Effects of Achyranthes Bidentata Polysaccharide on Growth Performance , Immunological , Adrenal , and Somatotropic Responses of Weaned Pigs Challenged with Escherichia coli Lipopolysaccharide

A study was conducted with 48 weaned barrows (28±3 d, 8.45±0.14 kg) to determine the effect of Achyranthes bidentata polysaccharide (ABPS) supplementation on pig performance, immunological, adrenal and somatotropic responses following Escherichia coli lipopolysaccharide (LPS) challenge. The experiment was a 2×2 factorial design; the main factors included diet (supplementation with 0 or 500 mg/kg ABPS) and immunological challenge (LPS or saline). On d 14 and 21 of the trial, pigs were given an intraperitoneal injection with either 100 μg/kg BW of LPS or an equivalent amount of sterile saline. Blood samples were obtained 3 h after injection for analysis of tumor necrosis factor-α (TNF-α), prostaglandin E2 (PGE2), cortisol, growth hormone (GH), insulin-like growth factor (IGF)-I and immunoglobulin G (IgG). On d 2 after LPS challenge, peripheral blood lymphocyte proliferation (PBLP) was measured. LPS administration decreased average daily feed intake (ADFI) (p<0.05), had a tendency to decrease average daily gain (ADG) (p<0.10) during both the first and second challenge periods and increased (p<0.05) feed:gain ratio only during the first challenge period. ABPS tended to improve ADG (p<0.10) during the first challenge period, and improved ADG (p<0.05) and tended to improve ADFI (p<0.10) during the second challenge period. ABPS did not affect feed:gain ratio. An interaction (p<0.05) between LPS challenge and diet was observed for the plasma concentrations of TNF-α, PGE2 and cortisol after both LPS challenges such that, among LPStreated pigs, pigs fed the ABPS diet were lower for these indices than those receiving the control diet. In contrast, pigs fed the ABPS diet had higher IGF-I (p<0.05) compared with those fed the control diet. No effect of diet, LPS challenge or both on GH and IgG was observed after both LPS administrations. LPS challenge increased PBLP when these cells were incubated with 8 μg/ml of LPS during both the challenge periods, and did likewise when incubated with 8 μg/ml of concanavalin A only after the first challenge. ABPS had no effect on PBLP. These data demonstrate that ABPS alters the release of pro-inflammatory cytokines following an immunological challenge, which might enable pigs to achieve better performance. (


INTRODUCTION
In swine production, the continual exposure of weanling piglets to a wide variety of microorganisms will lead to an immunological challenge, which can result in a series of physiological changes including temporary fever, depressed feed intake and activation of the immune system (Kelley et al., 1994).Consequently, the immunological challenge results in poor growth on animals and increases economic loss for pig producers.Johnson (1997) and Webel et al. (1997) propose that these changes are mostly ascribed to the release of pro-inflammatory cytokines, including tumor necrosis factor (TNF), interleukin (IL)-1 and IL-6.Overproduction of these pro-inflammatory cytokines has negative effects on growth and feed efficiency.Therefore, using immunomodulators to modulate the secretion of these cytokines is considered as a potential means to mitigate the negative effects induced by an immunological challenge (Lang et al., 1996).
Achyranthes bidentata polysaccharide (ABPS), a graywhite powder, which is isolated from the root of Chinese medicinal herb Achyranthes bidentata Blume, is composed of fructose and glucose residues and the molar ratio is 8:1 (Chen et al., 2005).ABPS is a graminans-type fructan that contains a β-D fructofuranosyl backbone having residues linked (2→1)-and (2→6)-with branches and an α-Dglucopyranose residue on the nonreducing end of the fructan chain (Jin et al., 2007).Compared with other polysaccharides, ABPS has a smaller molecular mass.In humans and rodents, it has been used as an immune modulator (Li and Li, 1997;Shao et al., 2002;Jin et al., 2007).Because of its traits of natural origin, as well as diverse pharmacological effects, producing no drug residues and low side effects, ABPS is an attractive alternative to antibiotics.Within our knowledge, no study has been conducted to observe the effect which ABPS exerts its activities in pigs.Therefore, this study was conducted to evaluate the effects of dietary ABPS supplementation on performance, as well as the immunological, adrenal, and somatotropic response in weaned pigs challenged with Escherichia coli lipopolysaccharide (LPS).

