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ANIMAL NUTRITION |
National Key Lab of Animal Nutrition, China Agricultural University, Beijing, P. R. China 100094
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Key Words: Astragalus membranaceus • ß-Glucan • Immunity • Inflammatory Response • Piglet
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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). Previous studies advanced the concept that pigs injected with Escherichia coli could imitate the response of piglets exposed to microorganisms under a conventional environment (Johnson, 1997; Webel et al., 1997). Klasing (1988) illustrated that an injection of lipopolysaccharide (LPS) resulted in decreased growth rate, accelerated degradation of body protein, and a glucose production peak caused by an abundant release of proinflammatory cytokines such as IL-1. In several species, including catfish and mice, feeding ß-glucans has been shown to stimulate both specific and nonspecific immune responses (Chen and Ainsworth, 1992; Yun et al., 1997). Additionally, polysaccharides from aloe also have been shown to decrease the inflammatory response (McAnalley et al., 1995), although other polysaccharides also were observed to induce the production of IL-1 and tumor necrosis factor via stimulation of glucan receptor (Abel and Czop, 1992).
In China, Astragalus membranaceus (AM), which is originally from Mongolia, has been reported to contain a relatively high concentration of ß-glucan (Fang, 1988). In broilers fed the glucan from AM, an increase in peripheral T-lymphocyte proliferation has been noted (Tang et al., 1998). Because of their attributes of natural origin, producing no drug residues and low side effects (Lu et al., 2003), herbal medicines such as AM are an attractive alternative to antibiotics. Therefore, the objective of the current study was to explore the effects of Astragalus membranaceus ß-glucan (AMG) on the performance as well as immunological, adrenal, and somatotropic responses of weaned piglets after LPS challenge.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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A total of 108 weanling pigs (Large White x Landrace x Pietran) weaned at 28 d of age (7.75 ± 0.24 kg of BW) were randomly allotted to one of six treatments by initial BW, ancestry, and sex in a 2 x 3 factorial arrangement; main effects were LPS challenge (with or without) and level of AMG (0, 500, or 1,000 mg/kg; as-fed basis). Piglets were housed in six replicate pens per treatment (three pens of females and three pens of males) with three pigs per pen. The pens were equipped with metal slotted floors and measured 1.8 m x 1.25 m. Each pen had a feeder and a nipple waterer to allow pigs ad libitum access to feed and water. The initial temperature was 29°C, and it was lowered 1°C each week during the study. Piglet weight and feed consumption were determined weekly. The experimental protocol in this study was approved by the Animal Care and Use Committee of China Agricultural University.
Experimental Diets
The experimental diets (Table 1) were formulated to meet or exceed NRC (1998) requirements for all nutrients. The experimental diets were formulated using primarily corn and soybean meal with smaller amounts of fish meal, whey powder, and spray-dried porcine plasma. A basal diet was formulated, and then the remaining two diets were supplemented with 500 and 1,000 mg of AMG/kg. Astragalus membranaceus was purchased from the HongSheng Herbal Drug Store (Beijing, China), and the glucan was extracted as described subsequently.
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Extraction of AMG was conducted according to the method of Ni (1998) with some modifications. In summary, 12 L of water were added to 1 kg of AM and boiled for 90 min. After this procedure, 8 L of water were added into the residual post-filtration. The mixture was again boiled for 1 h and then filtered. The two filtrates were combined and concentrated to 10 L in a vacuum desiccator (Model FZG-15; Changzhou XianFeng Drying Equipment Co., Changzhou, China) at 70°C. A solution of 95% ethanol was added to precipitate the polysaccharide, which was then dissolved in distilled water. Following another addition of 95% ethanol to the polysaccharide solution, the solution was vacuum-dried, and the resulting powder consisting of the polysaccharides was removed.
Immune Challenge Model
Lipopolysaccharide (E. coli: Serotype O55:B5; Sigma Chemical Co., St. Louis, MO) was injected intramuscularly (200 µg/kg of BW) on d 7 and 21 of the trial. The LPS dose was based on the results of three preliminary studies (data not reported). The concentration of the LPS solution (200 µg/mL) was made by dilution with endotoxin-free Hank’s Balanced Salt Solution (HBSS; Sigma Chemical Co.). The control pigs were injected with an equal volume of endotoxin-free HBSS solution.
