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Dietary Apostichopus japonicus enhances the respiratory and intestinal mucosal immunity in immunosuppressive mice a
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Rong Zheng , Xuemin Li , Binbin Cao , Tao Zuo , Juan Wu , Jingfeng Wang , Changhu a
Xue & Qingjuan Tang
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College of Food Science and Engineering, Ocean University of China, Qingdao, P.R. China Published online: 04 Sep 2014.
Click for updates To cite this article: Rong Zheng, Xuemin Li, Binbin Cao, Tao Zuo, Juan Wu, Jingfeng Wang, Changhu Xue & Qingjuan Tang (2015) Dietary Apostichopus japonicus enhances the respiratory and intestinal mucosal immunity in immunosuppressive mice, Bioscience, Biotechnology, and Biochemistry, 79:2, 253-259, DOI: 10.1080/09168451.2014.955454 To link to this article: http://dx.doi.org/10.1080/09168451.2014.955454
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Bioscience, Biotechnology, and Biochemistry, 2015 Vol. 79, No. 2, 253–259
Dietary Apostichopus japonicus enhances the respiratory and intestinal mucosal immunity in immunosuppressive mice Rong Zheng, Xuemin Li, Binbin Cao, Tao Zuo, Juan Wu, Jingfeng Wang, Changhu Xue and Qingjuan Tang* College of Food Science and Engineering, Ocean University of China, Qingdao, P.R. China Received June 9, 2014; accepted July 28, 2014
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http://dx.doi.org/10.1080/09168451.2014.955454
Although Apostichopus japonicus is recognized as a food and drug resource with significant immunomodulatory activity, its role in regulating the mucosal immunity remains unclear. This study aimed to explore the effects of dietary A. japonicus on mucosal immunity with an immunosuppressive mouse model. The expression of lysozyme, secretory immunoglobulin A(sIgA), and immunoglobulin A(IgA) as well as polymeric immunoglobulin receptor(pIgR) in respiratory and intestine organs was investigated. The results showed that A. japonicus could improve both the systematic and mucosal immunity. The expression of lysozyme, sIgA, and IgA in the respiratory organ was increased more significantly. Consumption of A. japonicus with the dose of 512 mg kg−1, which equals to 1/2 sea cucumber per day for adults, showed better effects. This study elucidated positive effects of A. japonicus on mucosal immunity for the first time, suggesting that moderate consumption of A. japonicus is helpful in improving mucosal immunity and preventing exogenous infection. mucosal immunity; lysozyme; sIgA; Apostichopus japonicus; immunosuppressive The mucosal immune system provides the first defense line of the inner body surfaces to against the harmful exotic pathogens. In the human body, mucosal surfaces of the intestinal, respiratory, and urogenital tracts have a combined surface area of at least 400 m2.1) It consists of many immunocompetent cells and molecules scattered throughout lamina propria and organized lymphatic tissues.2) Under some stress-related conditions, such as chemotherapy, burn, and fatigue, host mucosal defense will be undermined.3–5) Mucosal immune dysregulation can cause harmful substances, such as bacteria and antigens, to enter the body and cause diseases, including persistent stomachache, diarrhea, inflammatory bowel disease, respiratory infections, bronchitis, etc. Thus, it is extremely important to enhance mucosal immunity to prevent infection disease. Key words:
*Corresponding author. Email: [email protected] © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry
Both innate and adaptive mucosal immunities contribute to the host health. Lysozyme, working as the innate immune effector, is predominantly located at mucosal surfaces and secretions and plays an important role in preventing infection.6) It can kill bacteria by hydrolyzing the β-1,4 glycosidic bonds, enhance the phagocytic activity of both polymorphonuclear leukocytes and macrophages, and activate the immune system of the host.7,8) In addition, the secretory IgA (sIgA), as hallmark of the adaptive mucosal system, can prevent infection and remove antigens crossing the mucosal barrier. Dimeric IgA(dIgA), after binding on pIgR, is carried to the apical cell surface and the extracelluar region of pIgR(SC) is cleaved, followed by release of sIgA into the lumen. It has been proposed that pIgR plays a critical role for secretion of sIgA.9) Studies confirmed that enhanced secretion of sIgA and lysozyme could improve mucosal immunity, and thus prevent or treat mucosal dysfunction-related diseases.10,11) Sea cucumbers (phylum Echinodermata), generally consumed in Asia, are high-valued food and drug resources and have good immunomodulatory efficacy. They are rich in abundant bioactive materials such as polysaccharides, phospholipids, and saponins, which play unique roles in the regulation of immune function. Apostichopus japonicus belongs to the phylum Echinodermata and is a precious traditional delicacy, which is accepted widely among older persons, pregnant women, and especially persons who are recovering from surgery.12,13) There are numerous studies focusing on the immunomodulatory effects of A. japonicus,14,15) while their effect on mucosal immunity remains unclear. Therefore, this study aimed to investigate the effects of dietary A. japonicus on mucosal immunity with an immunosuppressive mouse model, and find the optimal edible dose of A. japonicus.
