牛膝多糖对猪的营养效应和免疫调控机理研究
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摘要
本论文通过牛膝多糖的提取和分离纯化、动物在体试验及体外细胞培养试验,探讨了牛膝多糖(Achyranthes bidentata polysaccharides,ABP)对猪的营养效应和免疫调控及其机理。
试验一研究了ABP的提取工艺并分析了ABP组分。选用中药牛膝(achyranthesbidentata)的块根经热水提取、乙醇分级沉淀,鞣酸去除蛋白质,再经DEAE-Sepharosefast-flow柱和Sephadex G-150凝胶过滤,洗脱液用蒸馏水透析后,冻干。结果显示,牛膝中ABP的含量为28.86%,ABP的分子量为1300-1400 d,纯度为98.62%,经HPLC分析ABP为均一多糖,结构分析表明ABP是由果糖和葡萄糖组成,两者的摩尔比为8:1。
试验二研究了ABP对猪生长性能和血清学指标的影响。试验选用160头28d胎次相同、体重相近(8.898±0.742 kg BW)、健康的杜洛克×长白×大白(Duroc×Landrace×Yorkshire)断奶仔猪,随机分为5个处理,每个处理设4个重复(栏),每个重复8头猪,分别饲喂基础日粮、添加金霉素100 mg/kg和ABP 500 mg/kg,1000 mg/kg,1500mg/kg日粮,预试期7天,正式期为126d。分别测定猪的生长性能、腹泻率、喘气病发生率以及血清尿素氮(BUN)、血糖(GLU)、碱性磷酸酶(ALP)和免疫球蛋白(Ig)、激素等指标。结果显示:ABP显著提高28-60d阶段猪的平均日增重和饲料转化率(P＜0.05或P＜0.01),效果优于金霉素;ABP有改善61-154d阶段猪的生长性能的趋势,但差异不明显(P＞0.05);整个试验期(28-154d),与对照组相比,试验Ⅰ、Ⅱ、Ⅲ、Ⅳ组分别提高平均日增重2.11%(P＜0.05),3.90%(P＜0.01),5.53%(P＜0.01)和2.44%(P＜0.05),降低料肉比4.69%(P＜0.05),3.25%(P＞0.05),7.22%(P＜0.05)和5.78%(P＜0.05);ABP显著降低了各阶段猪的腹泻率和喘气病的发生率(P＜0.05或P＜0.01),其效果达到或超过金霉素的效果;ABP显著降低了血清中BUN、GLU的浓度(P＜0.05 or P＜0.01),提高了ALP的活性(P＜0.05 or P＜0.01),提高了血清IgM、IgA、IgG、补体C3、C4的浓度(P＞0.05,P＜0.05或P＜0.01),而金霉素对免疫球蛋白和补体的浓度没有显著影响(P＞0.05)。
试验三研究了ABP对猪肠道微生物区系和黏膜组织形态的影响。为探讨ABP促进仔猪生长和防治仔猪腹泻的机理,于动物饲养试验第35d时,每个重复组中随机选择1头进行屠宰,即每处理组宰杀4头,研究ABP对肠道pH、微生物菌群情况和黏膜组织形态等的影响。结果显示:ABP能使直肠、盲肠和结肠的pH有降低的趋势,其中试验Ⅲ、Ⅳ组显著降低了盲肠段和结肠段的pH值(P＜0.05或P＜0.01),但对直肠pH没有显著性的差异(P＞0.05),而抗生素组与对照组没有差异(P＞0.05);ABP具有显著抑制肠道大肠杆菌和提高肠道双歧杆菌、乳酸杆菌的作用(P＜0.05或P＜0.01),效果达到或显著好于金霉素(P＞0.05,P＜0.05或P＜0.01),随着ABP添加量的增加,其抑制肠道大肠杆菌和促进肠道有益菌的效果逐渐提高,试验Ⅱ和Ⅳ组之间差异显著或极显著(P＜0.05或P＜0.01);试验Ⅰ、Ⅱ、Ⅲ、Ⅳ组之间的十二指肠段上皮细胞层厚度无显著性差异(P＞0.05),随着ABP添加量的增加,回肠段和空肠段的上皮细胞层厚度增加,显著或极显著大于对照组和抗生素组(P＜0.05或P＜0.01);ABP显著增加十二指肠段肠绒毛高度和降低隐窝深度(P＜0.05或P＜0.01),不同浓度ABP对回肠和空肠的隐窝深度的影响没有显著性差异,试验Ⅲ、Ⅳ组各测定指标之间差异不显著(P＞0.05);ABP显著增加十二指肠、回肠、空肠肠段的绒毛高度与隐窝深度比值(VH/CD值)(P＜0.05或P＜0.01),进而改善营养物质的消化吸收,提高猪的生长性能。