Blood Sampling and Analyses
On d 7 and 21, 3 h after the LPS challenge, blood samples (one piglet per pen) were collected into 10-mL heparinized vacuum tubes (Becton Dickinson Vacutainer System; Franklin Lake, NJ) and centrifuged (800 x g for 10 min) to separate plasma, which was stored at –20°C until analysis. A second 10-mL sample was taken from the same pig for the lymphocyte proliferation study. All blood responses were analyzed in duplicate.
Plasma glucose was assayed using a Technicon Automatic Biochemical Analyzer (Technicon RA1000; Miles Inc., Diagnostics Division, Tarrytown, NY). Plasma IL-1ß was analyzed using a commercially available swine IL-1ß ELISA kit (BioSource International Inc., Camarillo, CA). Minimum detectability of swine IL-1ß was 15 pg/mL, and the intraassay CV was <10%.
Plasma cortisol was analyzed using a commercially available 125I RIA kit (Beijing Beimian Dongya Institute of Biological Technology, Beijing, China). The minimum detectable concentration of cortisol was 1 ng/mL, and the intraassay CV was <5%.
Prostaglandin E2 was analyzed using a commercially available 125I RIA kit (College of Medical Science of Suzhou University, Jiangsu, China). Minimum detectability of porcine PGE2 was 6.25 pg/mL, and the intraassay CV was <10%.
Plasma GH was measured using a commercially available 125I RIA kit (Beijing North Institute of Biological Technology, Beijing, China). The assay used human GH and antibodies against human GH as the standard. Minimum detectability of GH was 0.1 ng/mL, and the intraassay CV was <10%.
Plasma IGF-I was analyzed using a commercially available 125I RIA kit (Biocode S. A., Brussels, Belgium). In the assay, recombinant human IGF-I and mouse antiIGF-I monoclonal antibody were used as the standard. Recovery rate ranged from 92.3 to 110.0%. The intraassay CV was <10%, and the minimum detectable concentration of IGF-I was 5 ng/mL.
In Vitro Lymphocyte Proliferation Response to Concanavalin A
In vitro cellular immune response was measured using a lymphocyte blastogenesis assay. Peripheral T-lymphocyte proliferation was assayed according to a previously described method (Qiao et al., 2001). In brief, blood was layered onto a Ficoll-Hypaque solution (density = 1.077; Tianjin Blood Research Center, Tianjin, China). The lymphocytes were collected by density-gradient centrifugation at 686 x g for 30 min and washed three times with HBSS. The lymphocytes were then resuspended in 10 mL of RPMI 1640 complete culture medium (Gibco BRL, Grand Island, NY) supplemented with 10% (vol/vol) of heat-inactivated fetal calf serum, 100 U of penicillin /mL, 100 µg of streptomycin/mL, and 25 mM of N-(2-hydroxyethyl)-piperazine-N-2-ethane-sulfonic acid. The cells were detected by trypan blue dye exclusion and counted to adjust the density of the cells to 2 x 106 cells of culture medium/mL. Then, 100 µL of cell suspension and the lymphocyte mitogen concanavalin A (Sigma Chemical Co.) were added to a 96-well microtiter plate to provide a final concentration of 5 µg of concanavalin A/mL. Preliminary validation assays using concanavalin A at 2.5, 5, 10, and 20 µg/ mL were conducted to assess an optimal lymphocyte stimulation level based on our experimental conditions and genetic background of the piglets. Cells then were incubated at 37°C with 5% CO2 in an incubator. After 66 h of incubation, 10 µL of MTT (3- [4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium) were added to each well, and the plates were incubated for another 6 h. Subsequently, 100 µL of 10% sodium dodecyl sulfate dissolved in 0.04 M HCl solution were added into plates to lyse the cells and solubilize the MTT crystals. Finally, the plates were read via an automated ELISA reader (Sunrise; Tecan, Salzburg, Austria) at 570 nm.
Assay of IL-2 Biological Activity of Peripheral Blood
The IL-2 production was determined via the biological activity assay method according to the method of previous studies (Liu and Li, 1999), which is described in the following three sections.