Materials and methods Materials. The dried sea cucumbers A. japonicus were purchased from Nanshan markets in Qingdao. A. japonicus was mainly composed of polysaccharide,
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protein, lipids, mineral, saponin, etc. The sea cucumbers were soaked in distilled water for 2 days and rinse thoroughly with water. Then the sea cucumbers were cooked slightly for 30 min and stored at 4 °C in distilled water after which they were cooled for 2 days. Finally, the sea cucumbers were cut them into small pieces and lyophilized. All the reagents used in the animal experiment were of analytical purity. Animal maintenance. Male balb/c mice (18–20 g, 4 weeks old) were obtained from Vital River (Beijing, China). During the experimental period, the mice were housed in a room maintained under a 12 h light/dark cycle at 24 °C. Mice had free access to standard laboratory pellet chow (Ingredients: corn, bean pulp, fish meal, flour, bran, salt, calcium hydrophosphate, vitamins, microelements, amino acid, etc. The chow meets the Chinese national standard GB 13078 and GB 14924.2) (Kangda, Jinan, China) and fresh water. Animal treatment. The mice were randomly assigned to six groups with 10 mice per group, normal control group, cyclophosphamide model group, positive control group, and A. japonicus groups of three different dosages. The A. japonicus groups were given sea cucumber-lyophilized powder followed by 256, 512, and 1024 mg kg−1 by gavage for 28 days, which equaled to consumption of 1/4, 1/2, and one sea cucumber (the lyophilized powder was 3 g) per person (60 kg) every day (the conversion factor between person and mouse is 12.33). The positive control group was given Zhenqifuzheng particle (4.55 g kg−1), a Chinese medicine commonly used in clinic to improve chemotherapy patients’ immunity, while the normal and model groups were given normal saline as control. Except for the normal group, the others were treated with cyclophosphamide (50 mg kg−1) through intraperitoneal injection on the 26th and 27th feeding days. The normal mice were injected with saline as control. The body weights of mice were weighed every two days. The mice were sacrificed by cervical dislocation on the 29th day and the thymus, spleen, lung, and small intestine were collected for analysis. All experimental procedures were conducted according to the guidelines provided by the ethical committee of experimental animal care at Ocean University of China (Qingdao, China). Western blot analysis. The expression levels of lysozyme, immunoglobulin A (IgA), and polymeric immunoglobulin receptor (pIgR) of lung and small intestine were investigated by western blot analysis. Briefly, the tissues were homogenized in RIPA buffer (50 mM Tris, 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, pH 7.4) and put on ice for 20 min and then centrifuged at 12,000 ×g for 5 min at 4 °C. The protein concentration of the supernatant was measured using the BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China) with bovine serum albumin as the standard sample. The proteins were separated using sodium dodecyl sulfate–polyacrylamide
gel electrophoresis (SDS–PAGE) and transferred to polyvinyl difluoride (PVDF) membranes (GE, Fairfield, CT, USA). The membrane was blocked with TBST (10 mM Tris, 150 mM NaCl, and 0.1% Tween 20, pH 7.6) containing 5% skim milk (BD, Franklin Lakes, NJ, USA) and incubated with the indicated primary antibody in TBST overnight at 4 °C. Then, the membrane was incubated with the secondary antibody and subsequently visualized by the enhanced chemiluminescence kit (GE, Fairfield, CT, USA). Equal lane loading was assessed using β-actin. Elisa for sIgA. The mice’s lungs were lavaged thrice with 0.8 ml 0.01 M PBS and the total liquid was collected. Then, the solution was centrifuged at 200 ×g for 10 min at 4 °C and the supernatant for respiratory sIgA analysis was collected. The first part of jejunum (about 4 cm) was used for analysis of intestinal sIgA. It was weighed prior to scrapping the mucosa. The mucosa was then dissolved in 0.01 M PBS on ice. Then the solution was mixed thoroughly and centrifuged at 3200 ×g for 5 min at 4 °C. The supernatant was collected. SIgA levels were measured by Elisa kit (USCN Life Science, Inc, Wuhan, China) according to the manufacturer’s instructions. Statistical analysis. Tukey’s post hoc test (ANOVA) with SPSS v18.0 software was used to compare the intergroup differences in the mean values. Statistical significance was considered if p value was less than 0.05. All values in the tables and figures were expressed as mean ± standard error.
Results A. japonicus enhanced host immunity of immunosuppressive mice significantly After cyclophosphamide i.p. treatment, the body weight of mice in model group decreased significantly compared to that in normal group mice (Fig. 1(A)). However, oral administration of A. japonicus was helpful to mitigate the body weight changes caused by cyclophosphamide. The result might indicate the immunomodulatory effect of dietary A. japonicus. To further testify our hypothesis, thymus and spleen index were evaluated in this study. Thymus and spleen are important immune organs in host. The main function of the thymus is to produce T lymphocytes and secret thymosin and spleen is rich in lymphocytes and macrophages.17,18) The organ/bodyweight index reflects the host immune function on the whole. It showed that cyclophosphamide injection seriously damaged the thymus (p < 0.05) and spleen (p < 0.05) in model group mice (Fig. 1(B) and (C)) compared to that of normal group mice. Nevertheless, the thymus/bodyweight index of 512 mg kg−1 A. japonicus group increased significantly compared with the model group (p < 0.05), even better than that of positive control group mice. Similar to the effect of A. japonicus on thymus/bodyweight index, the spleen/bodyweight index of all A. japonicus dose group mice got improved
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Fig. 1. Mice’s systematic immunity after administration of A. japonicus. Notes: (A) The body weight was weighed every two days. After injecting cyclophosphamide, the body weight was weighed every day. (B) and (C) Thymus/BW and spleen/BW index. Each value represents the mean ± SEM of eight mice in each group. ##Significantly different from normal group, p < 0.01. *Significantly different from model group, p < 0.05. **Significantly different from model group, p < 0.01.
