significant when p < 0.05. *p < 0.05 compared with control group.
PEP. Enhancement of AKP activity was observed in thymus and spleen of PEP-treated mice (Fig. 2a). The activities of AKP were 43.04 ± 1.19, 75.05 ± 3.21, 58.09 ± 1.58, and 58.33 ± 3.53 in thymus of mice treated with 0, 200, 400, and 800 mg kg−1 PEP, respectively, whereas the activities of AKP in spleen of PEP-treated mice were 42.27 ± 1.75, 59.14 ± 1.89, 53.02 ± 2.91, and 55.90 ± 4.98, respectively. There was no significant difference between the experimental groups and the control group in ACP activity of thymus and spleen (Fig. 2b). Moreover, PEP significantly enhances the activity of SOD in thymus and spleen compared with the control group (Fig. 2c). Fig. 2d shows that PEP enhances the activity of LDH in thymus and spleen; however, there was no statistically significant difference in thymus and spleen of mice treated with low dose of PEP (200 mg kg−1 ) compared with the control group. These results indicate that PEP affects the activities of AKP, SOD, and LDH in the thymus and spleen of mice without altering the activity of ACP.
not in a dose-dependent manner, the results also suggest that NFB plays an important role in the immune response of mice treated with PEP. 4. Discussion In recent decades, polysaccharides from different sources can be used as an immune-regulator because of their fewer side effect and higher efficiency [14,18]. In my study, we found polysaccharides from E. prolifera could enhance immune response of normal mice. However, the immune-enhancement response is not in a dose-dependent manner. Sun et al. have reported that polysaccharides from Porphyridium cruentum could significantly increase proliferation of peritoneal acrophages. In the concentration range 25–200 g/mL, the absorbance of the treatment groups were significantly higher than that of the control group, among which 100 g/mL polysaccharides had the most positive effect on
3.7. PEP regulates the level of NF-B of the thymus and spleen The level of NF-B was determined by western blot analysis. The results show that PEP significantly increases the level of NF-B in thymus (Fig. 3a) and spleen (Fig. 3b). Although these increases were Table 4 Effect of PEP on body weight, thymus index, and spleen index of mice. Group
Dose (mg kg−1 )
Body weight (g)
Control 1 2 3
– 200 400 800
46.9 47.6 46.0 48.6
± ± ± ±
3.9 4.2 3.9 3.7
Thymus index (mg g−1 ) 2.08 2.34 2.05 2.00
± ± ± ±
0.51 0.81 0.56 0.38
Spleen index (mg g−1 ) 4.44 4.90 5.54 4.60
± ± ± ±
1.15 1.54 1.21* 1.83
Note: Data are presented as mean ± SD (n = 12) and evaluated by one-way ANOVA using SPSS version 11.5. Differences were considered to be statistically significant when p < 0.05. * p < 0.05 compared with control group.
Fig. 3. Effect of polysaccharides from Enteromorpha prolifera on the level of NF-B in murine thymus (a) and spleen (b). Organs treated with 200, 400, and 800 mg kg−1 were analyzed by western blot as described in Section 2. Results shown are representative of three independent experiments. *p < 0.05 compared with control group.
J. Wei et al. / International Journal of Biological Macromolecules 64 (2014) 1–5
proliferation. When polysaccharides concentration was >100 g/mL, the ability to promote proliferation decreased [19]. Chen et al. have reported the immunomodulatory activity of polysaccharide from Potentilla anserine. The result showed that polysaccharide could enhance pinocytic activity of mouse peritoneal macrophages, thymus indices and levels of some cytokines and that the increase was not in a dose-dependent manner [14]. Cheng et al. [18] and Bai et al. [20] got the similar result. So we inferred that the non-dose dependent manner was the common way of action of some polysaccharide. The possible reason is that polysaccharide has the characteristics of structural diversity complicated composition and that the immune response is only related to the effective dose, and is not related to the actual dose. Splenocyte proliferation is an effective way to detect the immune response. Previous results showed that sulphated polysaccharides from E. prolifera significantly increased Con A-induced splenocyte proliferation [10]. In this study, we confirmed that PEP increased Con A-induced splenocytes proliferation (Fig. 1). The data shows that PEP promotes splenocyte comitogenic ability. In the subsequent in vivo tests, we found that the spleen indexes of mice treated with PEP did not increase accordingly (Table 3). One possible reason is that the actual PEP level achieved in vivo was not high enough to produce detectable effects on spleen weight. To further investigate the mechanism of