Acquirement of IL-2 Sample by Astragalus Membranaceus Glucan Treatments
Lymphocytes were prepared as described previously, suspended in RPMI 1640 complete media, and adjusted to a cell density of 2 x 107 cells/mL. The combination of 1 mL of cell suspension and 1 mL of RPMI 1640 supplemented with 5 µg of concanavalin A/mL was added into 24-well plates and incubated for 24 h at 37 °C with 5% CO2. The cell culture was transferred into a cube and centrifuged at 572 x g for 15 min. Then, the supernatant fraction was collected and stored at – 20°C for determination of IL-2 activity.
Preparation of Target Cells
The combination of 1 mL of cell suspension with a cell density of 2 x 107 cells/mL and 1 mL of 20 µg of concanavalin solution/mL was added into a 24-well plate and incubated for 48 h at 37°C with 5% CO2 in the incubator. The cell culture was then obtained and purified by density-gradient centrifugation at 686 x g for 30 min. The precipitate obtained was washed immediately with RPMI 1640 and centrifuged twice. Finally, the precipitate was cultured in the RPMI 1640, in which the cell density was adjusted to 1 x 107 cells/mL.
Determination of IL-2 Activity
The combination of 50 µL of target cell culture, 50 µL of IL-2 sample supernatant fraction, and 50 µL of RPMI 1640 was added into a 96-well plate and incubated at 37°C with 5% CO2 for 24 h. Salt of MTT (final concentration of 5 µg/mL) was added into the plate for assessment of cell proliferation. Finally, 50 µL of a 10% sodium dodecyl sulfate in 0.04 M HCl solution were added to lyse the cells and solubilize the MTT crystals. The absorbance of each sample was obtained on an automatic ELISA reader at 570 nm.
Statistical Analyses
Data were analyzed by ANOVA using the GLM procedures of SAS (SAS Inst., Inc., Cary, NC.). The initial statistical model included the effects of sex (barrow vs. gilt), challenge (HBSS vs. LPS), level of AMG (0, 500, or 1,000 mg/kg), and their appropriate interactions. Because of the fact that the effect of sex on all responses was not significant, this variable was excluded in subsequent analyses. Linear and quadratic effects of AMG on all indices were determined according to the regression program of SAS. Pen was used as experimental unit for the performance data, whereas individual pig was used as the experimental unit for plasma responses, lymphocyte proliferation, and IL-2 bioactivity analysis. An 
level of 0.05 was used as a criterion for statistical significance, whereas a level of 0.10 was taken to indicate a statistical trend.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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The results of the entire performance trial showed no improvement in weight gain caused by dietary inclusion of AMG (Table 2). Pigs challenged with LPS had lower weight gain (P < 0.01) than did controls in the week immediately after injection with LPS. Feed intake increased quadratically (P < 0.01) with increasing addition of AMG only during the weeks after the challenge of pigs with LPS. Pigs fed 500 mg of AMG/kg had the greatest feed intake. Feed conversion rate (P < 0.01) was only affected by LPS after the first LPS challenge, but it was not affected by the second challenge or inclusion of AMG.
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After both LPS challenges, an interaction (P = 0.01 to 0.09; Table 3) between AMG and LPS was observed for all plasma indices with the exception of IGF-I after the second challenge and growth hormone after both challenges. Plasma concentrations of glucose, IL-1ß, PGE2, and cortisol were all increased (P < 0.05) after both LPS challenges compared with the control; the responses generally were more pronounced in pigs fed 0 mg of AMG/kg. The IGF-I concentrations were less (P < 0.05) for LPS-challenged pigs than for control pigs (again with a greater response in pigs fed 0 mg of AMG/ kg), whereas GH concentrations were unaffected by LPS. Astragalus membranaceus glucan had a quadratic effect on the plasma concentrations of glucose, IL-1ß, PGE2, and cortisol (P < 0.05) after both LPS challenges; pigs fed the 500-mg of AMG/kg diet generally had a more muted response to the LPS challenge than those fed 1,000-mg/kg diet after both immune challenges. However, GH concentrations were unaffected by AMG.