significantly (p < 0.05), compared to that of model group mice. The results implicate that dietary A. japonicus has an enhancement effect on the immune system against chemotherapeutic injury. A. japonicus ameliorated lysozyme expression level in immunosuppressive mice, and showed better effect on the respiratory organ than that on intestine Lysozyme is an important bactericidal substance in body fluids, which works as mucosal chemical barrier and represents the innate mucosal immunity effector. Therefore, we detected lysozyme expression to investigate whether A. japonicus could protect the mucosal barrier against chemotherapeutic injury. After administration of A. japonicus, the lysozyme expression in lungs of positive control and dose groups increased significantly (p < 0.01) compared with that in model group (Fig. 2(A)). But it has no apparent effect on intestinal lysozyme expression, except for that in 1024 mg kg−1 group in which the lysozyme enhanced significantly (p < 0.05) (Fig. 2(B)). A. japonicus enhanced respiratory sIgA secretion significantly, but showed no ameliorative effect on intestinal sIgA To investigate the effects of A. japonicus on the mucosal-adaptive immunity, the respiratory and intestinal mucosal sIgA were examined, respectively. It shows that
cyclophosphamide i.p. treatment significantly lowers the sIgA levels in both respiratory and small intestine mucosa in model group mice, compared to that in normal control group mice (p < 0.05). Nonetheless, oral administration of A. japonicus significantly up-regulated the sIgA levels in respiratory tract (p < 0.05) compared with that of the model group (Table 1), and it was more obvious in 512 mg kg−1 group mice (p < 0.01). However, A. japonicus had no ameliorative effect on cyclophosphamide-induced sIgA decrease in intestinal mucosa. The different changes of sIgA in intestinal and respiratory organs were related to the changes of IgA and pIgR SIgA is composed of two immunoglobulin A (IgA) molecules, which are joined by a J-chain and a secretory component. And pIgR transporting dimeric IgA (dIgA) across the epithelial cell function is a necessary condition to the secretion of sIgA to exocrine fluid.19) Therefore, the protein expression of pIgR and IgA were studied to explore the mechanism of different sIgA changes in the respiratory organ and intestine. It shows that after administration of Zhenqifuzheng particles or A. japonicus, pIgR expression in lungs was increased compared with that of the model group’s, it being remarkable in 256 and 512 mg kg−1 groups (Fig. 3(A)). Similarly in intestine, the pIgR expression levels of 512 and 1024 mg kg−1 groups (p < 0.01) increased
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Fig. 2. Relative expression of lysozyme in respiratory and intestine. Notes: Lysozyme expression was analyzed by western blotting and β-actin was the loading control. Each value represents the mean ± SEM of eight mice in each group. #Significantly different from normal group, p < 0.05. ##Significantly different from normal group, p < 0.01. *Significantly different from model group, p < 0.05. **Significantly different from model group, p < 0.01. Table 1.
Effect of A. japonicus on sIgA levels in respiratory and intestine. Apostichopus japonicus
SIgA in respiratory (ng/ml) SIgA in intestine (mg/g)
Normal
Model
Positive control
8.90 ± 0.61 6.69 ± 0.45
6.87 ± 0.63# 4.01 ± 0.51##
10.27 ± 1.17 3.56 ± 0.27
−1
256 mg kg
12.00 ± 1.47* 3.36 ± 0.38
512 mg kg−1
1024 mg kg−1
13.95 ± 2.45** 3.67 ± 0.27
12.81 ± 2.18* 3.21 ± 0.28
Notes: Data are presented as means ± SEM (n = 8). Statistical analyses were performed using ANOVA between the normal and the control groups, and analysis of variance among the model, positive control, A. japonicus 256, 512, and 1024 mg kg−1 groups. # Significantly different from normal group, p < 0.05. ## Significantly different from normal group, p < 0.01 *Significantly different from model group, p < 0.05. **Significantly different from model group, p < 0.01.
Fig. 3. Different expressions of pIgR in respiratory and intestine. Notes: Protein expression was analyzed by western blotting and β-actin was the loading control. Each value represents the mean ± SEM of eight mice in each group. #Significantly different from normal group, p < 0.05. *Significantly different from model group, p < 0.05. **Significantly different from model group, p < 0.01.
markedly (Fig. 3(B)). The results suggest that A. japonicus enhanced the expression of pIgR in both respiratory and intestinal organs. Meanwhile, the IgA expressions in respiratory and intestinal organs were detected. The variation of IgA expression showed the comparative result with the
pIgR in the respiratory organ (p < 0.01) (Fig. 4(A)). Like on sIgA, oral administration of 512 mg kg−1 A. japonicus showed the most dramatic effect on IgA and pIgR expression. But in intestine, it showed no significant effect on intestinal IgA of dose groups compared with that of the model group (Fig. 4(B)). These results
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Fig. 4. Different expressions of IgA in respiratory and intestine. Notes: Protein expression was analyzed by western blotting and β-actin was the loading control. Each value represents the mean ± SEM of eight mice in each group. #Significantly different from normal group, p < 0.05. ##Significantly different from normal group, p < 0.01. *Significantly different from model group, p < 0.05. **Significantly different from model group, p < 0.01.
suggest that both enhanced IgA and pIgR expressions contribute to the sIgA secretion, which results in relatively higher sIgA response in the respiratory organ than that in intestine under A. japonicus administration.
