| This experiment was conducted to investigate the effects of dietary different tryptophan levels on antimicrobial peptides expression to regulate intestinal microflora in weaned piglets,and also study the prevention mechanism of supplementation tryptophan on Enterotoxigenic Escherichia coli injured SD rat's intestine tract.The main research content is divided into the following two research sections:Part 1:In this experiment,A total of 20 Rongchang ternary((Large White×Landrace×Rongchang)28-d weaned piglets(5.42±1.52 kg)were randomly allocated to 4 treatments with 10 barrows per group.Piglets in 4 treatments fed a dietary tryptophan containing 0.14%,0.21%,0.28%and 0.35%,respectively.The experiment was included 5 d pre-trial period and 28 d test period.Five piglets were slaughted at the29-th d test period and to collect intestinal tissue samples and chyme samples.The regulation of the level of tryptophan to the intestinal microflora of weanling piglets was investigated by detecting the intestinal morphologic structure,the level of intestinal protein and the expression of antimicrobial peptide gene as well as the structure index of the colonic microorganism.The results showed that:1)Improve the intestinal development of weand piglets.Increasing dietary tryptophan level were significantly increased the number of goblet cells in jejunum(P<0.05)and significantly increased villus height,crypth deep and the villus height to crypt deep ratio in ileum(P<0.05),and have trend to increased the villus height of jejunum(P=0.055)and the lymphocytes number of ileum(P=0.064)in weand piglets.2)Improve the level of intestinal protein and the expression of antimicrobial peptide.Increasing dietary tryptophan levels were significantly increased the levels of AKT,mTOR and?-defensin 2 protein(P<0.05)and the relative expression of PBD-2,ANG-1,ANG-4 and Fabp-2 gene mRNA(P<0.05)of jejunum and ileum in weaned piglets.3)Improve intestinal microbial diversity and improve intestinal flora structure.(1)According to the analysis of Alpha diversity,the number of OUT of microorganisms observed in the colon were increased.(2)At the phylum level,the dominant bacterial community of colon contents were mainly consisted of Proteobacteria,Firmicutes and Bacteroidetes in weaned piglets,as well as Verrucomicrobia,Spirochaetae and Euryarchaeota.Increasing dietary tryptophan level,the relative abdundance of Firmicutes,Spirochaetae and Euryarchaeota microorganisms were decreased and the relative abdundance of Bacteroidetes and Verrucomicrobia microorganisms were increased,and the relative abdundance of Proteobacteria microorganisms were increased firstly and then decreased.(3)At the class level,the dominant bacterial community of colon contents were mainly consisted of Clostridium,Bacilli,Bacteroidetes and Proteobacteria microorganisms in weaned piglets.Increasing dietary tryptophan level,the relative abundance of Bacilli and Bacteroidetes were increased and the relative abundance of?-Proteobacteria,Spirochaetes,Methanobacteria and Clostridium were decreased.(4)At the family and genus level,the relative abundance of Lactobacillus,Lachnospira,Prevotella,Ruminococcaceae and Anaerovibrio and Blautia in the colon contents were gradually increased with the increase of dietary tryptophan levels,and the relative abundance of Succinivibrio,Bulleidia,Mitsuokella,Desulfovibrio,Enterobacteriaceae,Paraprevotellaceae and Methanobacteriaceae were gradually decreased.Part 2:A total of 40 female SD rats(28 d)with an average body weight of68.41±0.29 g were randomly allocated to 4 groups with 10 rats per group.Two groups were randomly selected from four groups to oral challenge ETEC(1.0×10~8 CFU/mL),and the remaining groups were unchallenge.And then randomly seleted one group from challenge groups and unchallenge groups to oral inhibitor solution(0.1 mL/d),and the remaining groups were oral normal saline(0.1 mL/d).The grouping were:control group(C),ETEC challenge and tryptophan deficiency group(E+I),ETEC challenge group(E)and tryptophan deficiency group(I).The experiment was