试验四研究了ABP对猪屠宰性能和肉品质的影响。动物试验结束时,每重复栏中随机选择1头猪,即每处理组4头猪,进行屠宰性能测定以及肉品质分析。结果显示:ABP能显著提高猪的眼肌面积和瘦肉率(P＜0.05),但对屠宰率和背膘厚没有显著影响(P＞0.05);ABP能显著提高肌肉肉色评分、大理石纹评分、熟肉率和肌内脂肪含量(P＜0.05),对肌肉pH、滴水损失和失水率效果不显著(P＞0.05);ABP在肉色评分和大理石评分项中显著优于金霉素(P＜0.05);随着ABP添加量的增加,肌肉中氨基酸的含量有提高的趋势,但各组各氨基酸含量之间没有显著性差异(P＞0.05);金霉素和ABP有提高肌肉上清液中超氧化物歧化酶(SOD)活性的趋势,但各组之间的SOD活性差异不显著(P＞0.05),ABP能提高谷胱甘肽过氧化物酶(GSH-Px)活性,试验Ⅳ组的GSH-Px活性测定值显著高于对照组和试验Ⅰ组(P＜0.05),同时,ABP有降低丙二醛(MDA)的趋势,试验Ⅱ、Ⅲ、Ⅳ组分别降低了6.60%(P＞0.05),10.76%(P＞0.05)和18.53%(P＜0.05),说明ABP具有增强猪肌肉抗氧化机能的作用。
试验五研究了ABP对猪IGF-Ⅰ和IL-1β基因表达的影响。在饲养试验第35d时的屠宰猪体内,取出肝脏、空肠黏膜及肠系膜淋巴结,采用实时荧光定量PCR研究ABP对猪的IGF-1和IL-1β基因表达水平的影响。结果显示:ABP对肝脏、空肠黏膜、肠系淋巴结IGF-1mRNA的转录水平有一定的提高,随着ABP添加量的增加,IGF-1mRNA的转录水平逐渐增强,但各组之间未达到显著性差异水平(P＞0.05);肝脏和肠系膜淋巴结IL-1βmRNA的转录水平随着ABP添加量的增加而逐渐增加,其中试验Ⅳ组IL-1βmRNA表达量显著高于对照组和试验Ⅰ组(P＜0.05);ABP对空肠黏膜IL-1βmRNA表达量没有显著影响(P＞0.05)。试验结果显示,ABP促进IL-1β基因表达水平比IGF-1基因表达水平高,提示ABP增强免疫功能强于促生长性能。
试验六研究了ABP对仔猪淋巴细胞增殖作用和细胞因子分泌量的影响。在动物试验第14d和28d时,采集仔猪血液研究ABP对仔猪外周血淋巴细胞增殖作用和细胞因子分泌量的影响,同时,采取体外培养方式,在培养体系中加入不同浓度的ABP(0,25,50,100,200,400,800μg/mL),体外培养5种免疫细胞(猪脾、外周血淋巴细胞单核细胞,分离的猪外周血T、B细胞以及无胸腺裸鼠脾细胞),观测ABP对体外培养不同淋巴细胞增殖作用的影响,并探讨ABP对体外培养仔猪外周血淋巴细胞释放细胞因子IL-2、IL-4及IFN-γ的影响。结果显示:ABP能显著提高猪的脾脏和胸腺指数以及仔猪外周血淋巴细胞转化率和仔猪血清中TNF、IL-1β、IL-2和IL-6等细胞因子的含量,进而提高动物的免疫性能;在体外培养试验中发现ABP能剂量依赖性地促进猪外周血淋巴细胞、脾淋巴细胞和外周血T细胞的增殖,而对猪外周血B细胞以及无胸腺鼠脾细胞(B细胞)的体外增殖无显著影响(P＞0.05),提示T细胞是ABP直接作用的靶细胞之一。体外培养仔猪外周血淋巴细胞释放细胞因子含量的测定结果显示:ABP可促进细胞因子IL-2、IFN-γ的分泌,该作用呈时间、剂量依赖性变化,而ABP抑制IL-4的分泌。
以上试验结果表明:牛膝多糖为果糖-葡萄糖聚合的活性多糖,对猪具有促生长效应和免疫调控机能,能提高猪的抗病能力,降低猪腹泻率和猪哮喘病发生率,完全可以替代金霉素;牛膝多糖能改善猪肉品质,提高猪肉商业价值;牛膝多糖提高猪的免疫机能效果强于促生长性能效果;T细胞是ABP直接作用的靶细胞之一,牛膝多糖是通过增强Th1类细胞因子的表达,抑制Th2类细胞因子的表达,促进Th1类免疫应答而起着免疫调控作用。
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.