Discussion Cyclophosphamide is an antineoplastic chemotherapy drug commonly used in clinic to treat tumor, leukemia, rheumatoid arthritis, nephrotic syndrome, etc. It can cause mucosal damage and immunodeficiency, even bacterial translocation.20) Chemotherapy patients often suffered from diarrhea or upper respiratory tract infection. In current contribution, for the first time, we compared the protective effect of bioactive A. japonicus on mucosal immunity, both in respiratory and intestinal organs, of mice intraperitoneally injected with cyclophosphamide. The data show that, dietary A. japonicus enhanced the respiratory mucosal immunity by increasing lysozyme expression and sIgA secretion apparently. This is particularly obvious in 512 mg kg−1 A. japonicus group, while the effect of highest dose group was not ideal. The composition of sea cucumber is complicated, including polysaccharide, protein, lipid, saponin, etc. Mice with higher dose of A. japonicus had relatively higher intake of these sea cucumber components. Combined effects of multiple components are speculated to make sea cucumbers have different physiological activities. Apart from that, some research reported that saponin had hemolytic activity and certain cytotoxicity.21) Therefore, the side effect of high-dose administration of A. japonicus may be caused by over intake of saponin or other toxic substances in sea cucumber while the underlying mechanism needs further study. But as for the intestine, little mucosal immunomodulatary effect was shown, with only pIgR and lysozyme expressions improved by A. japonicus intake. Cyclophosphamide mainly damages the fast self-renewing cells. Intestinal villi, as rapidly growing tissues, are more easily attacked by cyclophosphamide. The dam-
age on the intestine caused by cyclophosphamide was more severe than other tissues in many cases. Therefore, administration of A. japonicus didn’t show significant effect on intestinal mucosal immunity while it has significant effects on the respiratory organ. As for normal mice, we detected sIgA, IgA, and pIgR to research the effect of dietary A. japonicus on normal mice (data are not shown). Nonetheless, the results showed no apparent effects. Furthermore, lots of studies showed that polysaccharide and peptide of sea cucumber play a role in immunomodulatory effects.22,23) Nevertheless, it is still not clear what constituents of the sea cucumber are mainly responsible for these effects. For further work, it is necessary to extract these bioactive components individually and then investigate what constituents are responsible for the immune effects. Lysozyme is an important part of mucosal defense system. It has many functions such as phagocytosis, antibacterium, anti-virus, and anti-tumour, and plays an important role in the clearance of exogenous foreign bodies and endogenous residues. It is abundant in a number of secretions, such as tears, saliva, human milk, and mucus. Cyclophosphamide could reduce the activity of lysozyme.24,25) Local lysozyme deficiency may contribute to the pathogenesis of recurrent sinusitis, hyaline membrane disease, and early stage cystic fibrosis. Our study showed that cyclophosphamide caused the decrease of lysozyme expression in both lungs and guts. But meanwhile, A. japonicus altered the situation. In respiratory, the lysozyme expressions of all A. japonicus dose groups were augmented and the effect on 512 mg kg−1 group was most significant. But in intestine, only the protective effect at the dosage of 1024 mg kg−1 was significant in upregulating lysozyme expression. Secretory IgA (sIgA) is the most characteristic component of the mucosal immune system to protect mucosal surfaces. It prevents pathogens from invading hosts through the mucosa and contributes to maintenance of mucosal homeostasis.26,27) When the secretion of sIgA reduced, the immune barrier is weakened. Foreign
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micro-organisms, such as bacteria, viruses, and parasites, invaded easily. Thus, bowel disease or respiratory infections will happen. It’s even life-threatening if the infection is serious.28,29) After administration of A. japonicus, the sIgA level in respiratory tract was significantly enhanced, which indicates that A. japonicus is helpful in reducing the damage caused by cyclophosphamide. And in the 512 mg kg−1 group, which was equivalent to eat 1/2 sea cucumber for a person every day, it showed the best result. The results provide a theoretical guidance on consumption of sea cucumber. But the mechanisms underlying the different effects of A. japonicus on respiratory and intestine need further study. Fukatsu et al.30,31) reported that interleukin-7 intaking could increase the GALT cell numbers but had no effect on secretory IgA levels. So, it is possible that the gut regulates immunity through other ways. After binding to the polymeric Ig receptor (pIgR), dIgA is transported to the apical cell surface and then forms the sIgA.32,33) PIgR plays a critical role in transepithelial transport of dIgA.9,27) Our study demonstrated that both the IgA and pIgR in lungs of A. japonicus groups got increased, especially in the 256 and 512 mg kg−1 group, which were responsible for the increase of sIgA in respiratory. Although the pIgR expression in intestine of 512 and 1024 mg kg−1 group was increased markedly, the IgA showed no significant differences. Thus, it can be speculated that the unchanged IgA level caused the invariance of sIgA level in intestine. In conclusion, consumption of A. japonicus could improve the mucosal immunity and has better effect on respiratory than intestine. This study elucidated the positive effects of A. japonicus on mucosal immunity for the first time, suggesting that moderate consumption of A. japonicus is helpful in improving mucosal immunity and preventing exogenous infection, which provides an experimental basis for chemotherapeutic patients.
Funding This work was supported by the National Natural Science Foundation of China [grant number 31101281]; Program for Changjiang Scholars and Innovative Research Team in University [grant number PCSIRT: IRT1188]; Special funds for “Taishan Scholar” construction project.
