Food & Function

View Article Online View Journal

Accepted Manuscript

This article can be cited before page numbers have been issued, to do this please use: D. Qiao, C. Wei, C. Ke and X. Zeng, Food Funct., 2015, DOI: 10.1039/C4FO01121J.

This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.

www.rsc.org/foodfunction

Page 1 of 27

Food & Function View Article Online

DOI: 10.1039/C4FO01121J

Effects of Hyriopsis cumingii polysaccharides on angiogenesis, macrophage chemotaxis, proliferation and phagocytosis

Published on 05 January 2015. Downloaded by Selcuk University on 17/01/2015 08:41:33.

Food & Function Accepted Manuscript

Deliang Qiao,* ab Chuanbao Wei,a Chunlin Keb and Xiaoxiong Zeng* b

a

College of Biological and Pharmaceutical Engineering, West Anhui University, Lu’an 237012,

China. E-mail: [email protected]; Fax: +86-564-3305033; Tel: +86-564-3305073 b

College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095,

China. E-mail: [email protected]; Fax: +86-25-84396791; Tel: +86-25-84396791 1

Food & Function

Page 2 of 27 View Article Online

DOI: 10.1039/C4FO01121J

Abstract Anti-angiogenic activity of crude Hyriopsis cumingii polysaccharides (HCPS) and its purified fractions (HCPS-1, HCPS-2 and HCPS-3) were evaluated in vivo by

Published on 05 January 2015. Downloaded by Selcuk University on 17/01/2015 08:41:33.

of crude HCPS and its purified fractions on the chemotaxis, proliferation and phagocytosis of peritoneal macrophage were tested by cell model in vitro and cyclophosphamide-induced immuno-suppression animal model in vivo. The results showed that HCPS could significantly suppress the neovascularization of chicken embryo CAM, promote peritoneal macrophage migrating to monocyte chemotactic protein-1 (MCP-1), propagating and devouring sheep red blood cell (SRBC) in a dose-dependent manner. In addition, HCPS-3 showed stronger immunostimulatory activities in vitro than crude HCPS, HCPS-1 and HCPS-2. The beneficial effects of HCPS on the immune system might be, at least in part, attributed to the improvement of chemotaxis, proliferation and phagocytosis of peritoneal macrophage. All these results suggested that HCPS was a potential immunoenhancing and anti-tumor agent. Keywords: Hyriopsis cumingii; Polysaccharides; Anti-angiogenesis; Chemotaxis; Cell proliferation; Phagocytosis

2

Food & Function Accepted Manuscript

chicken embryo chorioallantoic membrane (CAM) assay. And the promoting effects

Page 3 of 27

Food & Function View Article Online

DOI: 10.1039/C4FO01121J

1. Introduction Immunostimulatory activities of polysaccharides, such as increasing index of immune organ, activating immunocyte, promoting production of cytokine and

Published on 05 January 2015. Downloaded by Selcuk University on 17/01/2015 08:41:33.

blood vessels, is recognized as a fundamental process and an essential component of tumor growth and metastasis. Anti-angiogenesis has become a strategy for the treatment of cancer,6 and anti-angiogenic tumour therapy has gained much interest in preclinical and clinical assessments.7 Recently, some natural polysaccharides with immuno-modulating and anti-angiogenic activity were isolated and characterized.8,9 Hyriopsis cumingii, a member of freshwater pearl mussels, is widely cultivated in China for producing quality pearls. In addition, it is also one of the animals that are used as medicine and food simultaneously in traditional Chinese medicine. Recent studies demonstrated that the H. cumingii flesh was rich in polysaccharides,10,11,12 which resulted in nutrient and pharmacological functions such as anti-tumor, antiinflammation, anti-oxidation and anti-aging etc.10,13,14,15 In previous studies, we optimized the extraction of Hyriopsis cumingii polysaccharides (HCPS), and purified the crude HCPS. Three purified fractions of HCPS (HCPS-1, HCPS-2 and HCPS-3) were obtained.12 Furthermore, we evaluated the antioxidant activities of crude HCPS, HCPS-1, HCPS-2 and HCPS-3.15 In this paper, we report the anti-angiogenesis, stimulating macrophage chemotaxis, proliferation and phagocytosis of crude HCPS in vitro and in vivo, as well as its purified fractions (HCPS-1, HCPS-2 and HCPS-3) in vitro. 2. Results and discussion 2.1. Anti-angiogenic activity in vivo of HCPS on chick embryo chorioallantoic membrane (CAM) 3

Food & Function Accepted Manuscript

antibody et al, have been reported widely.1,2,3,4,5 Angiogenesis, the formation of new

Food & Function

Page 4 of 27 View Article Online

DOI: 10.1039/C4FO01121J

The CAM-assay has been showed to be a suitable in vivo model in studying many mechanisms relevant for physiological and pathological angiogenesis.16 So, the potential anti-angiogenic activity of HCPS was examined in vivo by using the chicken

Published on 05 January 2015. Downloaded by Selcuk University on 17/01/2015 08:41:33.

with branches and vascular networks formed densely (Fig. 1A). By compared with the control group, following results were obtained. At the concentration of 2.5 mg/mL, HCPS-1 did not inhibited the neovascularization (Fig. 1, C-1), and the crude HCPS and HCPS-2 suppressed slightly (Fig. 1, B-1, D-1), whereas HCPS-3 presented the severe inhibition on the neovascularization (Fig. 1, E-1). At 5.0 mg/mL, crude HCPS, HCPS-2 and HCPS-3 could inhibit the neovascularization severely (Fig. 1, B-2, D-2, E-2), only HCPS-1 showed slight inhibition (Fig. 1, C-2). When sample concentration was 10 mg/mL, crude HCPS and its purified fractions (HCPS-1, HCPS-2 and HCPS-3) all presented the severe inhibition on the neovascularization (Fig. 1, B-3, C-3, D-3, E3). It was also obtained that HCPS-3 exhibited the strongest anti-angiogenic activity, while HCPS-1 showed the weakest ability of anti angiogenesis. And the antiangiogenic activity of crude HCPS was stronger than that of HCPS-2. These results implied that crude HCPS and its purified fractions (HCPS-1, HCPS-2 and HCPS-3) could suppress the neovascularization of chicken embryo CAM in a dose-dependent manner.