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The lymphocyte proliferation response to 5 µg of concanavalin A/mL was increased linearly (P < 0.01) with increasing AMG after both LPS challenges (Table 4). Pigs challenged with LPS had greater (P = 0.06) T-lymphocyte proliferation than unchallenged pigs after the first immunological challenge but not after the second. The IL-2 bioactivity of peripheral blood increased linearly (P < 0.05) with increasing AMG after both LPS challenges. Pigs challenged with LPS had greater (P = 0.07) IL-2 bioactivity than unchallenged pigs after the first but not after the second LPS challenge.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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, who reported no improvement in piglet performance, in the first of a series of experiments, when ß-glucan was included at 1,000 ppm. A subsequent experiment using 250 ppm produced modest improvement in piglet performance. Schoenherr et al. (1994) concluded that the optimum inclusion level of ß-glucan to maximize piglet performance was between 250 and 500 ppm when fed throughout the entire nursery period. Therefore, the optimum level of AMG needs to be further validated; however, the levels in the current study were chosen to allow for effects on factors other than growth performance. The LPS challenge significantly decreased piglet weight gain, and this finding agrees with some previous studies in pigs (Johnson and von Borell, 1994; Liu et al., 2003).
The circulating concentrations of the inflammatory cytokine IL-1ß and PGE2 were increased by LPS challenge, but pigs fed AMG had lower concentrations of IL-1 and PGE2 than did pigs fed the control diet. The increase in IL-1 and PGE2 in the presence of the LPS challenge is consistent with earlier work, which has shown that, during an immune challenge, one of the first responses of an animal is to release cytokines such as interleukin-1ß from macrophages (Spurlock, 1997). The release of these cytokines stimulates the production of PGE2 in muscle (Hellerstein et al., 1989) and initiates a wasting process (Rodermann and Goldberg, 1982). The end result of this process is that nutrients are directed away from tissue growth to support immune function (Spurlock, 1997). Dritz et al. (1996) observed that an LPS challenge resulted in temporarily decreased feed intake, but residual effects could be observed. Those researchers also illustrated that decreased growth performance resulting from LPS challenge was attributable to two parts: approximately two-thirds of the decrease was due to decrease in nutrient intake, and one-third was due to decrease in efficiency of nutrient use for growth.
Astragalus membranaceus ß-glucan decreased the release of IL-1, which was consistent with the reports of Tzianabos (2000). This result would presumably allow the animal to continue directing resources toward tissue growth, which may explain the improved growth response observed by Dritz et al. (1995) and Schoenherr et al. (1994) to lower levels of ß-glucan than those used in the current study. The anti-inflammatory effects of AMG could be attributed to three possible mechanisms. First, ß-glucan may inhibit pro-inflammatory cytokine synthesis through the synthesis of anti-inflammatory cytokines such as IL-10, secreted by lymphocytes to maintain the balance between the pro- and anti-inflammatory mediators (Hoqaboam et al., 1998). Second, ß-glucan may promote the secretion of IL-1ß receptor antagonist (Poutsiaka et al., 1993), which is a specific inhibitor of IL-1ß receptor. Third, Castro et al. (1994) observed that the release of macrophage arachidonic acid metabolites (prostaglandin) in response to Candida albicans was inhibited to a significant, but lesser, degree by soluble ß-glucan. The AMG may inhibit IL-1 synthesis because of its inhibition of arachidonic acid metabolite production, a mechanism suggested by Belury (2002); however, the mechanisms related to a possible interaction between AMG were not explored.
In the current study, the inconsistent response of the GH/ IGF-I axis was in agreement with the reports of Thissen and Verniers (1997), who suggested that the resistance to GH decrease induced by IL-1ß might be mediated by a decrease of GH receptors. Hasselgren (1993) suggested that the decreases in IGF-I were indicative of the repartitioning of nutrients away from normal growth to the activation of immune system after LPS challenge. Soto et al. (1998) also concluded that the decrease of IGF-I and GH release was an important causative factor of decreased growth during an inflammatory challenge; however, low IGF-I was not always concomitant with a corresponding decreased concentration of GH (Gianotti et al., 1998). Anorexia induced by LPS inflammatory challenge was associated with the decreased release of IGF-I (Fan et al., 1994). Accordingly, in the current study, AMG, which had the effect of mitigating the decrease in feed intake, might have caused an attenuated decrease of IGF-I release.