included 3 d pre-trial period and 23 d test period.All SD rats were slaughter at the 24-th d test period and to be collected organ tisse,intestinal tissue samples and feces samples.The preventive effect of tryptophan on the intestinal tract of SD rats damaged by ETEC was investigated by detecting the intestinal morphology,the level of intestinal protein and the expression of the antimicrobial peptide,the level of cytokine,the amount of ETEC in the fecesas well as the structure index of the cecum microorganism.The results showed that:1)Tryptophan deficiency and ETEC challenge were decrease growth preformance of SD rats.Tryptophan deficiency was significantly decreased final weight and average daily gain of SD rats(P>0.05).ETEC challenge was significantly reduced the final weight,average daily gain and average daily feed intake of SD rats(P<0.05).2)Tryptophan deficiency and ETEC challenge were impaired intestinal development.Tryptophan deficiency was significantly reduced villus height,lymphocytes amount,villus height to crypt depth ratio and significantly increased crypt depth of SD rats in jejunum and significantly reduced villus height,lymphocytes amount,villus height to crypt depth ratio of SD rats in ileum(P<0.05).ETEC challenge was siginificantly increased crypt depth,goblet cells and siginificantly decreased lymphocytes amount,villus height to crypt depth ratio of SD rats in jejunum(P<0.05),and significantly reduced villus height,lymphocytes amount,villus height to crypt depth ratio of SD rats in ileum(P<0.05).3)Tryptophan deficiency and ETEC challenge cause intestinal up-regulation proinflammatory cytokines of concentration and down-regulation anti-inflammatory cytokines of concentration.Tryptophan deficiency was significantly increased the cytokine concentration of IL-6 in jejunum and ileum(P<0.05),and had trend to increased the cytokine concentration of TNF-?(P=0.059)and reduced the cytokine concentration of TGF-?(P=0.056)in jejunum.ETEC challenge was significantly increased the cytokine concentration of IL-6(P<0.05)in jejunum and the cytokine concentration of IL-6,IL-8,TNF-?in ileum(P<0.05),and had trend to decreased the cytokine concentration of TGF-?(P=0.059)in jejunum.4)Tryptophan deficiency or ETEC challenge were significantly decreased the level of?-defensin 2 and increased the the level of TLR-4 receptor.Tryptophan deficiency or ETEC challenge were significantly decreased the level of mTOR,?-defensin 2 and AKT protein and increased the level of TLR-4 of jejunum in SD rats(P<0.05),and significantly decreased the level of AKT,?-defensin 2 of ileum in SD rats(P<0.05).ETEC challenge was significantly decreased the level of mTOR,?-defensin 2,AKT of jejunum in SD rats(P<0.05)and significantly increased the level of TLR-4 of jejunum in SD rats(P<0.05),and significantly decreased the level of AKT,?-defensin 2 of ileum in SD rats(P<0.05).5)Tryptophan deficiency or ETEC challenge were significantly decreased antibacterial peptide expression.Tryptophan deficiency was significantly decreased Defa-5,BD-2 gene mRNA expression and significantly increased ACE-2 gene mRNA expression of jejunum and ileum in SD rats(P<0.05).ETEC challenge was significantly increased Defa-5,IDO gene mRNA expression of jejunum and ileum in SD rats(P<0.05).6)Tryptophan deficiency and ETEC challenge were reduces cecal microbial diversity in SD rats,which were mainly affecting the diversity of Bacteroidetes microorganisms through analysis of DGGE results.7)ETEC challenge was increase the number of intestinal ETEC.From 0 d to 6 d,the number of ETEC were large and constant in ETEC challenge groups,ETEC challenge groups feces of 7~18 d were gradually decreased and level off to unchallenge group.In summary,tryptophan was contributed to promote the development and repair of intestinal mucosa,increase the expression of antibacterial peptide genes and the level of protein and improving the structure of intestinal microflora,which play a positive role in prevention the invasion of pathogens and maintenance of intestinal health. |
PDF Full Text Request