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Bioscience, Biotechnology, and Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbbb20
Dietary Apostichopus japonicus enhances the respiratory and intestinal mucosal immunity in immunosuppressive mice a
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Rong Zheng , Xuemin Li , Binbin Cao , Tao Zuo , Juan Wu , Jingfeng Wang , Changhu a
Xue & Qingjuan Tang
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College of Food Science and Engineering, Ocean University of China, Qingdao, P.R. China Published online: 04 Sep 2014.
Click for updates To cite this article: Rong Zheng, Xuemin Li, Binbin Cao, Tao Zuo, Juan Wu, Jingfeng Wang, Changhu Xue & Qingjuan Tang (2015) Dietary Apostichopus japonicus enhances the respiratory and intestinal mucosal immunity in immunosuppressive mice, Bioscience, Biotechnology, and Biochemistry, 79:2, 253-259, DOI: 10.1080/09168451.2014.955454 To link to this article: http://dx.doi.org/10.1080/09168451.2014.955454
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Bioscience, Biotechnology, and Biochemistry, 2015 Vol. 79, No. 2, 253–259
Dietary Apostichopus japonicus enhances the respiratory and intestinal mucosal immunity in immunosuppressive mice Rong Zheng, Xuemin Li, Binbin Cao, Tao Zuo, Juan Wu, Jingfeng Wang, Changhu Xue and Qingjuan Tang* College of Food Science and Engineering, Ocean University of China, Qingdao, P.R. China Received June 9, 2014; accepted July 28, 2014
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http://dx.doi.org/10.1080/09168451.2014.955454
Although Apostichopus japonicus is recognized as a food and drug resource with significant immunomodulatory activity, its role in regulating the mucosal immunity remains unclear. This study aimed to explore the effects of dietary A. japonicus on mucosal immunity with an immunosuppressive mouse model. The expression of lysozyme, secretory immunoglobulin A(sIgA), and immunoglobulin A(IgA) as well as polymeric immunoglobulin receptor(pIgR) in respiratory and intestine organs was investigated. The results showed that A. japonicus could improve both the systematic and mucosal immunity. The expression of lysozyme, sIgA, and IgA in the respiratory organ was increased more significantly. Consumption of A. japonicus with the dose of 512 mg kg−1, which equals to 1/2 sea cucumber per day for adults, showed better effects. This study elucidated positive effects of A. japonicus on mucosal immunity for the first time, suggesting that moderate consumption of A. japonicus is helpful in improving mucosal immunity and preventing exogenous infection. mucosal immunity; lysozyme; sIgA; Apostichopus japonicus; immunosuppressive The mucosal immune system provides the first defense line of the inner body surfaces to against the harmful exotic pathogens. In the human body, mucosal surfaces of the intestinal, respiratory, and urogenital tracts have a combined surface area of at least 400 m2.1) It consists of many immunocompetent cells and molecules scattered throughout lamina propria and organized lymphatic tissues.2) Under some stress-related conditions, such as chemotherapy, burn, and fatigue, host mucosal defense will be undermined.3–5) Mucosal immune dysregulation can cause harmful substances, such as bacteria and antigens, to enter the body and cause diseases, including persistent stomachache, diarrhea, inflammatory bowel disease, respiratory infections, bronchitis, etc. Thus, it is extremely important to enhance mucosal immunity to prevent infection disease. Key words:
*Corresponding author. Email: [email protected] © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry
Both innate and adaptive mucosal immunities contribute to the host health. Lysozyme, working as the innate immune effector, is predominantly located at mucosal surfaces and secretions and plays an important role in preventing infection.6) It can kill bacteria by hydrolyzing the β-1,4 glycosidic bonds, enhance the phagocytic activity of both polymorphonuclear leukocytes and macrophages, and activate the immune system of the host.7,8) In addition, the secretory IgA (sIgA), as hallmark of the adaptive mucosal system, can prevent infection and remove antigens crossing the mucosal barrier. Dimeric IgA(dIgA), after binding on pIgR, is carried to the apical cell surface and the extracelluar region of pIgR(SC) is cleaved, followed by release of sIgA into the lumen. It has been proposed that pIgR plays a critical role for secretion of sIgA.9) Studies confirmed that enhanced secretion of sIgA and lysozyme could improve mucosal immunity, and thus prevent or treat mucosal dysfunction-related diseases.10,11) Sea cucumbers (phylum Echinodermata), generally consumed in Asia, are high-valued food and drug resources and have good immunomodulatory efficacy. They are rich in abundant bioactive materials such as polysaccharides, phospholipids, and saponins, which play unique roles in the regulation of immune function. Apostichopus japonicus belongs to the phylum Echinodermata and is a precious traditional delicacy, which is accepted widely among older persons, pregnant women, and especially persons who are recovering from surgery.12,13) There are numerous studies focusing on the immunomodulatory effects of A. japonicus,14,15) while their effect on mucosal immunity remains unclear. Therefore, this study aimed to investigate the effects of dietary A. japonicus on mucosal immunity with an immunosuppressive mouse model, and find the optimal edible dose of A. japonicus.