试验一研究了ABP的提取工艺并分析了ABP组分。选用中药牛膝(achyranthesbidentata)的块根经热水提取、乙醇分级沉淀,鞣酸去除蛋白质,再经DEAE-Sepharosefast-flow柱和Sephadex G-150凝胶过滤,洗脱液用蒸馏水透析后,冻干。结果显示,牛膝中ABP的含量为28.86%,ABP的分子量为1300-1400 d,纯度为98.62%,经HPLC分析ABP为均一多糖,结构分析表明ABP是由果糖和葡萄糖组成,两者的摩尔比为8:1。
试验二研究了ABP对猪生长性能和血清学指标的影响。试验选用160头28d胎次相同、体重相近(8.898±0.742 kg BW)、健康的杜洛克×长白×大白(Duroc×Landrace×Yorkshire)断奶仔猪,随机分为5个处理,每个处理设4个重复(栏),每个重复8头猪,分别饲喂基础日粮、添加金霉素100 mg/kg和ABP 500 mg/kg,1000 mg/kg,1500mg/kg日粮,预试期7天,正式期为126d。分别测定猪的生长性能、腹泻率、喘气病发生率以及血清尿素氮(BUN)、血糖(GLU)、碱性磷酸酶(ALP)和免疫球蛋白(Ig)、激素等指标。结果显示:ABP显著提高28-60d阶段猪的平均日增重和饲料转化率(P＜0.05或P＜0.01),效果优于金霉素;ABP有改善61-154d阶段猪的生长性能的趋势,但差异不明显(P＞0.05);整个试验期(28-154d),与对照组相比,试验Ⅰ、Ⅱ、Ⅲ、Ⅳ组分别提高平均日增重2.11%(P＜0.05),3.90%(P＜0.01),5.53%(P＜0.01)和2.44%(P＜0.05),降低料肉比4.69%(P＜0.05),3.25%(P＞0.05),7.22%(P＜0.05)和5.78%(P＜0.05);ABP显著降低了各阶段猪的腹泻率和喘气病的发生率(P＜0.05或P＜0.01),其效果达到或超过金霉素的效果;ABP显著降低了血清中BUN、GLU的浓度(P＜0.05 or P＜0.01),提高了ALP的活性(P＜0.05 or P＜0.01),提高了血清IgM、IgA、IgG、补体C3、C4的浓度(P＞0.05,P＜0.05或P＜0.01),而金霉素对免疫球蛋白和补体的浓度没有显著影响(P＞0.05)。
试验三研究了ABP对猪肠道微生物区系和黏膜组织形态的影响。为探讨ABP促进仔猪生长和防治仔猪腹泻的机理,于动物饲养试验第35d时,每个重复组中随机选择1头进行屠宰,即每处理组宰杀4头,研究ABP对肠道pH、微生物菌群情况和黏膜组织形态等的影响。结果显示:ABP能使直肠、盲肠和结肠的pH有降低的趋势,其中试验Ⅲ、Ⅳ组显著降低了盲肠段和结肠段的pH值(P＜0.05或P＜0.01),但对直肠pH没有显著性的差异(P＞0.05),而抗生素组与对照组没有差异(P＞0.05);ABP具有显著抑制肠道大肠杆菌和提高肠道双歧杆菌、乳酸杆菌的作用(P＜0.05或P＜0.01),效果达到或显著好于金霉素(P＞0.05,P＜0.05或P＜0.01),随着ABP添加量的增加,其抑制肠道大肠杆菌和促进肠道有益菌的效果逐渐提高,试验Ⅱ和Ⅳ组之间差异显著或极显著(P＜0.05或P＜0.01);试验Ⅰ、Ⅱ、Ⅲ、Ⅳ组之间的十二指肠段上皮细胞层厚度无显著性差异(P＞0.05),随着ABP添加量的增加,回肠段和空肠段的上皮细胞层厚度增加,显著或极显著大于对照组和抗生素组(P＜0.05或P＜0.01);ABP显著增加十二指肠段肠绒毛高度和降低隐窝深度(P＜0.05或P＜0.01),不同浓度ABP对回肠和空肠的隐窝深度的影响没有显著性差异,试验Ⅲ、Ⅳ组各测定指标之间差异不显著(P＞0.05);ABP显著增加十二指肠、回肠、空肠肠段的绒毛高度与隐窝深度比值(VH/CD值)(P＜0.05或P＜0.01),进而改善营养物质的消化吸收,提高猪的生长性能。