Experimental design
Our experimental protocol was approved by the Animal Care and Use Committee of Hubei Province.A total of 48 crossbred (Duroc×Large White×Landrace) male pigs weaned at 28±3 d of age (8.45±0.14kg) were randomly allotted to one of four treatments by initial BW in a 2×2 factorial arrangement that included a dietary addition of ABPS (0 or 500 mg/kg) and LPS challenge (with or without).
Pigs were housed in 1.20×1.10m pens with six replicate pens per treatment with two pigs per pen.Each pen was equipped with plastic slotted floor, and a feeder and a nipple waterer to allow pigs ad libitum access to feed and water.The temperature in the room was controlled at 25-27°C by air condition.The basal diet (Table 1) was formulated to meet or exceed NRC (1998) requirements for all nutrients.ABPS, which contained polysaccharide no less than 90% and had a molecular weight of 2,680, was purchased from Zhejiang Jingxin Pharmaceutical Company (Zhejiang, China).Pigs were weighed and feed disappearance was measured on days 0, 14, 21 and 28 throughout the 28-day trial.

Immunological challenge model
On d 14 and 21 of the trial, the challenged group was injected intraperitoneally with LPS (Escherichia coli Serotype O55:B5; Sigma Chemical Co., St. Louis, MO, USA) at 100 μg/kg BW and the unchallenged group was administrated an equivalent amount of 0.9% (wt/vol) NaCl solution.The LPS was dissolved in sterile 0.9% NaCl solution such that 0.2 ml of solution/kg of BW would achieve the desired dosage.

Blood sample collections and assays
On d 14 and 21, 3 h after the LPS or saline administration, blood samples (all pigs) were respectively collected into heparinized vacuum tubes (Becton Dickinson Vacutainer System; Franklin Lake, NJ) and centrifuged (3,500×g for 10 min) to separate plasma, and then stored at -80°C until analysis.
Plasma prostaglandin E 2 (PGE 2 ) was analyzed using a commercially available 125 I RIA kit (College of Medical Science of Suzhou University, Jiansu, China), which we have also previously validated in pigs (Liu et al., 2003).Minimum detectability of PGE 2 was 6.25 pg/ml with an intra-assay CV less than 10%.
Plasma cortisol was analyzed using a Coat-a-CountTM assay kit (Diagnostic Products, Los Angeles, CA).Minimum detectable dose of cortisol was 2 ng/ml.Intraand inter-assay CV were 4.6% and 9.0%, respectively.
Plasma growth hormone (GH) was measured using a commercially available porcine GH 125 I RIA kit (Linco Research, Inc., St. Charles, MO).The minimum detection limit was 1 ng/ml, with an intra-assay CV of 4.0%.
Plasma insulin-like growth factor-I (IGF-I) was determined by using a commercially available porcine IGF-1 IRMA kit (Diagnostic Systems Laboratories, Inc.).The minimum detection limit was 2 ng/ml, with an intra-assay CV of 3.9%.
IgG concentration was determined using a radial immunodiffusion test kit according to the methods of Hicks et al. (1998) as a representative humoral immune response.Five microliters of standard solutions and diluted plasma samples were pipetted to a separately identified well of the test plates.The plate was securely covered and placed in a 37°C, humidified incubator for 48 to 72 h.After incubation, plates were removed and placed over a source of illumination to clearly see precipitin rings.The external diameters of the rings were measured to the nearest 0.1 mm using the scale provided.A reference curve was plotted using the diameters measured from standard solutions.From the reference curve, the IgG concentration of each diluted test sample was calculated by multiplying the concentration read from the curve by the dilution factor to obtain the actual concentration.
All blood related measurements were analyzed in duplicate.