4

Food & Function Accepted Manuscript

embryo CAM model, and Fig. 1 showed the result. In control group, blood vessels

Page 5 of 27

Food & Function View Article Online

Fig. 1 Antiangiogenic activity of HCPS on the chick embryo CAM in vitro. A: double distilled water (control); B: crude HCPS (B1: 2.5 mg/mL; B2: 5 mg/mL; B3: 10 mg/mL); C: HCPS-1 (C1: 2.5 mg/mL; C2: 5 mg/mL; C3: 10 mg/mL); D: HCPS-2 (D1: 2.5 mg/mL; D2: 5 mg/mL; D3: 10 mg/mL); E: HCPS-3 (E1: 2.5 mg/mL; E2: 5 mg/mL; E3: 10 mg/mL).

Angiogenesis, plays an important role in physiology and pathology, is influenced by the microenvironment and modulated by a multitude of pro- and anti-angiogenic 5

Food & Function Accepted Manuscript

Published on 05 January 2015. Downloaded by Selcuk University on 17/01/2015 08:41:33.

DOI: 10.1039/C4FO01121J

Food & Function

Page 6 of 27 View Article Online

DOI: 10.1039/C4FO01121J

factors. These factors, involved in different steps of the angiogenic process, include that affecting endothelial proliferation and migration, affecting blood coagulation and fibrinolysis and affecting the degradation of basement membranes and the extra-

Published on 05 January 2015. Downloaded by Selcuk University on 17/01/2015 08:41:33.

growth factors of the FGF family which stimulates endothelial cell proliferation in vitro and angiogenesis in vivo. FGF-2 exerts its biological effects after interaction with so-called low-affinity receptors (glycosaminoglycans, e.g., heparin, heparan sulfates) at the cell surface required for the binding to its high-affinity receptor tyrosine kinases.16 Studies have demonstrated roles for vascular endothelial growth factor (VEGF) in endothelial cell proliferation, migration, protease expression, cell adhesion, capillary tube formation, vessel maturation and vascular permeability.18 In angiogenesis, one of the most specific and important factors is VEGF, which links specifically to different membrane receptors of endothelial cells. In addition to stimulating angiogenesis, VEGF is also important for maintaining the integrity and permeability of blood vessels.19 In the present work, we found that crude HCPS and its purified fractions (HCPS-1, HCPS-2 and HCPS-3) exhibited potential antiangiogenic activities in a dose-dependent manner. About anti-angiogenic mechanism, whether HCPS inhibited the secretion and/or linkage of VEGF to membrane receptors of endothelial cells, or HCPS inhibited the secretion and/or binding of FGF-2 to receptors at the cell surface, and other, further and deep researches are needed. 2.2. Effect of HCPS on peritoneal macrophage chemotaxis in vitro The chemotaxis of macrophage, reflected by the migration of macrophage, was an important factor that impacting the macrophage’s function when body was of physiological or pathological.20 Monocyte chemotactic protein-1 (MCP-1), which is secreted by a number of cell types including neutrophils, macrophages, peritoneal 6

Food & Function Accepted Manuscript

cellular matrix.17 Fibroblast growth factor 2 (FGF-2) is one of the 19 heparin-binding

Page 7 of 27

Food & Function View Article Online

DOI: 10.1039/C4FO01121J

fluid mesothelial cells, and endometrial cells, is one of the key chemokines that regulate migration and infiltration of monocytes and macrophages.21 There, the action of HCPS on the migration of peritoneal macrophage to MCP-1 was investigated in

Published on 05 January 2015. Downloaded by Selcuk University on 17/01/2015 08:41:33.

HCPS-2 and HCPS-3) on the peritoneal macrophage chemotaxis were presented in Fig.2. In group II (50 µg/mL), there was no significantly difference (P > 0.05) in the ability of improving peritoneal macrophage chemotaxis among crude HCPS, HCPS-1, HCPS-2 and HCPS-3. But, the promoting effects of HCPS increased significantly (P < 0.05) with the increase of sample concentration ranging from 50 to 200 µg/mL. At the concentration of 200 µg/mL, the differences in the capability of strengthening chemotaxis among crude HCPS, HCPS-1, HCPS-2 and HCPS-3 were significant (P < 0.05), and the chemotaxis stimulating index of crude HCPS, HCPS-1, HCPS-2 and HCPS-3 was 1.93, 1.42, 1.62 and 2.17, respectively. About chemotaxis promoting activity, it was HCPS-3, crude HCPS, HCPS-2 and HCPS-1 in orderly from strongest to weakest. This result demonstrated that crude HCPS and its purified fractions (HCPS-1, HCPS-2 and HCPS-3) could promote peritoneal macrophage migrating to MCP-1 in a dose-dependent manner. Macrophage were known to have a variety of functions, these included not only the non-specific defense against bacteria and other exogenous matter but also the presentation of antigen information to lymphocytes in their immune responses and production of various immunoregulating factors.22 Chemotaxis was the directed movement of a cell toward a chemoattractant molecule and occurred when these chemoattractant signaling molecules were released either from bacteria or from cells stimulated by physical damage, neuropeptides, or UV radiation.23 The result, HCPS could promote the chemotaxis of peritoneal macrophage in a dose-dependent manner, 7

Food & Function Accepted Manuscript

vitro. And the promoting effects of crude HCPS and its purified fractions (HCPS-1,

Food & Function

Page 8 of 27 View Article Online

DOI: 10.1039/C4FO01121J

indicated that crude HCPS and purified fractions (HCPS-1, HCPS-2 and HCPS-3) might be used as chemoattractant cooperator to recruit peritoneal macrophage to

Fig. 2 Effects of HCPS on the peritoneal macrophage chemotaxis in vitro. Group II: low dose of HCPS (50 µg/mL); Group III: medium dose of HCPS (100 µg/mL); Group IV: high dose of HCPS (200 µg/mL).