immunomodulation, we evaluated the effects of PEP on cell-mediated immunity, humoral immunity, and mononuclear phagocytic system function. T lymphocyte is an important class of immunologically active cells and is mainly responsible for cellular immunity. PEP induces delayed type hypersensitivity reactions in mice, suggesting that PEP activated the cell-mediated immunity. One of the possible reasons is that polysaccharides from E. prolifera increased the secretion of IFN-␥ and IL-2 in T lymphocytes [10]. B lymphocyte is the only cell capable of producing antibodies for humoral immunity. In this study, we determined the level of serum serolysin, and the results showed that PEP enhances humoral immunity. In addition, phagocytes are key participants in the innate immune response, as they are among the earliest cell types in response to the invasion of pathogenic organisms. Macrophages, together with neutrophils, act as the first line of host defence [21,22]. The data indicate that PEP improves carbon clearance ability, which might be caused by the improvement of mononuclear phagocytic system function. Therefore, PEP should be considered as a potential natural health product to improve immunity. In this study, we investigated the activity of LDH and ACP in thymus and spleen of mice, as these enzymes are markers of macrophages. Previous studies indicated that activity of LDH and ACP increased with the activated macrophage and decreased with the inhibited macrophage [11]. Therefore, PEP increased the activity of LDH in the thymus and spleen of mice, suggesting that PEP activates macrophages. In addition, AKP and SOD play important roles as immune-related enzymes. They play an important part in enhancing the capability of phagocytic cells and improving immune function [23,24]. In the present study, activities of AKP and SOD in murine thymus and spleen were both increased. The NF-B family comprises structurally related proteins that modulate multiple physiological processes, ranging from immune responses to cell death and survival. These transcription factors are ubiquitously expressed and control the expression of a significant number of genes [25]. It was reported that NF-B proteins are master regulators of host immune responses in mammals [26] and plays a critical role in the development of innate immunity [27]. In this study, the results showed that treatment of mice with PEP resulted in a significant increase of NF-B level, suggesting that NF-B-mediated immune response plays a key role in the biological process induced by PEP in mice. It is well documented that
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activation of NF-B affects immune-related cytokines, including TNF-␣, IL-1, IL-6, and IL-8 [28]. It is feasible, therefore, that the increase of NF-B induced by PEP treatment might be related with immune response. Studies are in progress to search for more active ingredients and decipher the more complicated mechanism of this induction. 5. Conclusion In conclusion, our present study provides solid evidence that PEP improves the immune response of mice. The possible mechanism is that PEP upregulates immune-related enzymes like AKP, SOD, and LDH. In addition, transcription factors, such as NF-B, also play a significant role in immune response. Conflict of interest The authors report no declarations of interest. Acknowledgement This research project was financially supported by the National Natural Sciences Foundation of China (20974116 and 21175142). References [1] M. Aguilera-Morales, M. Casas-Valdez, S. Carrillo-Dominguez, B. GonzalezAcosta, F. Perez-Gil, Journal of Food Composition and Analysis 18 (2005) 79–88. [2] B.S. Mamatha, K.K. Namitha, S. Amudha, J. Smitha, G.A. Ravishankar, Food Chemistry 101 (2007) 1707–1713. [3] L. Jiao, X. Li, T. Li, P. Jiang, L. Zhang, M. Wu, L. Zhang, International Immunopharmacology 9 (2009) 324–329. [4] I. Michalak, K. Chojnacka, Applied Biochemistry and Biotechnology 160 (2010) 1540–1556. [5] M. Cho, H. Lee, I. Kang, M. Won, S. You, Food Chemistry 127 (2011) 999–1006. [6] X. Qi, W. Mao, Y. Gao, Y. Chen, Y. Chen, C. Zhao, N. Li, C. Wang, M. Yan, C. Lin, J. Shan, Carbohydrate Polymers 90 (2012) 1804–1810. [7] L. Jiao, P. Jiang, L. Zhang, M. Wu, Biotechnology and Bioprocess Engineering 15 (2010) 421–428. [8] J. Zhao, P. Jiang, Z. Liu, W. Wei, H. Lin, F. Li, J. Wang, S. Qin, Chinese Science Bulletin (2012), http://dx.doi.org/10.1007/s11434-012-5441-3. [9] M. Cho, C. Yang, S.M. Kim, S.G. You, Food Science and Biotechnology 19 (2010) 525–533. [10] J.K. Kim, M.L. Cho, S. Karnjanapratum, I.S. Shin, S.G. You, International Journal of Biological Macromolecules 49 (2011) 