Study On The Mechanism Of Nutrition And Immunomodultion Of Achyranthes Bidentata Polysaccharide In Pigs

The study including the extraction,separation and purification of Achyranthes bidentata polysaeeharide(ABP),animal feeding trial in vivo and cell culture in vitro, systematically investigated the mechanism of nutrition and immunomodultion of achyranthes bidentata polysacchadde in pigs.ExperimentⅠwas carried out to study the extract technics of ABP and to analyze the component of ABP.The roots of traditional Chinese medicine Achyranthes(achyranthes bidentata) were selected to be extracted by boiling water,precipitated fractionally by alcohol.The protein in the extract was removed by tannic acid and the resulting solution was pplied to a DEAE-Sepharose fast-flow column,then to a Sephadex G-150 column. The eluant was dialyzed against water and lyophilized,the result indicated that the content of ABP in achyranthes bidentata is 28.86 percent,and mean molecular weight of ABP was estimated to be 1300-1400 d,its purity was 98.6%.HPLC analysis showed that ABP was the homogeneous polysaccharide,and ABP was composed of fructose residues and glucose residues,the molar ratio of fructose to glucose was 8:1.ExperimentⅡwas used to evaluate the effect of ABP on growth performance, diarrhea,mycoplasmlpneumonia of swine(MPS) and serum biochemistry parameters in pigs,One hundred and sixty(8.898±0.742) kg,28 d,three-line crossbred(Duroc×Landrace×Yorkshire) weaning piglets were randomly assigned to 5 treatments and each treatment had 4 replicates with 8 piglet per replicate:control group:basal diet, groupⅠ:basal diet+100mg/kg chlortctraeyeline,groupⅡ:basal diet+500mg/kg ABP, groupⅢ:basal diet+1 000mg/kg ABP,groupⅣ:basal diet+1 500mg/kg ABP.The experiment was conducted for 133days.The results showed that ABP significantly increased the average daily gain and feed conversion rate(P＜0.05,or P＜0.01) of pre-trial pig(28-60d),better than chlortctracycline.In the late trial(61-153d),there were no significant effect for ABP to improve growth performance;During all trial period (28-154d),compared with the control group,groupⅠ,Ⅱ,Ⅲ,Ⅳraised respectively the average daily gain by 2.11%(P＜0.05),3.90%(P＜0.01),5.53%(P＜0.01) and 2.44% (P＜0.05),the feed/gain of groupⅠ,Ⅱ,Ⅲand were decreased by 4.69%(P＜0.05), 3.25%(P＞0.05),7.22%(P＜0.05) and 5.78%(P＜0.05).ABP reduced significantly the rate of pig diarrhea and mycoplasml pneumonio of swine(MPS)(P＜0.05,or P＜0.01),its effect reached or exceeded chlortctracycline.During all trial period(8-100 kg BW), compared with the control group,groupⅠ,Ⅱ,Ⅲ,Ⅳdeclined significantly the rate of diarrhea by 29.40%,54.12%,53.00%and 56.75%(P＜0.01),and decreased the rate of MPS by 46.38%,78.70%,83.72%and 89.72%(P＜0.01);ABP significantly decreased the concentration of BUN,GLU in serun(P＜0.05 or P＜0.01),while ABP increased the activity of ALP(P＜0.05 or P＜0.01) and the concentration of immunoglobulin IgM,IgA, IgG and complement C3,C4 in serum(P＞0.05,P＜0.05,or P＜0.01),but chlortctracycline had no significant impact on the concentration of immunoglobulin and complement in serum(P＞0.05).ExperimentⅢwas used to study the effects of ABP on intestinal microflora and mucous membrane of piglets.In order to investigate the mechanism of promoting growth and curing diarrhea of ABP for piglets,on the 35th day in animal feeding trial,one pig each replicate was randomly selected to slaughter and to evaluate the effect of ABP on intestinal pH,intestinal mieroflora and mucous membrane of pigs.The results showed that ABP had an up-trend to pH value of rectum,cecum and colon,and groupⅢand groupⅣsignificantly decreased the pH value of cecum and colon(P＜0.05,or P＜0.01), but didn't have significant difference on pH value of rectal(P＞0.05),pH value between antibioties group and control group didn't have significant differences(P＞0.05).Groups added with ABP had the same effect or had better effect on inhibition of intestinal E.coil and promotion of intestinal salutary bacteria in contrast with antibiotics group(P＞0.05,P＜0.05 or P＜0.01);With the increasing of the addition of ABP in groupⅡ,Ⅲ,Ⅳ,there were more inhibition of intestinal E.coli and promotion of intestinal salutary bacteria, and there were significant difference between groupⅡand groupⅣ(P＜0.05 or P＜0.01 ); The duodenal epithelial cell thickness in all experimental groups had no significant difference;With the increasing of the addition of ABP,the epithelial cell thickness of jejunum and ileum increased significantly than that of the control group and experimental groupⅠ(P＜0.05orP＜0.01);Compared with the control group,groupⅡ,ⅢandⅣimproved intestinal villi height and reduced recess depth significantly(P＜0.05 or P＜0.01);In the duodenum,Ⅱ,Ⅲ,Ⅳgroup improved intestinal villi height and reduced recess depth significantly than groupⅠ(P＜0.05 or P＜0.01);There were no significant difference for the depths of recess of jejunum and ileum in all experimental groups,and the intestinal mucous membrane had no significant difference between groupⅢandⅣ.ABP increased significantly villus height and depth of recess ratio(VH / CD