Branched-chain Amino Acids Modulate Intestinal Defensin Expression And Immune Barrier Function Of Piglets

Four experiments were conducted to investigate the effect of branched-chain amino acids on growth performance and intestinal immune barrier function in weaned piglets. The mechanism of β defensin stimulation by branched-chain amino acids was studied in vivo and in vitro.(1) Experiment1contained108weaned pigs which were allotted to3groups (control group (21%CP), low nitrogen group (17%CP) and BCAA group (17%CP+BCAA)). The results showed that the low nitrogen group had a significantly lower growth performance than the control group (P<0.01). Compared with the low nitrogen group, BCAA supplementation significantly improved the growth performance of weaned pigs. Meanwhile, the pigs offered BCAA diet had a higher plasma BCAA concentration and a lower urea nitrogen. Decreasing dietary nitrogen level declined the intestinal villous height (P<0.05) and immunoglobulins (P<0.05) and increased the number of intre-epithelial lymphocytes (P<0.05). BCAA had significantly improved the intestinal morphology and immune statues compare with the low nitrogen group. A interesting result was observed that β defensin expression in jejunum and ileum of weaned pigs was significant higher than the control and low nitrogen groups.(2) In Experiment2, the porcine intestinal epithelial cell line IPEC-J2was used as a in vitro model. At first, we found the expression of β defensins in IPEC-J2related with the time of exposure to isoleucine. After exposure to1.0mM of isoleucine, the expression of β defensins was highest in24h than other time. In three branched-chain amino acid, isoleucine is the strongest promoter of P-defensins expression in IPEC-J2and the highest β-defensins expression was seen in0.8mM L-isoleucine (P<0.05). The pEP2C expression was more sensitive to valine and leucine than pBD-2and pBD-3. The strongest effect of leucine and valine for stimulating β-defensin expression was0.1mM and0.2mM, respectively (P<0.05).(3) Experiment3studied the possible signaling pathway of defensin regulator, branched chain amino acids, using IPEC-J2cells. From western result, isoleucine, leucine and valine phosphorylated p44/42beginning from1to6hour after the start of treatment. The inhibitors splitomicin (a Sirtl inhibitor) and PD98059(a ERK inhibitor) completely blocked the activation of ERK/90RSK and decreased the mostly increasing of defensins by isoleucine (P<0.05). Although lipopolysaccharide (LPS) can induce some kinds of defensin in IPEC-J2, its pathway has no relate with ERK signaling as LPS reduced phosphorylated p44/42ERK and had no effect on the phosphorylation of90RSK. BCAA have the ability to stimulate the expression of porcine epithelial β-defensins through Sirt1/ERK/90RSK signaling. Furthermore, the β-defensins regulation of LPS is dependent on nuclear factor-κB (NF-κB) and has no relationship with ERK pathway.(4) In Experiment4, IPEC-J2and weaned piglets was taken to investigate the effect of isoleucine on intestinal epithelial cell against bacterial infection. The cell experiment illustrated IPEC-J2exposed isoleucine for24h significantly decreased the multiplication of Salmonella and Escherichia coli (E. coli). Animal experiment was conducted a2×2factorial design. Weaned pigs were randomly assigned to one of the four treatments (n=6), including (Ⅰ) Alanine+PBS challenge;(Ⅱ) Alanine+E. coli challenge;(Ⅲ) Isoleucine+PBS challenge;(Ⅳ) Isoleucine+E. coli challenge. We observed that compared with alanine diet, isoleucine significantly improved growth performance of weaned pigs neither challenge or not. Plasma leukocytes was not affected by diet and was increased by E. coli challenge. The plasma IgA concentration of isoleucine group was significant higher than that of alanine group (P<0.05). The dose of lipopolysaccharide in pig plasma was significantly affected by diet and challenge. Neither challenge or not, weaned piglet of isoleucine group had lower dose of lipopolysaccharide than alanine group (P<0.05). In two alanine diet groups, E. coli challenge increased plasma lipopolysaccharide level. After E. coli challenge, the concentration of IL-6and IL-1β were significant increased and the level of TNF-a was declined. Isoleucine supplementation in diet significantly increased expression of β defensins pBD-1, pBD-2, pBD-3, pBD-114and pBD-129in jejunum and ileum of weaned piglets. In conclusion, the present study reveals that BCAA have the ability to stimulate the expression of porcine epithelial β-defensins in vivo and in vitro and the possible pathway was the activation of Sirtl/ERK/90RSK signaling. The porcine intestinal immune barrier function against bacterial infection was improved by isoleucine also was proved.