1051–1058. [11] X. Chen, J. Lu, Y. Zhang, J. He, X. Guo, G. Tian, L. Jin, International Journal of Biological Macromolecules 43 (2008) 252–256. [12] H. Kuang, Y. Xia, B. Yang, Q. Wang, Y. Wang, Carbohydrate Polymers 83 (2011) 787–795. [13] H. Yuan, J. Song, X. Li, N. Li, J. Dai, Cancer Letters 243 (2006) 228–234. [14] J.R. Chen, Z.Q. Yang, T.J. Hu, Z.T. Yan, T.X. Niu, L. Wang, D.A. Cui, M. Wang, Fitoterapia 81 (2010) 1117–1124. [15] C. De Simone, P. Ferranti, G. Picariello, I. Scognamiglio, A. Dicitore, F. Addeo, L. Chianese, P. Stiuso, Molecular Nutrition & Food Research 55 (2011) 229–238. [16] M. Wang, X.Y. Meng, R.L. Yang, T. Qin, X.Y. Wang, K.Y. Zhang, C.Z. Fei, Y. Li, Y.L. Hu, F.Q. Xue, Carbohydrate Polymers 89 (2012) 461–466. [17] Q. Chen, S. Zhang, H. Ying, X. Dai, X. Li, C. Yu, H. Ye, Carbohydrate Polymers 88 (2012) 1476–1482. [18] X.Q. Cheng, H. Li, X.L. Yue, J.Y. Xie, Y.Y. Zhang, H.Y. Di, D.F. Chen, Journal of Ethnopharmacology 130 (2010) 363–368. [19] L. Sun, L. Wang, Y. Zhou, Carbohydrate Polymers 87 (2012) 1206–1210. [20] Y. Bai, P. Zhang, G. Chen, J. Cao, T. Huang, K. Chen, International Immunopharmacology 12 (2012) 611–617. [21] R.W. Birk, A. Gratchev, N. Hakiy, O. Politz, K. Schledzewski, P. Guillot, C.E. Orfanos, S. Goerdt, Hautarzt 52 (2001) 193–200. [22] M.W. Lingen, Archives of Pathology and Laboratory Medicine 125 (2001) 67–71. [23] M. Hermes-Lima, J.M. Storey, K.B. Storey, Comparative Biochemistry and Physiology 120 (1998) 437–448. [24] J. Du, H. Zhu, P. Liu, J. Chen, Y. Xiu, W. Yao, T. Wu, Q. Ren, Q. Meng, W. Gu, W. Wang, Fish & Shellfish Immunology 34 (2013) 315–323. [25] Y.M. Thu, A. Richmond, Cytokine & Growth Factor Reviews 21 (2010) 213–226. [26] S. Ganesan, K. Aggarwal, N. Paquette, N. Silverman, Current Topics in Microbiology and Immunology 349 (2011) 25–60. [27] A. Dev, S. Iyer, B. Razani, G. Cheng, Current Topics in Microbiology and Immunology 349 (2011) 115–143. [28] T.S. Blackwell, J.W. Christman, American Journal of Respiratory Cell and Molecular Biology 17 (1997) 13–19.
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Polysaccharides from Enteromorpha prolifera enhance the immunity of normal mice Jianteng Wei a,b , Shuxian Wang c , Ge Liu d , Dong Pei a,b , Yongfeng Liu a,b , Yi Liu a,b , Duolong Di a,b,∗ a Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, PR China b Center of Resource Chemical & New Material, Qingdao, Qingdao 266100, PR China c Mariculture Institute of Shandong Province, Qingdao 266002, PR China d Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, PR China
a r t i c l e
i n f o
Article history: Received 2 September 2013 Received in revised form 29 October 2013 Accepted 22 November 2013 Available online 1 December 2013 Keywords: Enteromorpha prolifera Polysaccharide Immune response Immune-related enzyme NF-B
a b s t r a c t The effects of polysaccharides from Enteromorpha prolifera (PEP) on cell-mediated immunity, humoral immunity and mononuclear phagocytic system function were evaluated to assess the immunomodulatory potential of these macromolecules. Relevant immunological mechanisms were verified by biochemical assays and western blot analysis. Results showed that PEP could induce splenocyte proliferation. In vivo experiments on Kunming mice confirmed that PEP could improve cell-mediated immunity, humoral immunity and mononuclear phagocytic system function. To illustrate the mechanism, we determined several immune-related enzymes in the thymus and spleen. The results indicated that PEP could enhance the activities of alkaline phosphatase, superoxide dismutase and lactate dehydrogenase. PEP could also increase the level of NF-B. These results suggested that PEP exhibited potent immunomodulatory properties and could be used as a novel potential immunostimulant in food and pharmaceutical industries. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Enteromorpha is a type of edible marine algae used as a traditional Chinese medicine. This alga exhibits a strong propagation capability and is widely distributed along the coast of China, particularly in the eastern regions. In ancient China, Enteromorpha was used as a natural health product to treat many diseases, including epistaxis and inflammation. Enteromorpha is also considered as a rich source of carbohydrates, protein, crude fibre, minerals, fats and vitamins [1,2]. Polysaccharides from Enteromorpha possess a wide range of pharmacological properties, such as antitumor [3], biosorption [4], antioxidant [5] and anticoagulant [6] properties. In recent years, much attention has been directed towards the natural immunoregulatory properties of polysaccharides from Enteromorpha [7].