2.3. Effect of HCPS on peritoneal macrophage proliferation in vitro Stimulatory effects of crude HCPS and its purified fractions (HCPS-1, HCPS-2 and HCPS-3) on the proliferation of peritoneal macrophage were tested in vitro by 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and presented in Fig. 3. The stimulating indices of crude HCPS and its purified fractions, all exceeded 1.0, indicated that all the HCPS samples had the activities to promote the proliferation of peritoneal macrophage. In addition, the promoting effects increased significantly (P

8

Food & Function Accepted Manuscript

Published on 05 January 2015. Downloaded by Selcuk University on 17/01/2015 08:41:33.

defend against exogenous invasion.

Page 9 of 27

Food & Function View Article Online

DOI: 10.1039/C4FO01121J

﹤0.05) with the increase of sample concentration ranging from 50 to 200 µg/mL. At the concentration of 200 µg/mL, the stimulating index was 3.38, 2.42, 2.83 and 3.70 for the crude HCPS, HCPS-1, HCPS-2 and HCPS-3, respectively. It was notably that

stimulating activity. And the stimulating activity of crude HCPS was stronger than that of HCPS-2. The result suggested that crude HCPS and its purified fractions of HCPS-1, HCPS-2 and HCPS-3 could improve the proliferation of peritoneal macrophage in a dose-dependent manner.

Fig. 3 Effects of HCPS on the peritoneal macrophage proliferation in vitro. Group II: low dose of HCPS (50 µg/mL); Group III: medium dose of HCPS (100 µg/mL); Group IV: high dose of HCPS (200 µg/mL).

Macrophages, is widely present in gastrointestinal tract, abdominal cavity and other tissues, play significant roles in the host defense mechanism. In order to effectively exercise their functions, mature macrophages must be activated. The activated 9

Food & Function Accepted Manuscript

Published on 05 January 2015. Downloaded by Selcuk University on 17/01/2015 08:41:33.

HCPS-3 presented the strongest promoting effect, while HCPS-1 showed the weakest

Food & Function

Page 10 of 27 View Article Online

DOI: 10.1039/C4FO01121J

macrophages show strong endocytosis towards infection agents.24 Immunomodulatory activity not only involves effects on macrophage activation but also on cell proliferation and differentiation.25 It has been reported that one of the pathways of

Published on 05 January 2015. Downloaded by Selcuk University on 17/01/2015 08:41:33.

proliferation of macrophages.24,25,26 Similar result, crude HCPS and its purified fractions (HCPS-1, HCPS-2 and HCPS-3) possessed stimulatory effects on the proliferation of peritoneal macrophage, was obtained in this work. 2.4. Effect of HCPS on peritoneal macrophage devouring sheep red blood cell (SRBC) in vitro One of the immune functions of macrophage is devouring extraneous material. In the present work, promoting effects of crude HCPS and its purified fractions (HCPS-1, HCPS-2 and HCPS-3) on the phagocytosis of peritoneal macrophage were measured in vitro by devouring SRBC assay. The stimulating indices, all above 1.0 (Fig. 4), indicating that all the HCPS samples had the activities of promoting peritoneal macrophages to devour SRBC. Furthermore, the improving effects increased with the increase of sample concentration ranging from 50 to 200 µg/mL. At the concentration of 200 µg/mL, the stimulating index was 4.05, 1.75, 2.98 and 4.75 for the crude HCPS, HCPS-1, HCPS-2 and HCPS-3, respectively. Notably, HCPS-3 presented the strongest promoting effect, while HCPS-1 had the weakest stimulating activity. The results demonstrated that crude HCPS and its purified fractions could strengthen the phagocytosis of peritoneal macrophages in a dose-dependent manner. Macrophage is the most important professional phagocyte and is able to clear large amounts of various materials.27 It is reported that macrophages can phagocytize extraneous materials to produce phagosome and then scavenge those extraneous matters by using some enzymes. Thus, the immune activity of macrophages can be 10

Food & Function Accepted Manuscript

polysaccharides exhibiting their immunostimulating activities was improving the

Page 11 of 27

Food & Function View Article Online

DOI: 10.1039/C4FO01121J

represented indirectly by its phagocytosis. The present result, crude HCPS and its purified fractions had the activity of stimulating peritoneal macrophages to devour SRBC, indicated that the beneficial effect of HCPS on immune system might be

Fig. 4 Effects of HCPS on the peritoneal macrophages devouring SRBC in vitro. Group II: low dose of HCPS (50 µg/mL); Group III: medium dose of HCPS (100 µg/mL); Group IV: high dose of HCPS (200 µg/mL).

2.5. Effect of crude HCPS on chemotaxis of peritoneal macrophage in vivo Immuno-suppressed

mice

were

induced

by

hypodermic

injection

of

cyclophosphamide (CPA) in order to investigate the effects of crude HCPS on the chemotaxis (migrating to MCP-1) and phagocytosis (devouring SRBC) of peritoneal macrophage in vivo, and the promoting effect of crude HCPS on the chemotaxis was showed in Table 1. A significant decrease of chemotaxis stimulating index was observed after treatment of CPA in Group II (model group) compared with Group I 11

Food & Function Accepted Manuscript

Published on 05 January 2015. Downloaded by Selcuk University on 17/01/2015 08:41:33.

partly attributed to the improvement of phagocytosis of peritoneal macrophage.

Food & Function

Page 12 of 27 View Article Online

DOI: 10.1039/C4FO01121J

(control group with stimulating index of 1.0). In group III (crude HCPS of low dose), there was a significant increase of stimulating index than that in Group II, although the stimulating index was not so many as that in Group I. From group III to group V

Published on 05 January 2015. Downloaded by Selcuk University on 17/01/2015 08:41:33.

with the increase of HCPS concentration. And, at the concentration of 800 mg/kg BW, the stimulating index was 1.94. This result demonstrated that hypodermic injection of CPA could suppress the chemotaxis of peritoneal macrophage, and gastric gavage of crude HCPS could improve the chemotaxis of peritoneal macrophage in a dosedependent manner. The crude HCPS was able to act against the chemotactic suppression induced by CPA in vivo.

Table 1 Effects of crude HCPS on the chemotaxis and devouring SRBC of peritoneal macrophage in vivo Group

Stimulating index of devouring SRBC

Stimulating index of chemotaxis



0.8125 ± 0.08133 a

0.8254 ± 0.10635 a



1.1042 ± 0.09826 b

0.9796 ± 0.09150 b



2.9375 ± 0.27792 c

1.4278 ± 0.09602 c



3.6042 ± 0.14583 d

1.9406 ± 0.10508 d

Different superscript (a, b, c, d) indicted there was significant difference (P < 0.05) between groups. Same superscript suggested difference between groups was not significant (P > 0.05).