Lymphocyte proliferation
Lymphocyte proliferation was measured using a colorimetric assay, with 3-(4,5-dimethlthiazol-2-yl)-2,5diphenyltetrazolium bromide (M-2128, Sigma Chemical Inc., St. Louis, USA) in cultures of purified peripheral blood mononuclear cells according to the method of Liu et al. (2003) and Kong et al. (2007).Briefly, mononuclear cells were collected by gradient centrifugation from the peripheral blood obtained d 2 after the first and the second LPS injection, at 3,000×g for 30 min.The cells were washed three times, and then suspended in RPMI-1640 complete culture medium supplemented with 10% (vol/vol) heat-inactivated fetal calf serum, 100 U/ml of penicillin, 100 μg/ml of streptomycin and 25 mM N-(2-hydroxyethyl)piperazine-N'-2-ethane-sulfonic acid.Cell activity was detected by trypan blue dye exclusion, and the cell density was counted to adjust to 2× 10 6 cells/ml culture medium.Then the cellular suspension was added to 96-well microtiter plates with a total culture volume of 200 μl.
Lymphocyte mitogen concanavalin A (ConA; Type IV, C-2010, Sigma Chemical Inc., St. Louis, USA) or LPS was added at a final concentration of 8 μg/ml culture medium, and then the plates were incubated at 37°C in a 5% CO 2 incubator.After 66 h of incubation, 10 μl MTT solution (5 mg MTT/ml in 1/15 M phosphate-buffered saline, pH 7.6) was added to each well and the plates were incubated at 37°C for another 6 h.At the end of incubation, 100 μl of a 10% sodium dodecyl sulfate in 0.04 M HCl solution was added to lyse the cells and solubilize the MTT crystals.Plates were read at 570 nm using an automated microplate reader (Bio-Rad, Model 550, Hercules, CA).Lymphocyte proliferation was expressed as a stimulation index, which was calculated as the absorbance of wells incubated with ConA or LPS divided by the absorbance of wells incubated without ConA or LPS.

Statistical analysis
All data were analyzed by ANOVA using the GLM procedures of SAS (SAS Inst.Inc., Cary, NC) appropriate for a factorial arrangement of treatments in a randomized complete block design.The statistical model consisted of the effects of challenge (saline or LPS), diet (ABPS or no ABPS) and their interactions.Pen was used as experimental unit for the performance data, whereas individual pig data were used as the experimental unit for blood analysis and measurement of lymphocyte proliferation.A level of p<0.05 was used as the criterion for statistical significance, whereas p<0.10 was taken to indicate a statistical trend.

Growth performance
There was no LPS challenge×diet interaction for average daily gain (ADG), average daily feed intake (ADFI) and feed/gain observed throughout the 28-d trial (Table 2).Prior to LPS challenge (from d 0 to 14), dietary treatment did not affect growth performance.During the first challenge period (from d 14 to 21), pigs fed ABPS tended to have a 10.0% higher ADG (p<0.10)than the control pigs, but ADFI and feed:gain were not affected.Correspondingly, LPS challenge reduced ADG (p<0.05) by 21.7% and ADFI (p<0.05) by 11.1%, and increased feed:gain (p<0.05) by 13.6% compared to the salineinjected pigs regardless of dietary treatment.During the second challenge period (from d 21 to 28), supplementation of ABPS increased ADG (p<0.05) by 8.9% and tended to increase ADFI (p<0.10) by 5.6%; whereas LPS challenge had a tendency to decrease ADG (p<0.10) by 7.7% and decreased ADFI (p<0.05) by 7.6%.In the whole performance trial (from d 0 to 28), there was no significant affect on ADG, ADFI and feed:gain observed by addition of ABPS, but LPS challenge reduced ADG (p<0.05) by 9.5% and ADFI (p = 0.05) by 6.2%, and had a tendency to increase feed:gain (p<0.10) by 3.7% compared with the saline-treated pigs.

Lymphocyte proliferative and humoral responses
The results of lymphocyte proliferation are shown in Table 3.There was no LPS challenge×diet interaction observed for lymphocyte proliferation when incubated with either LPS or ConA.Dietary treatment had no effect on lymphocyte proliferation.LPS challenge increased (p<0.05)lymphocyte proliferation when incubated with 8 μg/ml LPS after the first and second LPS challenges, and did likewise with 8 μg/ml ConA only after the first LPS challenge.
The concentration of serum IgG was measured to represent the humoral immune response, and the results are presented in Table 4.There was neither LPS challenge nor diet effect on the concentration of serum IgG after the first and the second LPS challenge.

Plasma TNF-α, PGE 2 , cortisol, GH, and IGF-I
As indicated by Table 5, after the first and second LPS challenges, an interaction between ABPS and LPS was observed for all plasma indices with the exception of GH.Plasma TNF-α (p<0.05),PGE 2 (p<0.10) and cortisol (p<0.10)responses to the LPS challenge were lower in pigs receiving the ABPS diet than the LPS-treated pigs fed the control diet, whereas there was no difference for these  indices response in the saline-injected pigs.In contrast, plasma IGF-I in pigs fed the ABPS diet was higher (p<0.05)than in those fed the control diet among LPS-injected pigs, whereas there was no difference among saline-injected pigs.
No effects of diet, LPS challenge or both on plasma GH concentration were observed following both the first and the second LPS challenge.