∗ Corresponding author at: Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, 18 Tianshui Middle Road, Lanzhou 730000, PR China. Tel.: +86 931 4968248; fax: +86 931 4968248. E-mail address: [email protected] (D. Di). 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.11.013
Enteromorpha prolifera is the dominant species in the Yellow Sea [8]. Previous studies investigated the immunomodulatory effects of polysaccharides from E. prolifera on the cellular level [9,10]. Watersoluble polysaccharides from E. prolifera can stimulate Raw 264.7 cells (mouse leukaemic monocyte macrophage cell line) by inducing considerable nitric oxide (NO) production [9]. The sulphated polysaccharides from E. prolifera stimulated Raw 264.7 to increase the production of various cytokines by upregulating mRNA expression. The results also showed that the sulphated polysaccharides significantly increase concanavalin A (Con A)-induced splenocyte proliferation, revealing the potential comitogenic activity of these polysaccharides [10]. These results confirmed the important function of polysaccharides from E. prolifera in triggering immune responses. In addition, polysaccharides from E. prolifera activated T cells by considerably increasing IFN-␥ and IL-2 secretions [10]. Despite extensive studies on the biological activities of polysaccharides from E. prolifera, few studies have been conducted on the immunoregulatory properties of polysaccharides from E. prolifera in normal mice. This study aimed to investigate the immunomodulatory effects of PEP on cell-mediated immunity, humoral immunity and mononuclear phagocytic system function in mice. Moreover, we investigated the effects of PEP on immunerelated enzymatic activities and NF-B levels.
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2. Methods and materials
2.5. Assessment of cell-mediated immunity by delayed type hypersensitivity test
2.1. Materials and chemicals E. prolifera was collected from Qingdao, China in July 2012. The collected sample was washed, dried and stored until use. RPMI1640 and foetal bovine serum (FBS) were purchased from HyClone Co., USA. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 2,4-dinitro-1-fluorobenzene (DNFB) were purchased from Sigma Co., USA. Concanavalin A (Con A) and antimouse/rabbit antibodies were purchased from Beijing Solarbio Science & Technology Co., Ltd., China. Alkaline phosphatase (AKP), acid phosphatase (ACP), superoxide dismutase (SOD), and lactate dehydrogenase (LDH) kits were purchased from Nanjing Jiancheng Bioengineering Institute, China. Antibody against NF-B was purchased from Santa Cruz Biotechnology, Inc. All of the other reagents used in this study were analytical grade chemicals. 2.2. Preparation of polysaccharides Polysaccharides were prepared as described previously [11]. In brief, the polysaccharides were extracted with boiled water. Proteins were removed by adding 15% (m/m) trichloroacetic acid. The filtrate was then neutralized with 1 mol L−1 NaOH and centrifuged to remove any insoluble particles. Subsequently, 95% ethanol was added to allow the polysaccharides to precipitate overnight. The precipitate was collected by centrifugation, washed with acetone thrice, dissolved in distilled water, applied to a Sephadex G-100 column (5.0 cm × 100 cm) and eluted with distilled water. The main fraction termed PEP was collected and lyophilized. 2.3. Experimental animals Kunming mice (six weeks old to eight weeks old) weighing 18–22 g were purchased from Qingdao Food and Drug Administration (Qingdao, China). The study was conducted in accordance with the standard guidelines for laboratory animal use and care by the Welfare Committee of the Centre of Experimental Animals, Qingdao, China. Upon arrival, the mice were acclimated for 3 d before the treatment. The mice were randomly divided into four groups and each group was housed in individual cages (twelve mice per cage). Pellets and tap water were provided ad libitum. The animals were intragastrically fed with 200, 400, and 800 mg kg−1 PEP for 30 d. The control group was fed with double-distilled water. 2.4. In vitro splenocyte proliferation assay The immunomodulating activity of PEP was evaluated in vitro by measuring the effect on spleen cell proliferation as described by Wang et al. [14]. The mouse spleens were aseptically removed from the sacrificed mice by using scissors and forceps in 0.1 mol l−1 cold PBS, gently homogenized and passed through a 40 m nylon cell strainer to obtain single-cell suspensions. The erythrocytes were removed; the splenocytes were washed and resuspended in RPMI1640 medium (2% FBS) at a cell concentration of 5 × 106 ml−1 . The cells (100 l/well) were seeded in a 96-well plate and stimulated with 50 l of Con A (5 g ml−1 ). PEP solution (100 l) was added to each well, and the final concentration was adjusted to 12.5, 25, 50, and 100 g ml−1 accordingly. After incubation for 48 h at 37 ◦ C in a 5% CO2 incubator, 20 l of 5% MTT solution was added to each well. After incubation for another 4 h, the formazan precipitate was dissolved in 150 l of DMSO, and the optical density at 570 nm was then measured using SpectraMax M5 Absorbance Microplate Reader (Molecular Devices, USA). The experiment was performed in triplicate.