Lipopolysaccharide (LPS), one of the main macrophage-activating factors, activates macrophage mainly by surface membrane receptor. And four kinds of 12

Food & Function Accepted Manuscript

(crude HCPS of high dose), the stimulating index increased significantly (P < 0.05)

Page 13 of 27

Food & Function View Article Online

DOI: 10.1039/C4FO01121J

receptors including scavenger, CD18, CD14, and TLR4 have been reported.24 In the present work, we speculated that gastric gavage of crude HCPS promoted peritoneal macrophage migrating to MCP-1 by increasing the expression of membrane receptors

Published on 05 January 2015. Downloaded by Selcuk University on 17/01/2015 08:41:33.

2.6. Effect of crude HCPS on peritoneal macrophage devouring SRBC in vivo Promoting effect in vivo of crude HCPS on the phagocytosis (devouring SRBC) of peritoneal macrophage was also presented in Table 1. There was a significant decrease of phagocytosis stimulating index in Group II (CPA model group) compared with Group I (control group with stimulating index of 1.0). In group III (crude HCPS of low dose), a significant increase of stimulating index was observed than that in Group II. From group III to group V (crude HCPS of high dose), the stimulating index increased significantly (P < 0.05) with the increase of HCPS concentration. And, at the concentration of 800 mg/kg BW, the stimulating index was 3.60. This result indicated that treatment of CPA could inhibit the phagocytosis of peritoneal macrophage, and administration of crude HCPS could promote the phagocytosis of peritoneal macrophage in a dose-dependent manner. The crude HCPS was able to retard the phagocytic suppression induced by CPA in vivo. Macrophages initiate the innate immune response by recognizing pathogens, phagocytosing

them,

and

secreting

inflammatory

mediators.27

The

immunomodulatory polysaccharides appeared to activate immune responses primarily by activation of macrophages, although direct activation of B cells and other immune cells has been implicated.28 The present result, gastric gavage of crude HCPS could promote peritoneal macrophage to devour SRBC in a dose-dependent manner, implied that crude HCPS might be used as activator to active peritoneal macrophages and initiate immune response subsequently. Macrophages not only can initiate immune 13

Food & Function Accepted Manuscript

of macrophage. But, it needs more researches to validate.

Food & Function

Page 14 of 27 View Article Online

DOI: 10.1039/C4FO01121J

responses, but also can serve as effector cells.28 After being activated, macrophages could secrete a variety of membrane receptors and cytokines, such as CD18, CD14, TLR4, IL-1, TNF-α, NO, and so on, which play key roles in the immune system. As

Published on 05 January 2015. Downloaded by Selcuk University on 17/01/2015 08:41:33.

cause the interaction between immune cells.24 After treated with HCPS, whether more receptors and cytokines were secreted by macrophages, it needs deep researches. It has been reported that sulfated polysaccharides exhibited higher biological activities such as anti-virus, immunity-enhancement, anti-coagulation and anti-tumor than unsulfated polysaccharides.29,30 In our previous report, we demonstrated that HCPS-3 had much higher content of sulfuric radical than crude HCPS, HCPS-1 and HCPS-2.12 Therefore, the results mentioned above that HCPS-3 showed stronger antiangiogenic activity and promoting effects on the macrophage chemotaxis, proliferation and phagocytosis than crude HCPS, HCPS-1 and HCPS-2 might be attributed to its higher content of sulfuric radical. The functions of macrophage including phagocytosis and chemotaxis are known to grow stronger with activation.22 The augmentation of phagocytosis and chemotaxis of macrophage is enough to suggest that other functions of macrophage in the body were simultaneously augmented and the immunological function was also strengthened.22 The administration of HCPS significantly increased the stimulating index of chemotaxis, proliferation and phagocytosis of peritoneal macrophage in a dosedependent manner. Furthermore, the HCPS could suppress the neovascularization of chicken embryo CAM in a dose-dependent manner. All these results suggested that HCPS was a potent immunoenhancing and anti-tumor agent.

3. Experimental 14

Food & Function Accepted Manuscript

endogenous signals, they could induce secondary production of other cytokines and

Page 15 of 27

Food & Function View Article Online

DOI: 10.1039/C4FO01121J

3.1. Materials and reagents Crude HCPS and its purified fractions of HCPS-1, HCPS-2 and HCPS-3 were prepared from H. cumingii according to our previous method.12 Mussel H. cumingii

Published on 05 January 2015. Downloaded by Selcuk University on 17/01/2015 08:41:33.

extracted by using methods of hot water lixiviation, ethanol precipitation and Sevag’s deproteination. The crude HCPS was separated firstly through DEAE-cellulose 52 chromatography column and further purified through chromatography column of Sephadex G-100. Then three purified fractions, named as HCPS-1, HCPS-2 and HCPS-3, were obtained. Preliminary characterizations of crude HCPS and its purified fractions were reported simultaneously in our earlier article.12 Sugar content of crude HCPS, HCPS-1, HPS-2 and HCPS-3 was 76.43%, 98.88%, 96.60% and 80.06% respectively. Relative molecular weight of HCPS-1, HPS-2 and HCPS-3 was 432.2 kDa, 457.9 kDa and 503.1 kDa respectively. Relative viscosity (to water) of crude HCPS, HCPS-1, HPS-2 and HCPS-3 was 1.1810, 1.1592, 1.1631 and 1.3080 respectively. About monosaccharide composition, crude HCPS was composed of rhamnose, arabinose, fucose, mannose, glucose and galactose with a molar ratio of 5.71 : 2.21 : 1.69 : 3.40 : 82.21 : 4.78. HCPS-1 was composed of arabinose and glucose with a molar ratio of 2.12 : 97.88. HCPS-2 was composed only glucose. HCPS-3 was composed of rhamnose, fucose, mannose, glucose and galactose with a molar ratio of 13.80 : 4.51 : 7.70 : 64.92 : 9.07. The Kunming mice (8-weeks-old), grade of specific pathogen frees with body weight (BW) of 20 ± 2 g, were purchased from the Experimental Animal Center of Anhui Medical University (Hefei, China). All experiments that involved the use of live animals were performed in compliance with the relevant laws and institutional guidelines. Chick embryos were obtained from Xingdian Poultry Co. (Nanjing, China). 15

Food & Function Accepted Manuscript

were purchased from Jiangning Pearl Farm (Nanjing, China), and crude HCPS was

Food & Function

Page 16 of 27 View Article Online

DOI: 10.1039/C4FO01121J

MTT were purchased from Sigma Chemical Co., USA. Fetal bovine serum (FBS), Wright staining solution and RPMI-1640 medium were obtained from Shanghai Sangon Biological Engineering Technology & Services Co., China. SRBC and rabbit

Published on 05 January 2015. Downloaded by Selcuk University on 17/01/2015 08:41:33.