value) in duodenum,ileum and jejunum(P＜0.05 or P＜0.01),therefore ABP could improve digestion and absorption of nutrients,and then improve the growth perfonnance for pigs.In experimentⅣ,tweenty pigs were selected randomly(one pig each replicate )to analysis slaughter performance,carcass traits and meat quality at the end of the animal feeding trial.The content of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px) and malondialdehyde(MDA) in muscles was determinatd to investigate the mechanism of ABP on pork quality.The results showed that ABP can significantly improved loin eye area and lean meat percentage(P＜0.05),but didn't significantly impact dressing percentage and baekfat thickness(P＞0.05);ABP can significantly improved meat color score,marbling score and the content of intramuscular fat and the rate of cooked meat (P＜0.05),but there were no significant effect on muscle pH,drip loss and rate of water loss(P＞0.05);For meat color score and marbling score,ABP was significantly better than that of chlortctracycline(P＜0.05);Amino acids in the muscles had an enhancive trend in groups added with ABP,while there were no significant differentenee among all groups(P＞0.05).There was an active uptrend of SOD and GSH-Px by ABP or chlortctracycline compared with control group,and GSH-Px activity in groupⅣwas significantly higher than control group and groupⅠ(P＜0.05).Meanwhile,ABP had an up-trend to decrease the content of MAD in muscle,and decreased by 6.60%(P＞0.05), 10.76%(P＞0.05) and 18.53%(P＜0.05) in groupⅡ,ⅢandⅣrespectively.These results suggested that ABP could enhance the activities of antioxidant enzyme including GSH-Px and decrease the content of MAD in muscle,these may be beneficial for pigs to maintain good carcass quality.ExperimentⅤwas used to further study mechanism of ABP on improving growth performance,the liver,jejunal mucosa and mesenteric lymph nodes were taken out from the slaughtering pigs on the 35th day in raising experiment,using real-time fluorescent quantitative PCR to study the effect of ABP on IGF-Ⅰand IL-1βgene expression.The results showed that ABP could increase IGF-ⅠmRNA transcription in the liver,jejunal mucosa and mesenteric lymph nodes,compared with control group.With the addition of ABP,the transcription levels of IGF-ⅠmRNA increased gradually,but there were no diffenence among all groups.(P＞0.05).Meanwhile,ABP had notable effects on IL-1βmRNA transcription level in liver,jejunal mucosa and mesenteric lymph nodes,with the addition of ABP,the transcription levels of IL-1βmRNA increased,and the content of IL-1βmRNA expression of liver and mesenteric lymph nodes in groupⅣwas higher than that of control group and groupⅠ(P＜0.05),but there were no difference in jejunal mucosa(P＞0.05).The results suggested that ABP can increase the expression level of IL-1βmRNA better than that of IGF-ⅠmRNA,So it is plausible that ABP were effective in improving immune function than in growth promotion for piglets.ExperimentⅥwas conducted to investigate the effects of ABP on the proliferation of lymphocytes and concentrations of cytokine in peripheral blood mononuclear cells (PBMC) of piglets.On the 14th and 28th day of animal feeding trial,the blood samples were collected to study the effect of ABP on proliferation of lymphocytes and concentrations of cytokine in PBMC of piglets.At the same time,different lymphocytes (blood lymphocytes,splenocytes,T cells,B cells of pigs and splenocytes of naked mice) were cultured in vitro with media containing series concentrations of ABP(0,25,50,100, 200,400,800μg/mL),and the proliferation of lymphocytes was determined.Furthermore, blood lymphocytes were cultured with media containing series concentrations of ABP(0, 100,200,400μg/mL) and the concentrations of IL-2,IL-4 and IFN-γwere measured.The results indicated that ABP could significantly improve the spleen index,thymus index and the conversion rate of peripheral blood lymphocytes in piglets,and also increase significantly the concentration of cytokine TNF,IL-1β,IL-2,IL-6 in PBMC of piglets. Therefore ABP can improve immune function of piglets effectively.The results also showed that ABP could improve the proliferation of pigs' blood lymphocytes,splenocytes and T cells(P＜0.05),but had no significant effect on the proliferation of pigs' B cells and naked mice's splenocytes(P＞0.05),which indicated that T cells may be one of target cells for ABP to act on directly.ABP can induce secretion of IFN-γ,and IL-2,and the effect is time and dose dependent,and ABP can anti-induce the expression of IL-4.The results suggested that ABP was one of active polysaccharide with fructose residues and glucose residues.ABP can promote growth and immunomodulation for pigs and it could be an alternative additive to replace chlortetracycline completely on increaseing disease resistance for pigs.ABP can improve meat quality and increase commercial valve for pork meat.T cells may be one of target cells for ABP to act on directly and ABP could promote Thl type immune response through enhancing the secretion of Th1 type cytokine and inhibiting secretion of Th2 cytokine.