Regulatory Effect Of Ala-gln On Humoral Immunity And The Intestinal Mucosa Cellular Immunity Of Weaned Piglets

The study was conducted to investigate the regulatory effect of Ala-Gln on humoralimmunity and the intestinal mucosa cellular immunity of early weaned piglets (21days),and to explore the regulatory mechanism of Ala-Gln in porcine epithelial cell proliferationand apoptosis using the intestinal porcine epithelial cells-1as the cell model.Experiment I: Effects of Ala-Gln on cytokines of serum and immune organs andT lymphocyte subpopulations in peripheral blood of weaned pigletsThe experiment was carried out as a single factor experimental design. A total of100castrated piglets (Landrace♂×Large White♀) with similar genetic background and BW(6.7±0.9kg), weaned at21±2d of age, were randomly selected and assigned to fourgroups with five replications, and five piglets in each replication. The control group wassubjected to receive the basal diet (free alanyl-glutamine), and the other group was fed thebasal diet supplementation with0.15%Ala-Gln,0.30%Ala-Gln or0.45%Ala-Gln. Theexperiment lasted21days. One piglet in each replication was selected randomly and bledvia anterior vena cava to collect blood sample, then anesthetized slaughtered to harvestimmune organs on d7, d14and d21of experiment. Samples were collected to elevateconcentration of interleukin in serum and immune organ, analysis of peripheral blood Tlymphocyte subsets to investigate the effect of Ala-Gln on weaned piglets’ humoralimmunity.The results showed that:(1) Dietary supplementation with0.15%and0.30%Ala-Glnsignificantly increased serum ALB, TP, IgA, IgG content and also significantly reducedCOR content of piglets on the7th of post-weaning (P<0.05). What’s more,0.30%Ala-Glnmarkedly elevated the concentration of TP, IgA, IgG and decreased COR level on d14ofpost-weaning (P<0.05), compared with the control.(2)0.15%~0.45%Ala-Gln couldsignificantly reduce the levels of serum cytokine IL-6on the experiment period (P<0.05).On d7and d14of post-weaning, The level of IL-2and TNF-α in0.30%Ala-Gln groupwere significantly higher than the control (P<0.05), while IL-5content in0.15%Ala-Glngroup was lower than the control (P<0.05). On d21after weaning, with the increasingaddition of Ala-Gln in diet, IL-5content showed a quadratically increasing trend, which in0.45%Ala-Gln group was to the top (P<0.05). In addition, dietary supplementation with0.30%Ala-Gln dramatically improved the IL-10level (P<0.05), while0.15%~0.45%Ala-Gln significantly decreased the TNF-α level in spleen and thymus comparing with thecontrol (P<0.05).(3) Ala-Gln could increase the expression of T lymphocyte subsets CD4+ and decreased CD8+expression in peripheral blood to some extent. The ratio ofCD4+/CD8+in Ala-Gln addition groups were higher than the control on d7ofpoet-weaning, and effects of which in0.45%Ala-Gln group were the most obvious on d14and d21of post-weaning (P<0.05). In conclusion, dietary supplementation with Ala-Glncould elevate serum immune globulin concentration, anti-inflammatory cytokine levels andreduce inflammatory cytokine levels to enhance the immunity of weaned piglets and easethe weaning stress.Experiment II: Effects of Ala-Gln on the number of SIgA plasma cells inintestinal lamina propria，the concentration of SIgA and cytokines in mucosal ofweaned pigletsThe experimental design was the same as Experiment I. One piglet in each replicationwas selected randomly and anesthetized slaughtered on d7, d14and d21of experiment.Intestinal segments and intestinal mucosa were collected and measured the SIgA plasmacells quantity, the concentration of SIgA and cytokines to explore the effect of Ala-Gln onthe intestinal mucosa cellular immunity of weaned piglets.The results indicated that:(1) Dietary supplementation with0.15%~0.45%Ala-Glnsignificantly elevated the number of SIgA plasma cells in duodenum and jejunum laminapropria on d7, d14and d21of post-weaning(P<0.05). Especially,0.30%Ala-Glnincreased the SIgA plasma cells quantity in jejunum by22.6%(P<0.05) on d14ofpost-weaning, compared with the control. In ileum, it was0.30%Ala-Gln that significantlyincreased SIgA cells quantity on d7and d14(P<0.05).(2) With the increasing addition ofAla-Gln in diet, the concentration of SIgA in jejunal mucosal quadratically increased, andthe SIgA content in0.30%Ala-Gln group was the highest(P<0.05). Moreover, the SIgAcontent in0.30%Ala-Gln group increased by70.4%(P<0.01) on d7of post-weaning.Both0.15%and0.30%Ala-Gln markedly improved SIgA content on d14and d21(P<0.05).(3) Dietary supplementation with0.15%or0.30%Ala-Gln significantlyincreased the level of IL-2and IL-5on d7of post-weaning (P<0.05). With the increasingof Ala-Gln, the level of IL-6showed a linear increasing trend, and was markedly higherthan the control (P<0.05). On d7and d21of experiment, the concentration of TNF-αdecreased linearly with the addition of Ala-Gln, which was the lowest in0.30%Ala-Glnand0.45%Ala-Gln group (P<0.05). In conclusion, dietary supplementation with Ala-Glncould elevate SIgA secreting cells’ quantity, SIgA, the level of IL-2and IL-6, decreaseTNF-α concentration in intestinal mucosa to enhance the immune response and improvethe intestinal mucosa cellular immunity of weaned piglets. Experiment III: Effect of Ala-Gln on intestinal porcine epithelial cells apoptosisThe intestinal epithelial cells (IPEC-1) were used as the model. IPEC-1was grown in (freeGln) DMEM-F12supplemented with5μg/L mEGF,1‰ITS,1%penicillin streptomycin invitro to simulate the weaning stress. Then for the dose-course study of Ala-Gln, IPEC-1was co-cultured with different concentration of Ala-Gln (0,0.25,0.5,1.0,2.0,4.0mM) for24h. The cell apoptosis ratio was detected by flow cytometry and the expression ofCaspase-3activation production and BCL-2were detected via Western Blot to explore theeffects of Ala-Gln on the intestinal porcine epithelial cells apoptosis and the regulatorymechanism of that.The results showed that,(1) IPEC-1cell apoptosis ratio in the Ala-Gln treatmentgroups were significantly lower than the control group (P<0.05). With the increasingconcentration of Ala-Gln, IPEC-1cell apoptosis ratio showed decreasing firstly and thenincreasing. When the concentration of Ala-Gln was1.0mM and2.0mM, the ratio ofIPEC-1cell apoptosis were lowest, which were significantly lower than the control group(P<0.05), but the difference between the two treatment groups was not marked (P>0.05).(2) It did not detect the expression of Caspase-3activation production and Bcl-2.