| To investgate the developmental rules of antibacterial peptides PR-39 andProtegrin-1(PG-1) gene expression, the effect of Glutamine(Gln) on its expressiveregulation and intestinal tract protection role of piglets, an optimizedsemi-quantitative RT-PCR was constructed to measure the effect of age, weaning andGln on expression of antibacterial peptides PR-39 and PG-1 mRNA of piglets, theprotective mechanism of Gln on intestinal tract of piglets was also discussed. Theseresults can support important guidance for the use and development of alternativeproducts for antibiotics or environment protection additives in the future. Results areas bellow:1.To build an optimized semi-quantitative RT-PCR, the special primers forPR-39, PG-1, and 18S rRNA were designed respectively according to the knownporcine PR-39, PG-1 and 18S rRNA gene sequences, and the effect of amplificationcycles on integrated optical density (IOD) was also investigated by selecting genePCR products from femur marrows, thymuses, spleens and mesenteric lymph node ofthe 21-day old non-weaning piglets as the subjects, respectively. The results showedthat an optimized semi-quantitative RT-PCR consisted of Mg2+ 1.5 mmol/L, annealingtemperature and amplification cycles ofPR-39, PG-1, 18S rRNA was 58℃, 58℃, 52℃and 31, 31, 25.2. Four healthy male piglets (Duroc×(Landrace×Yorkshine)) in each group withthe same parity, similar body weight and at the age of 1, 3, 14, 23, 28 day (keepmilking) and at the age of 23, 28 day (weaned on 21 day) were slaughtered, and thenthe femur marrows, thymuses, spleens, mesentery lymph node were taken foranalyzing the expression difference of PR-39 and PG-1 mRNA. The constructedsemi-quantitative RT-PCR was applied to analyze the relative expression of PR-39and PG-1 mRNA by using 18S rRNA as an inner marker. The results indicated that,the age of the piglets significantly affected the relative expression of PR-39 mRNA infemur marrow and thymus of piglets, with the lowest level at birth, a significantly increase after 2 d of sucking colostrum compared with the age of 1 d(p＜0.05), adecrease at 14th day compared with the age of 3rd day and a slowly increasethereafter, however the effect of age on the relative expression of PR-39 mRNA inspleen and mesentery lymph node were not significant(p＞0.05); the relativeexpression of PG-1 mRNA increased significantly in femur marrow and spleen withage increasing(p＜0.05), but it was not significant in thymus and mesentery lymphnode(p＞0.05); significant differences of the relative expression of PR-39 and PG-1mRNA existed among tissues of piglets at the same age,with the down order of femurmarrow, thymus, spleen and mesentery lymph node; weaning significantly reducedthe relative expression of PR-39 mRNA in femur marrow, thymus and PG-1 mRNA infemur marrow. The above results suggested that there were obvious tissue-specificand age-dependance of both PR-39 and PG-1 mRNA in the piglets, weaningdecreased the expression of PR-39 and PG-1 mRNA.3. Granulocytes of bone marrow were cultured in the basic media by addingdifferent concentrations of Gin (0, 300, 450, 600μg/mL) for 10 hours, the relativeexpressions of PR-39, PG-1 mRNA of each granulocyte-treated group and thebacteriostatic efficacy of supernatant fluids, alkaline phosphatase (ALP) activity,concentrations of IL-6 and TNF-αwere analyzed. The results showed that, the relativeexpressions of PR-39 and PG-1 in the granulocytes of femur marrow (p＜0.05)increased significantly by adding Gin to the media; compared with non-Gln media,the relative expression of PR-39 mRNA increased 61.1%(p＞0.05), 133.3 %(p＜0.05),51.9 %(p＞0.05) respectively when 300, 450, 600μg/mL Gln was added to media,PG-1 mRNA increased 13.9 %(p＞0.05), 32.4 %(p＜0.05), 22.2 %(p＜0.05)respectively; Correspondingly, bacteriostatic activity unit of the above supernatantfluid increased 154.0 %(p＜0.05), 55.1%(p＞0.05)and 14.4%(p＞0.05) respectively.ALP activity unit increased 51.9 %(p＜0.05), 42.6 %(p＜0.05), 14.0 %(p＞0.05)respectively; the concentrations of IL-6 increased 66.9 %(p＜0.05), 42.5 %(p＞0.05),33.2 %(p＞0. 