Materials and methods Materials. The dried sea cucumbers A. japonicus were purchased from Nanshan markets in Qingdao. A. japonicus was mainly composed of polysaccharide,
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protein, lipids, mineral, saponin, etc. The sea cucumbers were soaked in distilled water for 2 days and rinse thoroughly with water. Then the sea cucumbers were cooked slightly for 30 min and stored at 4 °C in distilled water after which they were cooled for 2 days. Finally, the sea cucumbers were cut them into small pieces and lyophilized. All the reagents used in the animal experiment were of analytical purity. Animal maintenance. Male balb/c mice (18–20 g, 4 weeks old) were obtained from Vital River (Beijing, China). During the experimental period, the mice were housed in a room maintained under a 12 h light/dark cycle at 24 °C. Mice had free access to standard laboratory pellet chow (Ingredients: corn, bean pulp, fish meal, flour, bran, salt, calcium hydrophosphate, vitamins, microelements, amino acid, etc. The chow meets the Chinese national standard GB 13078 and GB 14924.2) (Kangda, Jinan, China) and fresh water. Animal treatment. The mice were randomly assigned to six groups with 10 mice per group, normal control group, cyclophosphamide model group, positive control group, and A. japonicus groups of three different dosages. The A. japonicus groups were given sea cucumber-lyophilized powder followed by 256, 512, and 1024 mg kg−1 by gavage for 28 days, which equaled to consumption of 1/4, 1/2, and one sea cucumber (the lyophilized powder was 3 g) per person (60 kg) every day (the conversion factor between person and mouse is 12.33). The positive control group was given Zhenqifuzheng particle (4.55 g kg−1), a Chinese medicine commonly used in clinic to improve chemotherapy patients’ immunity, while the normal and model groups were given normal saline as control. Except for the normal group, the others were treated with cyclophosphamide (50 mg kg−1) through intraperitoneal injection on the 26th and 27th feeding days. The normal mice were injected with saline as control. The body weights of mice were weighed every two days. The mice were sacrificed by cervical dislocation on the 29th day and the thymus, spleen, lung, and small intestine were collected for analysis. All experimental procedures were conducted according to the guidelines provided by the ethical committee of experimental animal care at Ocean University of China (Qingdao, China). Western blot analysis. The expression levels of lysozyme, immunoglobulin A (IgA), and polymeric immunoglobulin receptor (pIgR) of lung and small intestine were investigated by western blot analysis. Briefly, the tissues were homogenized in RIPA buffer (50 mM Tris, 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, pH 7.4) and put on ice for 20 min and then centrifuged at 12,000 ×g for 5 min at 4 °C. The protein concentration of the supernatant was measured using the BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China) with bovine serum albumin as the standard sample. The proteins were separated using sodium dodecyl sulfate–polyacrylamide
gel electrophoresis (SDS–PAGE) and transferred to polyvinyl difluoride (PVDF) membranes (GE, Fairfield, CT, USA). The membrane was blocked with TBST (10 mM Tris, 150 mM NaCl, and 0.1% Tween 20, pH 7.6) containing 5% skim milk (BD, Franklin Lakes, NJ, USA) and incubated with the indicated primary antibody in TBST overnight at 4 °C. Then, the membrane was incubated with the secondary antibody and subsequently visualized by the enhanced chemiluminescence kit (GE, Fairfield, CT, USA). Equal lane loading was assessed using β-actin. Elisa for sIgA. The mice’s lungs were lavaged thrice with 0.8 ml 0.01 M PBS and the total liquid was collected. Then, the solution was centrifuged at 200 ×g for 10 min at 4 °C and the supernatant for respiratory sIgA analysis was collected. The first part of jejunum (about 4 cm) was used for analysis of intestinal sIgA. It was weighed prior to scrapping the mucosa. The mucosa was then dissolved in 0.01 M PBS on ice. Then the solution was mixed thoroughly and centrifuged at 3200 ×g for 5 min at 4 °C. The supernatant was collected. SIgA levels were measured by Elisa kit (USCN Life Science, Inc, Wuhan, China) according to the manufacturer’s instructions. Statistical analysis. Tukey’s post hoc test (ANOVA) with SPSS v18.0 software was used to compare the intergroup differences in the mean values. Statistical significance was considered if p value was less than 0.05. All values in the tables and figures were expressed as mean ± standard error.
Results A. japonicus enhanced host immunity of immunosuppressive mice significantly After cyclophosphamide i.p. treatment, the body weight of mice in model group decreased significantly compared to that in normal group mice (Fig. 1(A)). However, oral administration of A. japonicus was helpful to mitigate the body weight changes caused by cyclophosphamide. The result might indicate the immunomodulatory effect of dietary A. japonicus. To further testify our hypothesis, thymus and spleen index were evaluated in this study. Thymus and spleen are important immune organs in host. The main function of the thymus is to produce T lymphocytes and secret thymosin and spleen is rich in lymphocytes and macrophages.17,18) The organ/bodyweight index reflects the host immune function on the whole. It showed that cyclophosphamide injection seriously damaged the thymus (p < 0.05) and spleen (p < 0.05) in model group mice (Fig. 1(B) and (C)) compared to that of normal group mice. Nevertheless, the thymus/bodyweight index of 512 mg kg−1 A. japonicus group increased significantly compared with the model group (p < 0.05), even better than that of positive control group mice. Similar to the effect of A. japonicus on thymus/bodyweight index, the spleen/bodyweight index of all A. japonicus dose group mice got improved
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Fig. 1. Mice’s systematic immunity after administration of A. japonicus. Notes: (A) The body weight was weighed every two days. After injecting cyclophosphamide, the body weight was weighed every day. (B) and (C) Thymus/BW and spleen/BW index. Each value represents the mean ± SEM of eight mice in each group. ##Significantly different from normal group, p < 0.01. *Significantly different from model group, p < 0.05. **Significantly different from model group, p < 0.01.