DISCUSSION
In the present study, we used the well-recognized model for inducing sickness in pigs by injecting LPS (Johnson and von Borell, 1994;Lien et al., 2005) which is a component of the outer membrane of gram-negative bacteria, to determine the effect of ABPS supplementation on immunological challenge.Until now, few researches were conducted to evaluate the effect of ABPS on growth performance of livestock.In our study, before LPS challenge, dietary treatment had no effect on pig performance.This finding is similar to Chen (2002) who reported that there was no effect on broiler performance when ABPS added to 200 mg/kg.However, pigs fed 500 mg/kg ABPS had higher weight gain during both LPS challenge periods and higher feed intake during the first LPS challenge period compared with those fed the control diet, which indicates the importance of ABPS supplementation under stress, infection and diseases.The LPS challenge significantly decreased pig weight gain and feed intake, and this finding is consistent with some previous studies in pigs (Lee et al., 2000b;Liu et al., 2003;Mao et al., 2005).
In the current study, the circulating concentrations of the inflammatory indices TNF-α and PGE 2 were increased after LPS injection, which is consistent with Lee et al. (2000a) and Liu et al. (2008a, b).It is well known that, during an immunological challenge, one of the first responses of an animal is to release pro-inflammatory cytokines such as IL-1β and TNF-α from macrophages (Spurlock, 1997), which will induce an increase in the secretion of PGE 2 in muscle (Hellerstein et al., 1989) and an activation of the immune system.This response directs nutrients away from tissue growth to support immune function (Spurlock, 1997), and finally decreases the efficiency of nutrient utilization for growth.The pigs fed ABPS had lower concentrations of TNF-α and PGE 2 than did pigs fed the control diet among the LPS-challenged pigs, which indicates an antiinflammatory role of ABPS.Until now, no other research was conducted to evaluate the anti-inflammatory role of ABPS.The exact mechanism(s) by which ABPS exerts its anti-inflammatory role on the animals is unclear.However, there are three possible pathways for anti-inflammatory effects of ABPS.
First of all, ABPS may inhibit pro-inflammatory cytokine synthesis through the synthesis of antiinflammatory cytokines.Anti-inflammatory cytokines such as IL-10, by suppressing the activity of the signal transduction of nuclear transcription factor κB which is a major transcription factor of pro-inflammatory cytokines (Schottelius et al., 1999) inhibit the synthesis of proinflammatory cytokine to maintain the balance between the pro-and anti-inflammatory mediators (Hoqaboam et al., 1998).Secondly, ABPS may increase the synthesis of IL-1 receptor antagonist.IL-1 receptor antagonist is a competitive inhibitor of interleukin-1 that binds to type I interleukin-1 receptors (Poutsiaka et al., 1993).Regretfully, we did not measure the anti-inflammatory cytokines and interleukin receptor antagonists in the current study.Finally, ABPS may inhibit pro-inflammatory cytokines synthesis by decreasing the production of arachidonic acid metabolites such as prostaglandins (PGE 2 in the current study).
Prostaglandins play a major role in the inflammatory and immune responses and are capable to decrease cytokines production (Calder, 1997).
In our study, that ABPS supplementation significantly suppressed the increase of pro-inflammatory cytokine TNFα and PGE 2 release, which indicates less activation of the immune system, and consequently more nutrients to support tissue growth.This may partially explain why pigs fed 500 mg/kg ABPS had higher gain and feed intake compared with the control.
Besides depressed gain and feed intake, a reduction in plasma IGF-I was observed in pigs administrated with LPS.The study of Hasselgren (1993) suggested that the decrease in IGF-I was indicative of the repartitioning of nutrients away from normal growth to the immune response after LPS challenge.Soto et al. (1998) also proposed that the decrease in release of IGF-I was an important causative factor of decreased growth during an immunological challenge.Therefore, that the significantly mitigated reduction of plasma IGF-I due to addition of ABPS observed in our study may be an indication that ABPS supplementation alleviates the alterations in somatotropic axis after an LPS challenge, which might have been associated with the improved performance in pigs supplemented with ABPS compared with control pigs.
In the present study, in those LPS-challenged pigs, feeding ABPS partially attenuated the reduction of plasma IGF-I, concurrent with lower plasma TNF-α levels compared with the control.Therefore, the effect of ABPS on alleviating plasma IGF-I reduction seems to be closely associated with the decrease of pro-inflammatory cytokine release, such as TNF-α.McCarthy et al. (1995) proposed that anorexia induced by pro-inflammatory cytokines after LPS challenge was associated with the suppressed release of IGF-I.Accordingly, in our study, those LPS-challenged pigs fed with ABPS mitigated the decrease in feed intake, correspondingly attenuated the reduction of IGF-I.This may also explain why pigs fed ABPS had a higher feed intake compared with control pigs.
Pro-inflammatory cytokines are not only primarily associated with immune function, but also have the potential to change many aspects of neuroendocrine function, including the hypothalamic-pituitary-adrenal axis (HPAA) (Mandrup-Poulsen et al., 1995).Pro-inflammatory cytokines have been shown to stimulate neurons in the hypothalamus to release corticotropin-releasing hormone (Perlstein et al., 1993), which stimulated the adrenal cortex to produce cortisol (George and Chrousos, 1995).Concurred with aforesaid viewpoint, in the current study, we observed the increase in TNF-α induced by LPS challenge was concomitant with a rapid elevation of plasma cortisol, which is in agreement with previous study of Liu et al. (2003).However, feeding ABPS significantly decreased the level of plasma pro-inflammatory cytokine TNF-α and cortisol induced by LPS challenge.Therefore, ABPS may decrease the production of cortisol by suppressing proinflammatory cytokine TNF-α release.
In our study, ABPS supplementation decreased the release of pro-inflammatory cytokine TNF-α and PGE 2 in LPS-treated pigs, but did not reduce the production of TNFα and PGE 2 in saline-injected pigs, indicating that ABPS mitigated the excessive activation of immune system in animals subjected to immunological challenge, but did not affect the immune function of normal animals.Furthermore, we did not observe an effect of ABPS on the concentration of IgG and lymphocyte proliferation in the current study.So ABPS did not decrease the immune function of normal animals.Therefore, it is rational to say that it is not bad at least to animals to fed ABPS when pigs are challenged by infectious diseases.Further evaluation of effects of ABPS on growth and inflammatory response in pigs challenged by infectious diseases is warranted before it is used in commercial practice.