China). Mouse MCP-1 was purchased from Cloud-Clone Corp., China. CPA was purchased from Jiangsu Hengrui Medcine Co., China. Transwell insert plate and cell plate were purchased from Corning Incorporated, China. All other reagents were of analytical grade. Preparation of 6% starch medium: beef extract 0.3 g, peptone 1.0 g and NaCl 0.5 g were added into 100 mL distilled water, then 6.0 g soluble starch was appended and sterilized by autoclaving. 3.2. Determination of HCPS acting on angiogenesis in vivo Anti-angiogenic activity of HCPS was measured by chicken embryo CAM assay as described with slight modifications.8,31 A group of 7-day-old fertilized eggs was incubated at 37.5℃ with 55% relatively humidity. On day 8, eggs were rinsed with 75% ethanol and a window of 1 cm2 was carefully created with a puncher on the broad side of the egg. Double distilled water (20 µL/egg, control group) or test sample (20 µL/egg, crude or purified HCPS at a concentration of 2.5, 5 or 10 mg/mL) was added into a sterile cotton ball. After the cotton ball was placed on the CAM of chicken embryo, a permeable sticky tape was used to seal the window immediately. Incubated for 3 days, the eggshell was pushed aside around the window, and the blood vessels were photographed and analyzed in stereomicroscope (Stemi 2000-C, Zeiss, Germany). 3.3. Determination of HCPS acting on chemotaxis, proliferation and phagocytosis of macrophage in vitro 16

Food & Function Accepted Manuscript

anti SRBC hemolysin were purchased from Yuhuan Nanfang Regents Co. (Zhejiang,

Page 17 of 27

Food & Function View Article Online

DOI: 10.1039/C4FO01121J

3.3.1. Assay of macrophage chemotaxis The chemotaxis of peritoneal macrophage towards MCP-1 was determined by transwell insert assay according to the reported method with minor modification.20,32

Published on 05 January 2015. Downloaded by Selcuk University on 17/01/2015 08:41:33.

Kunming mice. Three days later, the mice were killed by cervical dislocation and peritoneal macrophages were harvested and adjusted to a density of 5×105 cell/mL in the RPMI 1640 medium supplemented with FBS (10%). Double distilled water (300 µL/well, control group) or test sample (300 µL/well, crude or purified HCPS at a concentration of 100, 200 or 400 µg/mL) was appended to lower chamber of transwell insert of 24-well transwell plate after 300 µL of MCP-1 (20 ng/mL) was added into lower chamber of transwell insert. Then, the peritoneal macrophages suspension (100 µL/well, 5×105 cell/mL) was added into upper chamber of transwell insert. After transwell plate was incubated in a humidified 5% CO2 incubator (3111, Thermo Scientific, USA) at 37 ℃ for 3 h, cotton wool with 0.9% NaCl was used to remove the cell in the upper chamber of transwell insert slightly. Then, the cell on the lower surface of microporous membrane, chemoattracted by HCPS, were immobilized with methanol and dyed with Wright staining. Finally, the chemoattracted cell on the lower surface of microporous membrane were photographed and counted in microscope (×400) (XD-202, Jiangnan Novel, China). The stimulating index of peritoneal macrophage chemotaxis was calculated by using following formula: Stimulating index = N1 / N0, where N1 is the accumulated cell number of 5 visual fields of sample group and N0 is the accumulated cell number of 5 visual fields of control group. 3.3.2. Assay of macrophage proliferation Measurement of peritoneal macrophage proliferation was done according to the MTT-based colorimetric method with slight modification.33,34 Briefly, the peritoneal 17

Food & Function Accepted Manuscript

In brief, sterile 6% starch medium (50 mL/kg BW) was injected intraperitoneally into

Food & Function

Page 18 of 27 View Article Online

DOI: 10.1039/C4FO01121J

macrophages of Kunming mice were prepared and adjusted to a density of 2×106 cell/mL in the RPMI 1640 medium with FBS (10%). Then, the peritoneal macrophages suspension (2×106 cell/mL) was seeded into a 96-well flat-bottom plate

in a humidified 5% CO2 incubator for 3 h. Non-adherent cells were removed by washing three times with RPMI 1640 medium. After 50 µL of RPMI 1640 medium with 10% FBS was added into each well, the RPMI 1640 medium with 10% FBS (50 µL/well, control group) or test sample (50 µL/well, crude or purified HCPS at a concentration of 100, 200 or 400 µg/mL) was appended to respective well. The cell plate was incubated in a 5% CO2 incubator at 37 ℃ for 48 h, then MTT solution (20 µL/well, 5 mg/mL) was added and the plate was further incubated for 4 h. Dimethyl sulfoxide of 150 µL was added into each well after medium in well was removed, then the absorbance of each well at 490 nm was measured by an enzyme-linked immunosorbent assay (ELISA) microplate reader (iMark, Bio-RAD, Japan). The stimulating index of peritoneal macrophage proliferation was calculated by the following equation: Stimulating index = A1 / A0, where A1 is the absorbance of sample group and A0 is the absorbance of control group. 3.3.3. Assay of macrophage devouring SRBC Devouring SRBC of peritoneal macrophages was measured according to the reported method with some modification.27,28 Briefly, the peritoneal macrophages were prepared, seeded in a 48-well flat-bottom plate (200 µL/well, 2×106 cell/mL) and allowed to adhere as described previously. Non-adherent cells were removed by washing three times with RPMI 1640 medium. The RPMI 1640 medium with 10% FBS (100 µL/well, control group) or test sample (100 µL/well, crude or purified HCPS at a concentration of 100, 200 or 400 µg/mL) was appended to respective well 18

Food & Function Accepted Manuscript

Published on 05 January 2015. Downloaded by Selcuk University on 17/01/2015 08:41:33.