significantly (p < 0.05), compared to that of model group mice. The results implicate that dietary A. japonicus has an enhancement effect on the immune system against chemotherapeutic injury. A. japonicus ameliorated lysozyme expression level in immunosuppressive mice, and showed better effect on the respiratory organ than that on intestine Lysozyme is an important bactericidal substance in body fluids, which works as mucosal chemical barrier and represents the innate mucosal immunity effector. Therefore, we detected lysozyme expression to investigate whether A. japonicus could protect the mucosal barrier against chemotherapeutic injury. After administration of A. japonicus, the lysozyme expression in lungs of positive control and dose groups increased significantly (p < 0.01) compared with that in model group (Fig. 2(A)). But it has no apparent effect on intestinal lysozyme expression, except for that in 1024 mg kg−1 group in which the lysozyme enhanced significantly (p < 0.05) (Fig. 2(B)). A. japonicus enhanced respiratory sIgA secretion significantly, but showed no ameliorative effect on intestinal sIgA To investigate the effects of A. japonicus on the mucosal-adaptive immunity, the respiratory and intestinal mucosal sIgA were examined, respectively. It shows that
cyclophosphamide i.p. treatment significantly lowers the sIgA levels in both respiratory and small intestine mucosa in model group mice, compared to that in normal control group mice (p < 0.05). Nonetheless, oral administration of A. japonicus significantly up-regulated the sIgA levels in respiratory tract (p < 0.05) compared with that of the model group (Table 1), and it was more obvious in 512 mg kg−1 group mice (p < 0.01). However, A. japonicus had no ameliorative effect on cyclophosphamide-induced sIgA decrease in intestinal mucosa. The different changes of sIgA in intestinal and respiratory organs were related to the changes of IgA and pIgR SIgA is composed of two immunoglobulin A (IgA) molecules, which are joined by a J-chain and a secretory component. And pIgR transporting dimeric IgA (dIgA) across the epithelial cell function is a necessary condition to the secretion of sIgA to exocrine fluid.19) Therefore, the protein expression of pIgR and IgA were studied to explore the mechanism of different sIgA changes in the respiratory organ and intestine. It shows that after administration of Zhenqifuzheng particles or A. japonicus, pIgR expression in lungs was increased compared with that of the model group’s, it being remarkable in 256 and 512 mg kg−1 groups (Fig. 3(A)). Similarly in intestine, the pIgR expression levels of 512 and 1024 mg kg−1 groups (p < 0.01) increased
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Fig. 2. Relative expression of lysozyme in respiratory and intestine. Notes: Lysozyme expression was analyzed by western blotting and β-actin was the loading control. Each value represents the mean ± SEM of eight mice in each group. #Significantly different from normal group, p < 0.05. ##Significantly different from normal group, p < 0.01. *Significantly different from model group, p < 0.05. **Significantly different from model group, p < 0.01. Table 1.
Effect of A. japonicus on sIgA levels in respiratory and intestine. Apostichopus japonicus
SIgA in respiratory (ng/ml) SIgA in intestine (mg/g)
Normal
Model
Positive control
8.90 ± 0.61 6.69 ± 0.45
6.87 ± 0.63# 4.01 ± 0.51##
10.27 ± 1.17 3.56 ± 0.27
−1
256 mg kg
12.00 ± 1.47* 3.36 ± 0.38
512 mg kg−1
1024 mg kg−1
13.95 ± 2.45** 3.67 ± 0.27
12.81 ± 2.18* 3.21 ± 0.28
Notes: Data are presented as means ± SEM (n = 8). Statistical analyses were performed using ANOVA between the normal and the control groups, and analysis of variance among the model, positive control, A. japonicus 256, 512, and 1024 mg kg−1 groups. # Significantly different from normal group, p < 0.05. ## Significantly different from normal group, p < 0.01 *Significantly different from model group, p < 0.05. **Significantly different from model group, p < 0.01.
Fig. 3. Different expressions of pIgR in respiratory and intestine. Notes: Protein expression was analyzed by western blotting and β-actin was the loading control. Each value represents the mean ± SEM of eight mice in each group. #Significantly different from normal group, p < 0.05. *Significantly different from model group, p < 0.05. **Significantly different from model group, p < 0.01.
markedly (Fig. 3(B)). The results suggest that A. japonicus enhanced the expression of pIgR in both respiratory and intestinal organs. Meanwhile, the IgA expressions in respiratory and intestinal organs were detected. The variation of IgA expression showed the comparative result with the
pIgR in the respiratory organ (p < 0.01) (Fig. 4(A)). Like on sIgA, oral administration of 512 mg kg−1 A. japonicus showed the most dramatic effect on IgA and pIgR expression. But in intestine, it showed no significant effect on intestinal IgA of dose groups compared with that of the model group (Fig. 4(B)). These results
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Fig. 4. Different expressions of IgA in respiratory and intestine. Notes: Protein expression was analyzed by western blotting and β-actin was the loading control. Each value represents the mean ± SEM of eight mice in each group. #Significantly different from normal group, p < 0.05. ##Significantly different from normal group, p < 0.01. *Significantly different from model group, p < 0.05. **Significantly different from model group, p < 0.01.
suggest that both enhanced IgA and pIgR expressions contribute to the sIgA secretion, which results in relatively higher sIgA response in the respiratory organ than that in intestine under A. japonicus administration.