Delayed-type hypersensitivity test (DTH) was carried out according to a previously described protocol [12]. Mice were smeared with 50 l of freshly prepared DNFB in acetone-sesame oil solution (1:1, v/v) on the belly 5 days prior to the assay. On Day 5 following immunization, the right ears of the mice were smeared with 10 l DNFB solution homogeneously. The mice were euthanized after DNFB treatment for 12 h. Ear punches (8 mm in diameter) were obtained and weighed. The difference between left and right ear weight indicates the extent of the delayed type hypersensitivity. 2.6. Assessment of humoral immunity by serolysin test Humoral immunity was evaluated by serolysin test as previously described [13]. In brief, 0.2 ml of 5% chicken red blood cells prepared in normal saline was intragastrically injected in mice 5 days prior to the assay. On Day 5 following immunization, 1.0 ml of 0.2% chicken red blood cells and 1.0 ml of mice serum were mixed with 1.0 ml of cell suspension and incubated for 1 h at 37 ◦ C. The optical density of the supernatant was measured at 415 nm. 2.7. Assessment of mononuclear phagocytic system function by carbon clearance test Mononuclear phagocytic system function assay was carried out as previously described [14]. Thirty days after PEP treatment, mice were injected with India ink (0.1 ml/10 g body weight) intravenously through a lateral tail vein. Blood samples (20 l) were taken from the retroorbital venous plexus at 2 min (t1 ) and 10 min (t2 ) intervals after India ink staining injection and mixed with 2 ml 0.1% Na2 CO3 . The optical densities of the samples were then determined at 600 nm (OD1 for 2 min and OD2 for 10 min). The phagocytic index (˛) was calculated according to the following formula: a=
body weight × K 1/3 liver weight + spleen weight
where K was calculated according to the following formula: K=
(lgOD1 − lgOD2 ) . t2 − t1
2.8. Biochemical assay The organ homogenates (including the thymus and spleen) were prepared in 0.1 g ml−1 wet weight of ice-cold physiological saline. The samples were centrifuged for 10 min at 5000 × g at 4 ◦ C, and the supernatants were used to measure AKP, ACP, SOD, and LDH levels. Enzyme activities were determined with the assay kits (Nanjing Jiancheng, China). The optical density of LDH was measured at 440 nm, the optical density of ACP and AKP was measured at 520 nm, and the optical density of SOD was measured at 550 nm [14–16]. 2.9. Western blot assay To determine the effect of PEP on NF-B, western blot was performed as previously described [17]. Briefly, the organs were washed twice with ice-cold PBS and then broken with homogenizer in RIPA buffer containing fresh protease inhibitor mixture (50 g ml−1 aprotinin, 0.5 mM phenylmethanesulfonyl fluoride, 1 mM sodium orthovanadate, 10 mM sodium fluoride, and 10 mM -glycerolphosphate). Total protein concentration was quantified using the BCA protein assay (Biocolor BioScience &
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Table 2 Effect of PEP on serum serolysin of mice. Group
Dose (mg kg−1 )
Absorbance of serum serolysin (A540 nm )
Control 1 2 3
– 200 400 800
0.56 0.83 0.82 0.91
± ± ± ±
0.31 0.18* 0.20* 0.15*
Note: Data are presented as mean ± SD (n = 12) and evaluated by one-way ANOVA using SPSS version 11.5. Differences were considered to be statistically significant when p < 0.05. * p < 0.05 compared with control group.
Fig. 1. Effect of polysaccharides from Enteromorpha prolifera on splenocyte proliferation. Splenocyte proliferation assay was carried out as described in Section 2. Data is presented as mean ± SD. The absorbance was obtained in more than three independent experiments. *p < 0.05 vs. control group with Con A group; #p < 0.05 vs. control group without Con A group.
Technology, China). Protein samples were resolved on 10–15% SDSpolyacrylamide gels. The separated protein samples were then transferred to nitrocellulose membranes and probed with protein specific antibodies followed by HRP-conjugated secondary antibody. 2.10. Statistical analysis The data were analyzed using SPSS version 11.5 and the difference was considered significant if p < 0.05. The data was presented as mean ± SD.