(100 µL/well) and the cells were allowed to adhere to the bottom of the plate at 37 ℃

Page 19 of 27

Food & Function View Article Online

DOI: 10.1039/C4FO01121J

after 100 µL of RPMI 1640 medium with 10% FBS was added into each well. The cell plate was incubated in a 5% CO2 incubator at 37 ℃ for 48 h and non-adherent cells were removed by RPMI 1640 medium, then 200 µL of hemolysin-opsonized

5% (V/V) SRBC suspension, then the mixture was water bathed at 37 ℃ for 30 min) was added into each well and further incubated for 120 min. After non-devoured SRBC were removed by RPMI 1640 medium, 100 µL of 0.08 mol/L NaCL (containing 0.18 mol/L glacial acetic acid) was added into each well for 10-15 min to swell the peritoneal macrophages. The fresh chromogenic solution (12.15 mL of 0.1 mol/L glacial acetic acid, 12.85 mL of 0.2 mol/L Na2HPO4, 20 mg of o-phenylene diamine and 25 mL of double distilled water were added into 150 mL of 8.82 mol/L H2O2)

of 200 µL was appended to each well and the cell plate was water bathed at 37 ℃ for 10 min, then 100 µL of l mol/L H2SO4 was added into each well to stop the reaction. Finally, the absorbance at 450 nm of each well was detected by an ELISA microplate reader. Stimulating index of devouring SRBC of peritoneal macrophages was calculated by the following equation: Stimulating index = A1 / A0, where A1 is the absorbance of sample group and A0 is the absorbance of control group. 3.4. Determination of crude HCPS acting on macrophage chemotaxis and phagocytosis in vivo 3.4.1. Animal grouping and experimental design Female Kunming mice were housed in an air-conditioned animal room with constant temperature (25 ± 1 ℃) and 60-70% relative humidity, then the mice were free to access to food and water and kept on a 12-h light/dark cycles during the experiments. After adapting to their environment for 1 week, these mice were randomly divided into five groups (10 for each) for experiment. Mice in Group I 19

Food & Function Accepted Manuscript

Published on 05 January 2015. Downloaded by Selcuk University on 17/01/2015 08:41:33.

SRBC (Equal volume of rabbit anti SRBC hemolysin, titer of 1:3000, was mixed with

Food & Function

Page 20 of 27 View Article Online

DOI: 10.1039/C4FO01121J

(control group) were treated with 0.9% NaCl by gastric gavage (25 mL/kg BW) once daily for seven days and by hypodermic injection (15 mL/kg BW) on days 1, 3, 5 and 7. Mice in Group II (model group) were administrated with gastric gavage of 0.9%

Published on 05 January 2015. Downloaded by Selcuk University on 17/01/2015 08:41:33.

0.5% CPA (15 mL/kg BW) on days 1, 3, 5 and 7. Mice in Group III (crude HCPS of low dose), Group IV (crude HCPS of medium dose) and Group V (crude HCPS of high dose) were treated with crude HCPS at dose of 200,400 and 800 mg/kg BW by gastric gavage (25 mL/kg BW), respectively once daily for seven days and with 0.5% CPA by hypodermic injection (15 mL/kg BW) on days 1, 3, 5 and 7. At the eighth day, sterile 6% starch medium (50 mL/kg BW) was injected intraperitoneally. Three days later, the mice were killed by cervical dislocation and peritoneal macrophages were harvested and adjusted to a density of 5×105 or 2×106 cell/mL in the RPMI 1640 medium supplemented with FBS (10%). 3.4.2. Assay of chemotaxis and phagocytosis of peritoneal macrophage The chemotaxis of peritoneal macrophages was detected by transwell insert assay. After MCP-1 of 10 ng/mL (600 µL/well) was added into lower chamber of transwell insert of 24-well transwell plate, the peritoneal macrophages suspension (100 µL/well, 5×105 cell/mL) was appended into upper chamber of transwell insert. Then, the incubation of transwell plate, removing of unchemoattracted cells, immobilization and dyeing of chemoattracted cells and photographing and counting of stained cells were done as described in section 2.3.1. The stimulating index of peritoneal macrophage chemotaxis was calculated by using following formula: Stimulating index = N1 / N0, where N1 is the accumulated cell number of 5 visual fields of model group or sample group and N0 is the accumulated cell number of 5 visual fields of control group.

20

Food & Function Accepted Manuscript

NaCl (25 mL/kg BW) once a day for seven days and with hypodermic injection of

Page 21 of 27

Food & Function View Article Online

DOI: 10.1039/C4FO01121J

Phagocytosis of peritoneal macrophages was determined by devouring SRBC assay. Briefly, the adhering of peritoneal macrophages, removing of non-adherent cells, devouring of hemolysin-opsonized SRBC, removing of non-devoured SRBC, clearage

Published on 05 January 2015. Downloaded by Selcuk University on 17/01/2015 08:41:33.

described in section 2.3.3. Stimulating index of peritoneal macrophages devouring SRBC was calculated by the following equation: Stimulating index = A1 / A0, where A1 is the absorbance of model group or sample group and A0 is the absorbance of control group. 3.5. Statistical analysis The data were reported as mean ± standard deviation (SD) and evaluated by oneway analysis of variance (ANOVA) followed by the Duncan’s multiple-range tests. Difference was considered to be statistically significant if P < 0.05. All statistical analyses were carried out by using SPSS for Windows, Version11.5 (SPSS, Chicago, IL).

4. Conclusion In the present study, the anti-angiogenic activity of crude HCPS and its purified fractions (HCPS-1, HCPS-2 and HCPS-3) were evaluated in vivo by chicken embryo CAM assay. And the promoting effects of crude HCPS and its purified fractions on the chemotaxis, proliferation and phagocytosis of peritoneal macrophage were tested by cell model in vitro and CPA-induced immuno-suppression animal model in vivo. The

results

demonstrated

that

HCPS

could

significantly

suppress

the

neovascularization of chicken embryo CAM, strengthen the chemotaxis (migrating to MCP-1), proliferation and phagocytosis (devouring SRBC) of peritoneal macrophage in

a

dose-dependent

manner.