In the current study, the finding that the circulating concentration of glucose was increased after LPS challenge was consistent with the results of Bieniek (1998). This increase, induced by an immune challenge, may be partly attributed to a change in the release of conventional hormones that regulate glucose metabolism. Some studies have shown that glucose production via gluconeogenesis and glycogenolysis, oxidation in extra-hepatic tissues, and recycling via the Cori cycle are increased during an immunological stress (Long, 1977; Meszaros et al., 1987). In rats, Roh et al. (1986) studied injection of a crude IL-1 preparation and observed an increased rate of alanine transport into hepatocytes, which was responsible for an increased gluconeogenesis from alanine. In addition, altered glucose metabolism was associated with release of some pro-inflammatory cytokines (Del Rey and Besedovsky, 1987). Rabinovitch et al. (1988) concluded that the glucose increase after a LPS challenge was related to the decreased secretion of insulin from rat islet cells. Astragalus membranaceus ß-glucan was able to attenuate the increase of glucose induced by the immunological stress, indicating that it may be capable of mitigating the overwhelming adrenal and somatotropic response to LPS challenge. However, the definite mechanism on how the ß-glucan acts to alter glucose metabolism needs to be elucidated further.
The T lymphocytes are the central regulatory cells of the immune system, and T-lymphocyte proliferation is an important indicator of lymphocyte function. Cantrell and Smith (1984) stated that ß-glucan could promote lymphocyte proliferation according to expression and secretion of cytokines in vivo and in vitro. They also illustrated that a key cytokine termed IL-2 was essential for T lymphocyte proliferation, in that it could promote the transition of activated T cells from cell cycle G1 to S phase. In the current study, AMG was found to promote IL-2 bioactivity, which agrees with some previous reports (Tang et al., 1998; Liu and Li, 1999). Accordingly, the increased bioactivity of IL-2 of peripheral blood lymphocytes may in part explain the increased lymphocyte proliferation.
The concentration of cortisol in plasma can be regarded as a criterion to reflect stress intensity (Webel et al., 1997). The proinflammatory cytokines such as IL-1 have the potential to change many aspects of neuroendocrine function, including the hypothalamic-pituitary-adrenal (HPA) axis (Maule and Vanderkooi, 1999). Interleukin-1 has been shown to stimulate neurons in the hypothalamus to secrete corticotropin-releasing hormone (CRH; Berkenbosch et al., 1987), which stimulated the adrenal cortex to produce cortisol (George and Chrousos, 1995). Consistent with these concepts, we observed that the increase in IL-1 due to LPS challenge was followed by an increase in plasma cortisol concentrations. Astragalus membranaceus ß-glucan decreased plasma cortisol concentrations induced by the LPS challenge, suggesting an effect of AMG in alleviating immune stress.
An excessive HPA response to inflammatory response induced by LPS was observed in the current study according to the increased concentration of IL-1 and cortisol, which resulted in immunosuppression. Accordingly, an interaction between the HPA axis and immune-mediated inflammatory response was observed. In addition, George and Chrousos (1995) stated that an excessive HPA response to inflammation could mimic the state of stress and, thus, increased susceptibility to infectious agents, but enhanced the resistance to inflammatory response; however, a quiescent system was not always resistant to pathogen intrusion. Blecha et al. (1994) illustrated that swine herds free from most common swine pathogens were more susceptible to clinical epidemics of disease caused by uncommon swine pathogens. Based on the above statements, a HPA response to immunological challenge may attenuate the inflammatory response but lead to more susceptibility to pathogen intrusion. In the current study, the evidence that AMG attenuated inflammatory response resulting from LPS challenge and simultaneously increased the immunity of pigs challenged with LPS seemed to contradict the above statements. Certainly, complicated mechanisms concerning effects of AMG on HPA axis and inflammatory response need to be explored further.
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Implications |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Footnotes |
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2 Correspondence: No. 2. Yuanmingyuan West Road (phone: 8610-62893588; fax: 8610-62893688; e-mail: Defali{at}public2.bta.net.cn).
Received for publication January 14, 2005. Accepted for publication August 16, 2005.
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Literature Cited |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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of the insulin-like growth factor-I messenger ribonucleic acid response to growth hormone in rat hepatocyte primary culture. Endocrinology 138:1078–1084.This article has been cited by other articles:
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