increases the difference between left and right ear weight, although it was not statistically significant compared with the control group. By contrast, significant differences were observed in mice treated with 400 and 800 mg kg−1 PEP (20.2 ± 2.8 mg and 22.0 ± 7.8 mg, respectively) compared with the control group. Therefore, the data suggest that PEP enhances cell-mediated immunity in mice. 3.3. PEP enhances humoral immunity Serolysin test was performed to evaluate whether PEP can enhance humoral immunity. The results show that PEP dramatically increases the level of serum hemolysin, but the increase was not in a dose-dependent manner (Table 2). The optical density of serum hemolysin was 0.56 ± 0.31 in the control group, whereas the optical densities of serum hemolysin were 0.83 ± 0.18, 0.82 ± 0.20, and 0.91 ± 0.15 in the experimental groups treated with 200, 400, and 800 mg kg−1 PEP, respectively. The data suggests that PEP enhances humoral immunity. 3.4. PEP improves carbon clearance ability
3. Results 3.1. PEP induces splenocyte proliferation Splenocyte proliferation plays an important role in the activation of both cellular and humoral immune responses. The effect of PEP on splenocyte proliferation was examined using MTT assay and the result is shown in Fig. 1. Con A triggers spleen splenocyte differentiation and the differentiated splenocytes of the Con A-treated group remarkably increased compared with those of the control group. However, the splenocytes in PEP-treated group displayed a marked increase at doses of 50 and 100 g ml−1 compared with the Con A-treated group. The data indicate that PEP has potential comitogenic activity. 3.2. PEP enhances cell-mediated immunity DTH was used as an in vivo assessment of cell-mediated immunity. We investigated whether DTH responsiveness was greater in the PEP-treated group compared with the control group. The results are shown in Table 1. Treatment of mice with 200 mg kg−1 PEP
The phagocytic indexes of mice treated with high doses of PEP (400 and 800 mg kg−1 ) were 5.32 ± 0.40 and 5.24 ± 0.54, respectively (Table 3). These indexes are significantly higher compared with the control group. These data indicate that PEP improves carbon clearance ability. 3.5. PEP affects spleen index of mice The spleen and thymus indexes of mice were quantified to examine the effect of PEP on these organs. The body weight and thymus index of mice were 46.9 ± 3.9 and 2.08 ± 0.51 in the control group, respectively, and there was no significant difference in PEP-treated group compared with the control group. Moreover, the intermediate dose of PEP (400 mg kg−1 ) increases the spleen index of mice, whereas no significant increase was observed in the other two groups (200 and 800 mg kg−1 ) compared with the control group (Table 4). 3.6. PEP affects enzyme activities of the thymus and the spleen The enzyme activities of AKP, ACP, SOD, and LDH of the thymus and spleen were investigated to assess the effect of
Table 1 Effect of PEP on delayed type hypersensitivity of mice. Group
Dose (mg kg−1 )
Difference between left and right ear weight (mg)
Control 1 2 3
– 200 400 800
14.3 18.4 20.2 22.0
± ± ± ±
5.6 5.8 2.8* 7.8*
Note: Data are presented as mean ± SD (n = 12) and evaluated by one-way ANOVA using SPSS version 11.5. Differences were considered to be statistically significant when p < 0.05. * p < 0.05 compared with control group.
Table 3 Effect of PEP on phagocytic index of mice. Group −1
Dose (mg kg ) Phagocytic index (˛)
Control
1
2
3
– 4.86 ± 0.42
200 5.05 ± 0.66
400 5.32 ± 0.40*
800 5.24 ± 0.54*
Note: Data are presented as mean ± SD (n = 12) and evaluated by one-way ANOVA using SPSS version 11.5. Differences were considered to be statistically significant when p < 0.05. * p < 0.05 compared with control group.
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Fig. 2. Effect of polysaccharides from Enteromorpha prolifera on activities of alkaline phosphatase (AKP) (a), acid phosphatase (ACP) (b), superoxide dismutase (SOD) (c) and lactate dehydrogenase (LDH) (d) in the murine thymus, spleen, and peritoneal macrophage. All data are presented as mean ± SD (n = 6). Differences were considered to be statistically significant when p < 0.05. *p < 0.05 compared with control group.
PEP. Enhancement of AKP activity was observed in thymus and spleen of PEP-treated mice (Fig. 2a). The activities of AKP were 43.04 ± 1.19, 75.05 ± 3.21, 58.09 ± 1.58, and 58.33 ± 3.53 in thymus of mice treated with 0, 200, 400, and 800 mg kg−1 PEP, respectively, whereas the activities of AKP in spleen of PEP-treated mice were 42.27 ± 1.75, 59.14 ± 1.89, 53.02 ± 2.91, and 55.90 ± 4.98, respectively. There was no significant difference between the experimental groups and the control group in ACP activity of thymus and spleen (Fig. 2b). Moreover, PEP significantly enhances the activity of SOD in thymus and spleen compared with the control group (Fig. 2c). Fig. 2d shows that PEP enhances the activity of LDH in thymus and spleen; however, there was no statistically significant difference in thymus and spleen of mice treated with low dose of PEP (200 mg kg−1 ) compared with the control group. These results indicate that PEP affects the activities of AKP, SOD, and LDH in the thymus and spleen of mice without altering the activity of ACP.
not in a dose-dependent manner, the results also suggest that NFB plays an important role in the immune response of mice treated with PEP. 4. Discussion In recent decades, polysaccharides from different sources can be used as an immune-regulator because of their fewer side effect and higher efficiency [14,18]. In my study, we found polysaccharides from E. prolifera could enhance immune response of normal mice. However, the immune-enhancement response is not in a dose-dependent manner. Sun et al. have reported that polysaccharides from Porphyridium cruentum could significantly increase proliferation of peritoneal acrophages. In the concentration range 25–200 g/mL, the absorbance of the treatment groups were significantly higher than that of the control group, among which 100 g/mL polysaccharides had the most positive effect on
3.7. PEP regulates the level of NF-B of the thymus and spleen The level of NF-B was determined by western blot analysis. The results show that PEP significantly increases the level of NF-B in thymus (Fig. 3a) and spleen (Fig. 3b). Although these increases were Table 4 Effect of PEP on body weight, thymus index, and spleen index of mice. Group
Dose (mg kg−1 )
Body weight (g)
Control 1 2 3
– 200 400 800
46.9 47.6 46.0 48.6
± ± ± ±
3.9 4.2 3.9 3.7
Thymus index (mg g−1 ) 2.08 2.34 2.05 2.00
± ± ± ±
0.51 0.81 0.56 0.38
Spleen index (mg g−1 ) 4.44 4.90 5.54 4.60
± ± ± ±
1.15 1.54 1.21* 1.83
Note: Data are presented as mean ± SD (n = 12) and evaluated by one-way ANOVA using SPSS version 11.5. Differences were considered to be statistically significant when p < 0.05. * p < 0.05 compared with control group.