试验四研究了ABP对猪屠宰性能和肉品质的影响。动物试验结束时,每重复栏中随机选择1头猪,即每处理组4头猪,进行屠宰性能测定以及肉品质分析。结果显示:ABP能显著提高猪的眼肌面积和瘦肉率(P＜0.05),但对屠宰率和背膘厚没有显著影响(P＞0.05);ABP能显著提高肌肉肉色评分、大理石纹评分、熟肉率和肌内脂肪含量(P＜0.05),对肌肉pH、滴水损失和失水率效果不显著(P＞0.05);ABP在肉色评分和大理石评分项中显著优于金霉素(P＜0.05);随着ABP添加量的增加,肌肉中氨基酸的含量有提高的趋势,但各组各氨基酸含量之间没有显著性差异(P＞0.05);金霉素和ABP有提高肌肉上清液中超氧化物歧化酶(SOD)活性的趋势,但各组之间的SOD活性差异不显著(P＞0.05),ABP能提高谷胱甘肽过氧化物酶(GSH-Px)活性,试验Ⅳ组的GSH-Px活性测定值显著高于对照组和试验Ⅰ组(P＜0.05),同时,ABP有降低丙二醛(MDA)的趋势,试验Ⅱ、Ⅲ、Ⅳ组分别降低了6.60%(P＞0.05),10.76%(P＞0.05)和18.53%(P＜0.05),说明ABP具有增强猪肌肉抗氧化机能的作用。
试验五研究了ABP对猪IGF-Ⅰ和IL-1β基因表达的影响。在饲养试验第35d时的屠宰猪体内,取出肝脏、空肠黏膜及肠系膜淋巴结,采用实时荧光定量PCR研究ABP对猪的IGF-1和IL-1β基因表达水平的影响。结果显示:ABP对肝脏、空肠黏膜、肠系淋巴结IGF-1mRNA的转录水平有一定的提高,随着ABP添加量的增加,IGF-1mRNA的转录水平逐渐增强,但各组之间未达到显著性差异水平(P＞0.05);肝脏和肠系膜淋巴结IL-1βmRNA的转录水平随着ABP添加量的增加而逐渐增加,其中试验Ⅳ组IL-1βmRNA表达量显著高于对照组和试验Ⅰ组(P＜0.05);ABP对空肠黏膜IL-1βmRNA表达量没有显著影响(P＞0.05)。试验结果显示,ABP促进IL-1β基因表达水平比IGF-1基因表达水平高,提示ABP增强免疫功能强于促生长性能。
试验六研究了ABP对仔猪淋巴细胞增殖作用和细胞因子分泌量的影响。在动物试验第14d和28d时,采集仔猪血液研究ABP对仔猪外周血淋巴细胞增殖作用和细胞因子分泌量的影响,同时,采取体外培养方式,在培养体系中加入不同浓度的ABP(0,25,50,100,200,400,800μg/mL),体外培养5种免疫细胞(猪脾、外周血淋巴细胞单核细胞,分离的猪外周血T、B细胞以及无胸腺裸鼠脾细胞),观测ABP对体外培养不同淋巴细胞增殖作用的影响,并探讨ABP对体外培养仔猪外周血淋巴细胞释放细胞因子IL-2、IL-4及IFN-γ的影响。结果显示:ABP能显著提高猪的脾脏和胸腺指数以及仔猪外周血淋巴细胞转化率和仔猪血清中TNF、IL-1β、IL-2和IL-6等细胞因子的含量,进而提高动物的免疫性能;在体外培养试验中发现ABP能剂量依赖性地促进猪外周血淋巴细胞、脾淋巴细胞和外周血T细胞的增殖,而对猪外周血B细胞以及无胸腺鼠脾细胞(B细胞)的体外增殖无显著影响(P＞0.05),提示T细胞是ABP直接作用的靶细胞之一。体外培养仔猪外周血淋巴细胞释放细胞因子含量的测定结果显示:ABP可促进细胞因子IL-2、IFN-γ的分泌,该作用呈时间、剂量依赖性变化,而ABP抑制IL-4的分泌。
以上试验结果表明:牛膝多糖为果糖-葡萄糖聚合的活性多糖,对猪具有促生长效应和免疫调控机能,能提高猪的抗病能力,降低猪腹泻率和猪哮喘病发生率,完全可以替代金霉素;牛膝多糖能改善猪肉品质,提高猪肉商业价值;牛膝多糖提高猪的免疫机能效果强于促生长性能效果;T细胞是ABP直接作用的靶细胞之一,牛膝多糖是通过增强Th1类细胞因子的表达,抑制Th2类细胞因子的表达,促进Th1类免疫应答而起着免疫调控作用。
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.
引文
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