Modulation Of Tight Junction Protein Expression In Chicken Intestine By Methionine Substitute And The Mechanism

It is well known about the beneficial effects of caloric restriction (CR) on prolonging life span and preventing aged-related diseases. These effects were due to methionine restriction (MR) that was revealed by some recent studies. A specific characteristic of MR is increase of tight junction (TJ) protein expression and improvement of epithelial barrier function. Two methionine analogues,2-hydroxy-4-(methylthio) butanoic acid (HMTBA) and2-hydroxy-4-(methylthio) butyrate calcium (HMTBA-Ca) are different from that of L-methionine in metabolism. They change into L-methionine gradually in vivo. This study was performed to prove the hypothesis that methionine analogues might play a role like MR and modulate TJ protein expression.Two hundred and fifty male broiler chickens (1day,42±3g) were randomly assigned into10groups:Control group, MR groups (MR1, MR2and MR3), HMTBA groups (H1, H2and H3) and HMTBA-Ca groups (HC1, HC2and HC3). All broilers were offered the same basal diet with added different levels of DLM, HMTBA and HMTBA-Ca. The growth performance of broilers was measured after they were killed at21st and46st day. The intestinal redox states (GSH/GSSG, CAT, T-AOC and MDA) were determined in broiler chickens. The composition of intestinal bacteria was analyzed by FISH-FC. The Fast Real-Time PCR was used to analyze the mRNA expression of Claudin-3, Occludin, TLRs, MyD88, NF-κB, AvBDs, IL-2, IL-8and IL-18in intestine.The results of21days showed that, compared with Control group, the T-AOC levels of intestinal tissues significantly enhanced, whereas the MDA levels significantly decreased in H2and H3group (P<0.05). The T-AOC levels of duodenum, jejunum and ileum significantly increased, while the MDA levels significantly decreased in HC2and HC3group (P<0.05). The GSH/GSSG of duodenum, jejunum and cecum significantly increased in H2group (P<0.05). Compared with control group, the mRNA expression levels of Claudin-3and Occludin in both duodenum and jejunum were significantly up-regulated in H3, HC3and MR3group (P<0.05). The mRNA expression levels of TLRs, MyD88, NF-κB and AvBDs in duodenum and in jejunum were significantly increased in H3and HC3group (P<0.05). H2increased the percents of Lactobacillus and Bifidobacteria and reduced Enterococcus faecalis and Escherichia coli dramatically (P<0.05). Ventral fat rate significantly decreased in H2and H3group (P<0.05). The mRNA expression levels of Claudin-3and Occludin were positively correlated with MyD88, NF-κB, TLR4and IL-2, et al., while they were negatively correlated with MDA in duodenum (P<0.01). They were also positively correlated with CAT(P<0.05). The mRNA expression levels of Claudin-3and Occludin were positively correlated with MyD88, NF-κB, TLR2, TLR4and IL-2, et al. in jejunum (P<0.01). The results of46days showed that, compared with Control group, the levels of T-AOC and GSH/GSSG in duodenum and ileum were significantly enhanced in H1and in HC1group (P<0.05), whereas the MDA levels of jejunum significantly decreased in H1, H2, HC1and HC2group (P<0.05). The MDA levels of ileum significantly decreased in H1and HC1group (P<0.05). Compared with Control group, the mRNA expression levels of Claudin-3, Occludin, TLRs, MyD88, NF-κB and AvBDs in duodenum increased significantly in H2and HC2group(P<0.05). The mRNA expression levels of Claudin-3, Occludin, TLR1, TLR2, TLR4, MyD88, NF-κB and AvBD7in jejunum increased significantly in H2and HC2group(P<0.05). The mRNA expression levels of Claudin-3and Occludin were positively correlated with GSH/GSSG, MyD88, NF-κB, TLR4and IL-2, et al., while they were negatively correlated with MDA in duodenum (P<0.01). The mRNA expression levels of Claudin-3and Occludin were positively correlated with MyD88, NF-κB, TLR4and TLR6, et al., while negativly correlated with MDA in jejunum (P<0.01). These results suggest that HMTBA increased mRNA expression of Claudin-3and Occludin through activation of TLRs-MyD88/NF-KB pathway, inhibition of oxidative stress, and modulation of intestinal microbiota.In conclusion,1) MR, HMTBA and HMTBA-Ca could increase the mRNA expression levels of TJ Claudin-3, Occludin in duodenum and jejunum in broiler chickens.2) HMTBA and HMTBA-Ca could decrease oxidative stress of intestinal tissues and increase their antioxidant capacity, which was better than MR. The antioxidant capacity of intestinal tissues was positively correlated with the mRNA expression levels of Claudin-3and Occludin.3) HMTBA and HMTBA-Ca increase the expression of Claudin-3, Occludin is partly by activating the TLR-MyD88/NF-κB-β-defensin signal system.4) Modulation of intestinal bacteria is a specific effect of HMTBA and HMTBA-Ca other than MR, which was related to change of Claudin-3and Occludin expression. This study is helpful for explanation mechanism that MR could modulate the expressions of TJ related protein and the results suggest a potential application of HMTBA and HMTBA-Ca in modulation of intestinal function.