Fig. 3. Effect of polysaccharides from Enteromorpha prolifera on the level of NF-B in murine thymus (a) and spleen (b). Organs treated with 200, 400, and 800 mg kg−1 were analyzed by western blot as described in Section 2. Results shown are representative of three independent experiments. *p < 0.05 compared with control group.
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proliferation. When polysaccharides concentration was >100 g/mL, the ability to promote proliferation decreased [19]. Chen et al. have reported the immunomodulatory activity of polysaccharide from Potentilla anserine. The result showed that polysaccharide could enhance pinocytic activity of mouse peritoneal macrophages, thymus indices and levels of some cytokines and that the increase was not in a dose-dependent manner [14]. Cheng et al. [18] and Bai et al. [20] got the similar result. So we inferred that the non-dose dependent manner was the common way of action of some polysaccharide. The possible reason is that polysaccharide has the characteristics of structural diversity complicated composition and that the immune response is only related to the effective dose, and is not related to the actual dose. Splenocyte proliferation is an effective way to detect the immune response. Previous results showed that sulphated polysaccharides from E. prolifera significantly increased Con A-induced splenocyte proliferation [10]. In this study, we confirmed that PEP increased Con A-induced splenocytes proliferation (Fig. 1). The data shows that PEP promotes splenocyte comitogenic ability. In the subsequent in vivo tests, we found that the spleen indexes of mice treated with PEP did not increase accordingly (Table 3). One possible reason is that the actual PEP level achieved in vivo was not high enough to produce detectable effects on spleen weight. To further investigate the mechanism of immunomodulation, we evaluated the effects of PEP on cell-mediated immunity, humoral immunity, and mononuclear phagocytic system function. T lymphocyte is an important class of immunologically active cells and is mainly responsible for cellular immunity. PEP induces delayed type hypersensitivity reactions in mice, suggesting that PEP activated the cell-mediated immunity. One of the possible reasons is that polysaccharides from E. prolifera increased the secretion of IFN-␥ and IL-2 in T lymphocytes [10]. B lymphocyte is the only cell capable of producing antibodies for humoral immunity. In this study, we determined the level of serum serolysin, and the results showed that PEP enhances humoral immunity. In addition, phagocytes are key participants in the innate immune response, as they are among the earliest cell types in response to the invasion of pathogenic organisms. Macrophages, together with neutrophils, act as the first line of host defence [21,22]. The data indicate that PEP improves carbon clearance ability, which might be caused by the improvement of mononuclear phagocytic system function. Therefore, PEP should be considered as a potential natural health product to improve immunity. In this study, we investigated the activity of LDH and ACP in thymus and spleen of mice, as these enzymes are markers of macrophages. Previous studies indicated that activity of LDH and ACP increased with the activated macrophage and decreased with the inhibited macrophage [11]. Therefore, PEP increased the activity of LDH in the thymus and spleen of mice, suggesting that PEP activates macrophages. In addition, AKP and SOD play important roles as immune-related enzymes. They play an important part in enhancing the capability of phagocytic cells and improving immune function [23,24]. In the present study, activities of AKP and SOD in murine thymus and spleen were both increased. The NF-B family comprises structurally related proteins that modulate multiple physiological processes, ranging from immune responses to cell death and survival. These transcription factors are ubiquitously expressed and control the expression of a significant number of genes [25]. It was reported that NF-B proteins are master regulators of host immune responses in mammals [26] and plays a critical role in the development of innate immunity [27]. In this study, the results showed that treatment of mice with PEP resulted in a significant increase of NF-B level, suggesting that NF-B-mediated immune response plays a key role in the biological process induced by PEP in mice. It is well documented that