In

addition, 21

HCPS-3

showed

stronger

Food & Function Accepted Manuscript

and coloration of peritoneal macrophages and detection of absorbance were done as

Food & Function

Page 22 of 27 View Article Online

DOI: 10.1039/C4FO01121J

immunostimulatory activities in vitro than crude HCPS, HCPS-1 and HCPS-2. The beneficial effects of HCPS on the immune system might be, at least in part, attributed to the improvement of chemotaxis, proliferation and phagocytosis of peritoneal

Published on 05 January 2015. Downloaded by Selcuk University on 17/01/2015 08:41:33.

and anti-tumor agent. Further works about possible mechanisms, which HCPS inhibit the secretion and/or binding of anti-angiogenic factors to membrane receptors of endothelial cells, increase the expression of membrane receptors and cytokines of macrophage, are in progress.

Conflict of Interest The authors declare that there are no conflicts of interest.

Acknowledgement This work was partly supported by a grant-in-aid for scientific research from National Natural Science Foundation of China (No. 31271853) and Anhui Provincial Natural Science Foundation (No. 1308085MC33).

References 1 G. G. L. Yue, B. C. L. Chan, P. M. Hon, E. J. Kennelly, S. K. Yeung, B. R. Cassileth, K. P. Fung, P. C. Leung, and C. B. S. Lau, Immunostimulatory activities of polysaccharide extract isolated from Curcuma longa, Int. J. Biol. Macromol., 2010, 47, 342-347. 2 H. Zhao, Y. Li, Y. Wang, J. Zhang, X. Ouyang, R. Peng, and J. Yang, Antitumor and immunostimulatory activity of a polysaccharide–protein complex from 22

Food & Function Accepted Manuscript

macrophage. All these results suggested that HCPS was a potential immunoenhancing

Page 23 of 27

Food & Function View Article Online

DOI: 10.1039/C4FO01121J

Scolopendra subspinipes mutilans L. Koch in tumor-bearing mice, Food Chem. Toxicol., 2012, 50, 2648-2655. 3

C. Jiang, L. Zhao, S. Li, X. Zhao, Q. Zhang, and Q. Xiong, Preliminary

Published on 05 January 2015. Downloaded by Selcuk University on 17/01/2015 08:41:33.

Glossaulax didyma, Food Chem. Toxicol., 2013, 62, 226-230. 4 S. J. Lee, H. K. Rim, J. Y. Jung, H. J. An, J. S. Shin, C. W. Cho, Y. K. Rhee, H. D. Hong, and K. T. Lee, Immunostimulatory activity of polysaccharides from Cheonggukjang, Food Chem. Toxicol., 2013, 59, 476-484. 5 M. Wang, C. Jiang, L. Ma, Z. Zhang, L. Cao, J. Liu, and X. Zeng, Preparation, preliminary characterization and immunostimulatory activity of polysaccharide fractions from the peduncles of Hovenia dulcis, Food Chem., 2013, 138, 41-47. 6

Z. Wang, L. Zheng, S. Yang, R. Niu, E. Chu, and X. Lin, NAcetylchitooligosaccharide is a potent angiogenic inhibitor both in vivo and in vitro, Biochem. Biophys. Res. Commun., 2007, 357, 26-31.

7 D. Ribatti, A. Vacca, B. Nico, G. De Falco, P. Giuseppe Montaldo, and M. Ponzoni, Angiogenesis and anti-angiogenesis in neuroblastoma, Eur. J. Cancer, 2002, 38, 750-757. 8 C. Ke, D. Wang, Y. Sun, D. Qiao, H. Ye, and X. Zeng, Immunostimulatory and antiangiogenic activities of low molecular weight hyaluronic acid, Food Chem. Toxicol., 2013, 58, 401-407. 9 A. Zong, T. Zhao, Y. Zhang, X. Song, Y. Shi, H. Cao, C. Liu, Y. Cheng, X. Qu, J. Cao, and F. Wang, Anti-metastatic and anti-angiogenic activities of sulfated polysaccharide of Sepiella maindroni ink, Carbohydr. Polym., 2013, 91, 403-409.

23

Food & Function Accepted Manuscript

characterization and immunostimulatory activity of polysaccharides from

Food & Function

Page 24 of 27 View Article Online

DOI: 10.1039/C4FO01121J

10 J. Hu, and M. Cao, Inhibitive effects of polysaccharide from Hyriopsis cumingii on tumor proliferation: an experimental study, Chin. J. Mod. Appl. Pharm., 2003, 20(1), 11-13 (in Chinese).

Published on 05 January 2015. Downloaded by Selcuk University on 17/01/2015 08:41:33.

immunostimulatory activity of a water-soluble polysaccharide from the calm of Hyriopsis cumingii Lea, Carbohyd. Polym., 2009, 77(2), 365-369. 12 D. Qiao, B. Hu, D. Gan, Y. Sun, H. Ye, and X. Zeng, Extraction optimized by using response surface methodology, purification and preliminary characterization of polysaccharides from Hyriopsis cumingii, Carbohyd. Polym., 2009, 76(3), 422429. 13 W. Cheng, H. Wu, Y. Lu, and Y. Chi, Effects of polysaccharides from Hyriopsis cumingii on experimental transplanted tumor in mice and on activity of NK cells in vitro, Chin. J. Mar. Drugs, 2007, 26(2), 30-33 (in Chinese). 14 Z. Zhang, H. Wu, L. Di, and L. Chen, A review about freshwater mussel (Unionidae), Chin. Arch. Tradit. Chin. Med., 2007, 25, 121-123 (in Chinese). 15 D. Qiao, C. Ke, B. Hu, J. Luo, H. Ye, Y. Sun, X. Yan, and X. Zeng, Anti-oxidant activities of the polysaccharides from Hyriopsis cumingii, Carbohyd. Polym., 2009, 78(2), 199-204. 16 J. Burgermeister, D. H. Paper, H. Vogl, R. J. Linhardt, and G. Franz, LaPSvS1, a (1→3)β-galactan sulfate and its effect on angiogenesis in vivo and in vitro, Carbohyd. Res., 2002, 337, 1459-1466. 17 D. Bouis, Y. Kusumanto, C. Meijer, N. H. Mulder, and G. A. P. Hospers, A review on pro- and anti-angiogenic factors as targets of clinical intervention, Pharmacol. Res., 2006, 53, 89-103.