IMPLICATIONS
The results of current study indicate that supplementation with achyranthes bidentata polysaccharide to pigs diet is able to relax the immunological and adrenal response to an Escherichaia coli lipopolysaccharide challenge by mediating the release of pro-inflammatory cytokines, and make pigs achieve better growth performance finally.However, source of achyranthes bidentata polysaccharide obtained from different methods or producers may vary in their structure, chemical composition, or both, and further evoke different effects on pigs.Accordingly, further investigation is warranted to focus on not only the performance and immune effect of achyranthes bidentata polysaccharide, but also the method producing the polysaccharide when it is added to animal diets.

Table 1 .
Ingredient composition of the basal diet (as-fed basis)

Table 3 .
Effect of feeding achyranthes bidentata polysaccharide (ABPS) and lipopolysaccharide (LPS) challenge on the proliferation of lymphocytes isolated from peripheral blood in weaned pigs after both lipopolysaccharide injection a, b Lipopolysaccharide was injected on d 14 and 21.Values are means (n = 12) for all pigs per treatment.Values are expressed as stimulation index, which is calculated as: absorbance of wells incubated with concanavalin A (ConA) or LPS divided by the absorbance of wells incubated without ConA or LPS.b Blood was obtained 2 d after the first and the second LPS administration.c LPS effect (p<0.05). a

Table 4 .
Effect of feeding achyranthes bidentata polysaccharide (ABPS) and lipopolysaccharide (LPS) challenge on serum immunoglobulin G (IgG) level after both lipopolysaccharide challenges in weaned pigs a, b a Lipopolysaccharide was injected on d 14 and 21.Values are means (n = 12) for all pigs per treatment.b Blood was obtained 3 h after the first and the second LPS administration.

Table 5 .
Effect of feeding achyranthes bidentata polysaccharide (ABPS) and lipopolysaccharide (LPS) challenge on plasma tumor necrosis factor-α (TNF-α), prostaglandin E 2 (PGE 2 ), cortisol, insulin-like growth factor (IGF)-I and growth hormone (GH) levels after both lipopolysaccharide challenges in weaned pigs a, b Lipopolysaccharide was injected on d 14 and 21.Values are means (n = 12) for all pigs per treatment.b Blood was obtained 3 h after the first and the second LPS administration. a