05) respectively; the concentrations of TNF-αincreased 81.3 %(p＜0.05),50.0 %(p＜0.05) respectively for 300 and 600μg/mL Gln group. Above resultsindicated that, gene expression of PR-39 and PG-1 of in vitro granulocytes in femur marrow increased remarkably by Gin.4. Sixty-four healthy 28-day old weaning piglets (Duroc×(Landrace×Yorkshine))with the same parity, similar body weight (about 7.5 kg)were randomly allotted to twogroups(namely the control and the 1% Gln-added group) with four replicates andwith eight weaning piglets(half male, half female) in each replicate and were fed forone mouth. One boar with the same weight from each replicate was chosen to collectblood on day 28, then lymphocytes and sera were separated for the analysis oflymphocyte proliferation rate and the level of IL-1β, IL-6 and TNF-α. One boar withthe same weight of each replicate was chosen to be slaughtered on day 30, then thecontents of recta, cecum and colon were obtained for the analysis of intestinalmicroflora; duodenum, jejunum and ileum were obtained for making optical scanningslice to view changes of intestinal villis and crypts; mucus and mucosal of jejunumwere obtained for analysis of S-IgA and the level of TNF-α, IL-2; femur marrow ofthe left thighbone and the mucosal of jejunum were obtained for analysis of therelative expression differences of PR-39 and PG-1 mRNA between the control and theGin-added group. The results indicated that, compared with the control, peripheralblood lymphocyte proliferation rate in respone to PHA increased 8.6 %(p＜0.05) forthe Gin-added group, the level of IL-1β, IL-6 and TNF-αin sera increased 28.6%(p＜0.05), 18.8%(p＞0.05) and 10.0%(p＞0.05) respectively; beneficial bateria inintestinal tract increased significantly and pathogenic bacteria reduced significantlyby Gin addition(p＜0.05), compared with the control, lactobacilluses in the contents ofrecta, cecum and colon increased 2.8 %(p＜0.05), 4.1%(p＜0.05) and 4.0%(p＜0.05)respectively in the Gln-adding group, bifidobacteria increased 9.4 %(p＜0.01), 9.7%(p＜0.01) and 9.6 %(p＜0.01) respectively, Escherichia coli reduced 3.0% (p＜0.05),2.9 %(p＜0.05) and 3.3 %(p＜0.05) respectively, the villus height of duodenum,jejunum and ileum elevated significantly(p＜0.01), while the crypt depthreduced(p＞0.05) with Gln supplementation; in contrast to control,the concentrationtsof S-IgA in mucus of jejunum increased 30.1%(p＜0.05), the concentrationts ofTNF-α, IL-2 in mucosal of jejunum increased 37.3 %(p＞0.05), 18.0 %(p＞0.05)respectively with Gln addition, the relative expressions of PR-39, PG-1 mRNA in femur marrow and PR-39 mRNA in the mucosal of jejunum increased 50.5％(p＜0.05), 27.6％(p＜0.05)and 24.0％(p＜0.05) respectively by Gin addition. Aboveresults suggested that, addition Gln to diets for weaning piglets could increase relativeexpression of PR-39 mRNA in both femur marrow and the mucosal of jejunum, andPG-1 mRNA in femur marrow. What's more, Gln addition could reduceimmunosuppression caused by weaning and maintain integrity of morphologicalfeature of the intestinal mucosal of the weaning piglets by increasing PR-39 and PG-1gene expression and improve intestinal microflora,... |
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