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activation of NF-B affects immune-related cytokines, including TNF-␣, IL-1, IL-6, and IL-8 [28]. It is feasible, therefore, that the increase of NF-B induced by PEP treatment might be related with immune response. Studies are in progress to search for more active ingredients and decipher the more complicated mechanism of this induction. 5. Conclusion In conclusion, our present study provides solid evidence that PEP improves the immune response of mice. The possible mechanism is that PEP upregulates immune-related enzymes like AKP, SOD, and LDH. In addition, transcription factors, such as NF-B, also play a significant role in immune response. Conflict of interest The authors report no declarations of interest. Acknowledgement This research project was financially supported by the National Natural Sciences Foundation of China (20974116 and 21175142). References [1] M. Aguilera-Morales, M. Casas-Valdez, S. Carrillo-Dominguez, B. GonzalezAcosta, F. Perez-Gil, Journal of Food Composition and Analysis 18 (2005) 79–88. [2] B.S. Mamatha, K.K. Namitha, S. Amudha, J. Smitha, G.A. Ravishankar, Food Chemistry 101 (2007) 1707–1713. [3] L. Jiao, X. Li, T. Li, P. Jiang, L. Zhang, M. Wu, L. Zhang, International Immunopharmacology 9 (2009) 324–329. [4] I. Michalak, K. Chojnacka, Applied Biochemistry and Biotechnology 160 (2010) 1540–1556. [5] M. Cho, H. Lee, I. Kang, M. Won, S. You, Food Chemistry 127 (2011) 999–1006. [6] X. Qi, W. Mao, Y. Gao, Y. Chen, Y. Chen, C. Zhao, N. Li, C. Wang, M. Yan, C. Lin, J. Shan, Carbohydrate Polymers 90 (2012) 1804–1810. [7] L. Jiao, P. Jiang, L. Zhang, M. Wu, Biotechnology and Bioprocess Engineering 15 (2010) 421–428. [8] J. Zhao, P. Jiang, Z. Liu, W. Wei, H. Lin, F. Li, J. Wang, S. Qin, Chinese Science Bulletin (2012), http://dx.doi.org/10.1007/s11434-012-5441-3. [9] M. Cho, C. Yang, S.M. Kim, S.G. You, Food Science and Biotechnology 19 (2010) 525–533. [10] J.K. Kim, M.L. Cho, S. Karnjanapratum, I.S. Shin, S.G. You, International Journal of Biological Macromolecules 49 (2011) 1051–1058. [11] X. Chen, J. Lu, Y. Zhang, J. He, X. Guo, G. Tian, L. Jin, International Journal of Biological Macromolecules 43 (2008) 252–256. [12] H. Kuang, Y. Xia, B. Yang, Q. Wang, Y. Wang, Carbohydrate Polymers 83 (2011) 787–795. [13] H. Yuan, J. Song, X. Li, N. Li, J. Dai, Cancer Letters 243 (2006) 228–234. [14] J.R. Chen, Z.Q. Yang, T.J. Hu, Z.T. Yan, T.X. Niu, L. Wang, D.A. Cui, M. Wang, Fitoterapia 81 (2010) 1117–1124. [15] C. De Simone, P. Ferranti, G. Picariello, I. Scognamiglio, A. Dicitore, F. Addeo, L. Chianese, P. Stiuso, Molecular Nutrition & Food Research 55 (2011) 229–238. [16] M. Wang, X.Y. Meng, R.L. Yang, T. Qin, X.Y. Wang, K.Y. Zhang, C.Z. Fei, Y. Li, Y.L. Hu, F.Q. Xue, Carbohydrate Polymers 89 (2012) 461–466. [17] Q. Chen, S. Zhang, H. Ying, X. Dai, X. Li, C. Yu, H. Ye, Carbohydrate Polymers 88 (2012) 1476–1482. [18] X.Q. Cheng, H. Li, X.L. Yue, J.Y. Xie, Y.Y. Zhang, H.Y. Di, D.F. Chen, Journal of Ethnopharmacology 130 (2010) 363–368. [19] L. Sun, L. Wang, Y. Zhou, Carbohydrate Polymers 87 (2012) 1206–1210. [20] Y. Bai, P. Zhang, G. Chen, J. Cao, T. Huang, K. Chen, International Immunopharmacology 12 (2012) 611–617. [21] R.W. Birk, A. Gratchev, N. Hakiy, O. Politz, K. Schledzewski, P. Guillot, C.E. Orfanos, S. Goerdt, Hautarzt 52 (2001) 193–200. [22] M.W. Lingen, Archives of Pathology and Laboratory Medicine 125 (2001) 67–71. [23] M. Hermes-Lima, J.M. Storey, K.B. Storey, Comparative Biochemistry and Physiology 120 (1998) 437–448. [24] J. Du, H. Zhu, P. Liu, J. Chen, Y. Xiu, W. Yao, T. Wu, Q. Ren, Q. Meng, W. Gu, W. Wang, Fish & Shellfish Immunology 34 (2013) 315–323. [25] Y.M. Thu, A. Richmond, Cytokine & Growth Factor Reviews 21 (2010) 213–226. [26] S. Ganesan, K. Aggarwal, N. Paquette, N. Silverman, Current Topics in Microbiology and Immunology 349 (2011) 25–60. [27] A. Dev, S. Iyer, B. Razani, G. Cheng, Current Topics in Microbiology and Immunology 349 (2011) 115–143. [28] T.S. Blackwell, J.W. Christman, American Journal of Respiratory Cell and Molecular Biology 17 (1997) 13–19.