24

Food & Function Accepted Manuscript

11 Z. Dai, H. Zhang, Y. Zhang, and H. Wang, Chemical properties and

Page 25 of 27

Food & Function View Article Online

DOI: 10.1039/C4FO01121J

18 X. Huang, Q. Y. Zhang, Q. Y. Jiang, X. M. Kang, and L. Zhao, Polysaccharides derived from Lycium barbarum suppress IGF-1-induced angiogenesis via PI3K/HIF-1α/VEGF signalling pathways in MCF-7 cells, Food Chem,, 2012, 131,

Published on 05 January 2015. Downloaded by Selcuk University on 17/01/2015 08:41:33.

19 C. M. P. Guerra Dore, M. G. C. Faustino Alves, N. D. Santos, A. K. M. Cruz, R. B. G. Camara, A. J. G. Castro, L. Guimaraes Alves, H. B. Nader, and E. Lisboa Leite, Antiangiogenic activity and direct antitumor effect from a sulfated polysaccharide isolated from seaweed, Microvas. Res., 2013, 88, 12-18. 20 L. Jiang, Y. Zhang, H. Li, and W. Weng, Effect of bulleyaconitin A on the chemotaxis of mouse peritoneal macrophages, Chin. J. Clin. Pharm., 2011, 20(1), 16-20 (in Chinese). 21 Y. J. Na, D. H. Lee, S. C. Kim, J. K. Joo, J. W. Wang, J. O. Jin, J. Y. Kwak, and K. S. Lee, Effects of peritoneal fluid from endometriosis patients on the release of monocyte-specific chemokines by leukocytes, Arch. Gynecol. Obstet., 2011, 283, 1333-1341. 22 T. Baha, Y. Itasaka, H. Kodama, M. Mukamoto, and S. Tsuji, Immunopotentiating activities of polysaccharide fraction from pine seed shell extract, J. Vet. Med. B, 1997, 44, 485-493. 23 M. Matsui, N. Muizzuddin, S. Arad, and K. Marenus, Sulfated polysaccharides from red microalgae have antiinflammatory properties in vitro and in vivo, Appl. Biochem. Biotechnol., 2003, 104, 13-22. 24 G. H. Wu, C. L. Lu, J. G. Jiang, Z. Y. Li, and Z. L. Huang, Regulation effect of polysaccharides from Pleurotus tuber-regium (Fr.) on the immune activity of mice macrophages, Food Funct., 2014, 5, 337-344.

25

Food & Function Accepted Manuscript

1479-1484.

Food & Function

Page 26 of 27 View Article Online

DOI: 10.1039/C4FO01121J

25 M. Shi, Y. Yang, D. Guan, Y. Zhang, and Z. Zhang, Bioactivity of the crude polysaccharides from fermented soybean curd residue by Flammulina velutipes, Carbohyd. Polym., 2012, 89, 1268-1276.

Published on 05 January 2015. Downloaded by Selcuk University on 17/01/2015 08:41:33.

on antioxidative and immunomodulatory activities of a Ganoderma lucidum polysaccharide originated from fermented soybean curd residue, Food Chem., 2014, 155, 50-56. 27 X. Q. Cheng, H. Li, X. L. Yue, J. Y. Xie, Y. Y. Zhang, H. Y. Di, and D. F. Chen, Macrophage immunomodulatory activity of the polysaccharides from the roots of Bupleurum smithii var. parvifolium, J. Ethnopharmacol., 2010, 130, 363-368. 28 S. A. Im, K. Kim, and C. K. Lee, Immunomodulatory activity of polysaccharides isolated from Salicornia herbacea, Int. Immunopharm., 2006, 6, 1451-1458. 29 X. Huang, X. Kong, D. Wang, and Y. Hu, Research progress on sulfating modification of polysaccharides and sulfated polysaccharides, Nat. Prod. Res. Develop., 2007, 19 (2), 328-332 (in Chinese). 30 V. H. Pomin, Review: An overview about the structure-function relationship of marine sulfated homopolysaccharides with regular chemical structures, Biopolym., 2009, 91, 601-609. 31 C. M. Yang, Y. J. Zhou, R. J. Wang, and M. L. Hu, Anti-angiogenic effects and mechanisms of polysaccharides from Antrodia cinnamomea with different molecular weights, J. Ethnopharmacol., 2009, 123, 407–412. 32 K. Tang, Y. Li, C. Xie, S. Huang, X. Li, and X. Yu, Expression of fractalkine in renal tubular epithelial cells mediate chemotaxis of macrophages, J. Sun Yat-Sen Univ. (Med. Sci.), 2005, 26(1), 42-47 (in Chinese).

26

Food & Function Accepted Manuscript

26 M. Shi, Y. Yang, X. Hu, and Z. Zhang, Effect of ultrasonic extraction conditions

Page 27 of 27

Food & Function View Article Online

DOI: 10.1039/C4FO01121J

33 T. Mosmann, Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays, J. Immunol. Meth., 1983, 65, 55–63.

Published on 05 January 2015. Downloaded by Selcuk University on 17/01/2015 08:41:33.

and

S.

Liang, Antitumor efficacy in

H22

tumor bearing

mice and

immunoregulatory activity on RAW 264.7 macrophages of polysaccharides from Talinum triangulare, Food Funct., 2014, 5, 2183-2193.

27

Food & Function Accepted Manuscript

34 L. Wang, Z. K. Nie, Q. Zhou, J. L. Zhang, J. J. Yin, W. Xu, Y. Qiu, Y. L. Ming,

Effects of Hyriopsis cumingii polysaccharides on angiogenesis, macrophage chemotaxis, proliferation and phagocytosis.

Anti-angiogenic activities of crude Hyriopsis cumingii polysaccharides (HCPS) and its purified fractions (HCPS-1, HCPS-2 and HCPS-3) were evaluated in...
1MB Sizes 0 Downloads 4 Views