Discussion Cyclophosphamide is an antineoplastic chemotherapy drug commonly used in clinic to treat tumor, leukemia, rheumatoid arthritis, nephrotic syndrome, etc. It can cause mucosal damage and immunodeficiency, even bacterial translocation.20) Chemotherapy patients often suffered from diarrhea or upper respiratory tract infection. In current contribution, for the first time, we compared the protective effect of bioactive A. japonicus on mucosal immunity, both in respiratory and intestinal organs, of mice intraperitoneally injected with cyclophosphamide. The data show that, dietary A. japonicus enhanced the respiratory mucosal immunity by increasing lysozyme expression and sIgA secretion apparently. This is particularly obvious in 512 mg kg−1 A. japonicus group, while the effect of highest dose group was not ideal. The composition of sea cucumber is complicated, including polysaccharide, protein, lipid, saponin, etc. Mice with higher dose of A. japonicus had relatively higher intake of these sea cucumber components. Combined effects of multiple components are speculated to make sea cucumbers have different physiological activities. Apart from that, some research reported that saponin had hemolytic activity and certain cytotoxicity.21) Therefore, the side effect of high-dose administration of A. japonicus may be caused by over intake of saponin or other toxic substances in sea cucumber while the underlying mechanism needs further study. But as for the intestine, little mucosal immunomodulatary effect was shown, with only pIgR and lysozyme expressions improved by A. japonicus intake. Cyclophosphamide mainly damages the fast self-renewing cells. Intestinal villi, as rapidly growing tissues, are more easily attacked by cyclophosphamide. The dam-
age on the intestine caused by cyclophosphamide was more severe than other tissues in many cases. Therefore, administration of A. japonicus didn’t show significant effect on intestinal mucosal immunity while it has significant effects on the respiratory organ. As for normal mice, we detected sIgA, IgA, and pIgR to research the effect of dietary A. japonicus on normal mice (data are not shown). Nonetheless, the results showed no apparent effects. Furthermore, lots of studies showed that polysaccharide and peptide of sea cucumber play a role in immunomodulatory effects.22,23) Nevertheless, it is still not clear what constituents of the sea cucumber are mainly responsible for these effects. For further work, it is necessary to extract these bioactive components individually and then investigate what constituents are responsible for the immune effects. Lysozyme is an important part of mucosal defense system. It has many functions such as phagocytosis, antibacterium, anti-virus, and anti-tumour, and plays an important role in the clearance of exogenous foreign bodies and endogenous residues. It is abundant in a number of secretions, such as tears, saliva, human milk, and mucus. Cyclophosphamide could reduce the activity of lysozyme.24,25) Local lysozyme deficiency may contribute to the pathogenesis of recurrent sinusitis, hyaline membrane disease, and early stage cystic fibrosis. Our study showed that cyclophosphamide caused the decrease of lysozyme expression in both lungs and guts. But meanwhile, A. japonicus altered the situation. In respiratory, the lysozyme expressions of all A. japonicus dose groups were augmented and the effect on 512 mg kg−1 group was most significant. But in intestine, only the protective effect at the dosage of 1024 mg kg−1 was significant in upregulating lysozyme expression. Secretory IgA (sIgA) is the most characteristic component of the mucosal immune system to protect mucosal surfaces. It prevents pathogens from invading hosts through the mucosa and contributes to maintenance of mucosal homeostasis.26,27) When the secretion of sIgA reduced, the immune barrier is weakened. Foreign
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micro-organisms, such as bacteria, viruses, and parasites, invaded easily. Thus, bowel disease or respiratory infections will happen. It’s even life-threatening if the infection is serious.28,29) After administration of A. japonicus, the sIgA level in respiratory tract was significantly enhanced, which indicates that A. japonicus is helpful in reducing the damage caused by cyclophosphamide. And in the 512 mg kg−1 group, which was equivalent to eat 1/2 sea cucumber for a person every day, it showed the best result. The results provide a theoretical guidance on consumption of sea cucumber. But the mechanisms underlying the different effects of A. japonicus on respiratory and intestine need further study. Fukatsu et al.30,31) reported that interleukin-7 intaking could increase the GALT cell numbers but had no effect on secretory IgA levels. So, it is possible that the gut regulates immunity through other ways. After binding to the polymeric Ig receptor (pIgR), dIgA is transported to the apical cell surface and then forms the sIgA.32,33) PIgR plays a critical role in transepithelial transport of dIgA.9,27) Our study demonstrated that both the IgA and pIgR in lungs of A. japonicus groups got increased, especially in the 256 and 512 mg kg−1 group, which were responsible for the increase of sIgA in respiratory. Although the pIgR expression in intestine of 512 and 1024 mg kg−1 group was increased markedly, the IgA showed no significant differences. Thus, it can be speculated that the unchanged IgA level caused the invariance of sIgA level in intestine. In conclusion, consumption of A. japonicus could improve the mucosal immunity and has better effect on respiratory than intestine. This study elucidated the positive effects of A. japonicus on mucosal immunity for the first time, suggesting that moderate consumption of A. japonicus is helpful in improving mucosal immunity and preventing exogenous infection, which provides an experimental basis for chemotherapeutic patients.
Funding This work was supported by the National Natural Science Foundation of China [grant number 31101281]; Program for Changjiang Scholars and Innovative Research Team in University [grant number PCSIRT: IRT1188]; Special funds for “Taishan Scholar” construction project.
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