Study For The Signal Pathway Of Tryptophan Promoting β-defensine Secretion In Intestine

In order to elucidate the relationship between the signal pathway of tryptophan promoting β-defensine secretion and the key proteins(protein indoleamine-2, 3-dioxygenase(IDO), tryptophan hydroxylase(TPH) and m TOR), the effect of orally administraed tryptophan on the expression of ileum mucosa β-defensine gene in rats Sprague-Dawley(SD) was studied, after 1-Methyltryptophan(1-MT), parachlorophenylalamine(PCPA) and rapamycin-1(RAPA) were applied to inhibit IDO, TPH and m TOR) of intestinal mucosa in rats, respectively in this research. Meanwhile, the effects of dietary tryptophan levels on intestinal immune and health in weaned piglets was conducted. This study included four experiments as follow:Trial 1 was conducted to study on the function of IDO in the signal pathway of tryptophan promoting β-defensine secretion. 40 female SD rats were randomly divided into 4 groups(10 rats per group): rats orally administrated 0.5 ml saline(CON group); rats orally administrated 0.5 ml saline containing 1-MT(50 mg/kg/d)(1-MT group); rats orally administrated Trp that feed with 0.5 ml saline containing Trp(260 mg/kg/d); rats orally administrated 0.5 ml saline containing 1-MT(50 mg/kg/d) and Trp(260 mg/kg/d)(1-MT+Trp group). Each group had 10 replicates, each of which was repeated in 1 rats. Pre trial period was 3 d, and the positive test period lasted 7 d. Four groups of rats were fed the diet without nitrogen. Rats in the 1-MT group and 1-MT+Trp group were orally administered 1-MT daily during the pre trial and the positive test period. Rats in Trp group and 1-MT+Trp group were orally administered L-Trp daily during the positive test period. At the 8th d, 6 rats were randomly selected from each group for slaughter and samples of blood, liver, jejunum, and ileum were collected. The ileum IL-22 concentrations of control group was higher than that of 1-MT group and 1-MT+Trp group in(P<0.05). The jejunum IL-22 and IL-17 concentrations in the 1-MT+Trp group was lower than that of the other groups(P<0.05). The defa4 gene expression in Trp group was significantly higher than that of the other groups(P<0.05). The r BD2 gene expression of Trp group was higher than that of 1-MT+Trp group in ileum(P<0.05). When IDO the key enzyme of tryptophan metabolism pathways has been inhibited by 1 – MT, the β-defensine secretion was significantly decreased in the rat guts.Trial 2 was conducted to study on the function of TPH in the signal pathway of tryptophan promoting β-defensine secretion. 40 female SD rats were randomly divided into 4 groups(10 rats per group): rats that orally administrated 0.5 ml saline(CON group); rats in group that were intraperitoneal injected 0.5 ml saline containing PCPA(50 mg/kg/d)(PCPA group); rats orally administrated Trp that feed with 0.5 ml saline containing Trp(260 mg/kg/d); rats in group that had intraperitoneal injected 0.5 ml saline containing PCPA(200 mg/kg/d) and orally administrated Trp(260 mg/kg/d)(PCPA+Trp). Each group had 10 replicates, each of which was repeated in 1 rats. Pre trial period was 3 d, and the positive test period lasted 7 d. Four groups of rats were fed the diet without nitrogen. Rats in the PCPA group and PCPA +Trp groupwere intraperitoneal injected PCPA daily during the pre trial and the positive test period. Rats in Trp group and PCPA +Trp group were orally administered L-Trp daily during the positive test period. At the 8th d, 6 rats were randomly selected from each group for slaughter and samples of blood, liver, jejunum, and ileum were collected. The jejunum 5-HT concentrations of control group was higher than that of the other groups(P<0.05). The ileum 5-HT concentrations of control group was higher than that of the PCPA group and PCPA+Trp group(P<0.05). The jejunum TPH concentrations of control group was higher than that of the PCPA group and PCPA+Trp group(P<0.05). The ileum defa4 gene and r BD2 gene expression was higher than that of the other groups(P<0.05). When the key enzyme TPH of tryptophan metabolism pathways has been inhibited by PCPA. The β-defensine secretion was significantly increased in the jejunum of rats.Trial 3 was conducted to study on the function of m TOR in the signal pathway of tryptophan promoting β-defensine secretion. 40 female SD rats were randomly divided into 4 groups(10 rats per group): rats orally administrated 0.5 ml saline(CON group); rats intraperitoneal injected 0.5 ml saline containing RAPA(1.5 mg/kg/d)(RAPA group); rats orally administrated Trp that feed with 0.5 ml saline containing Trp(260 mg/kg/d); rats intraperitoneal injected 0.5 ml saline containing RAPA(1.5 mg/kg/d) and orally administrated Trp(260 mg/kg/d)(RAPA+Trp). Each group had 10 replicates, each of which was repeated in 1 rats. Pre trial period was 3 d, and the positive test period lasted 7 d. Four groups of rats were fed the diet without nitrogen. Rats in the RAPA group and RAPA +Trp groupwere intraperitoneal injected RAPA daily during the pre trial and the positive test period. Rats in Trp group and RAPA +Trp group were orally administered L-Trp daily during the positive test period. At the 8th d, 6 rats were randomly selected from each group for slaughter and samples of blood, liver, jejunum, and ileum were collected. The results were shown that there was no defa4 expression of RAPA group in jejunum. And the AKT protein expression of RAPA group was lower than that of the other groups(P<0.05). The r BD2 gene expression of RAPA group was lower than that of the other groups(P<0.05). The r BD2 expression of Trp was higer than that of the other groups(P>0.05). When m TOR the key protein of tryptophan metabolism pathways has been inhibited by RAPA, the β-defensine secretion was significantly decreased in the rat guts.Trial 4 was conducted to study the effects of dietary tryptophan levels on intestinal immune in weaned piglets. 40 healthy Large White × Landrace × Rong Chang(LLR) piglets(5.42 ± 1.52 kg) were randomly allocated to 4 treatments with 10 piglets per group. Piglets in 4 treatments were fed a diet containing 0.14% Trp, 0.21% Trp, 0.28% Trp and 0.35% Trp. During the experiment, food and water were provided ad libitum. Piglets were fed at 08:00, 12:00 and 18:00, respectively. After 28 d experiments. Five piglets were randomly selected from each group for slaughter at the 29 th d and the tissue samples were collected. The average daily gain and average daily intake were increased with the growth of diet tryptophan levels. The diarrhea rate of each group was 2.9%, 1.4%, 1.8% and 3.6%. The jejunum weight index and jejunum length index were increased with the growth of diet tryptophan levels. The Trp concentrations in plasma was obviously increased with the growth of diet tryptophan levels(P<0.05). Those amino acid Asp, Thr, Ser, Glu, Gly, Ala, Val, Ile, Leu, Tyr, Phe, Lys, His, Arg and Pro concentrations of 0.21% group was lower than that of the other groups(P<0.05). The 5-HT and IDO concentrations of 0.35% group was higher than that of the other groups(P<0.05). The ileum IL-17 concentrations was decreased with the growth of diet tryptophan levels(P<0.05). The TNF-α concentrations was decreased with the growth of diet tryptophan levels(P>0.05). Dietary approciate tryptophan level could reduce the contents of inflammatory cytokines and improve immunity in piglets.