Mechanism Of Tryptophan On Intestinal Epithelial Barrier And Immune Functions In Chronic Unpredictable Stress-exposed Animals

Four experiments were conducted to study the effects of L-tryptophan on intestinal epithelial barrier and immune functions in chronic unpredictable mild stress exposed animals and the related mechanism.Exp.1 was conducted to test the hypothesis that L-tryptophan supplementation may regulate intestinal epithelial barrier dysfunctions in chronic unpredictable mild stress(CUMS)exposed broilers.320 one-day-old health male broilers(Arbor Acres)were fed basal diet supplemented with or without L-tryptophan in the absence or presence of CUMS for 6 weeks.The results showed that CUMS treatment caused a pronounced increase in plasma corticosterone,adrenaline,norepinephrine and resulted in poor growth performance compared with that of controls.CUMS administration induced a significant increase in intestinal permeability.These effects of CUMS were associated with decreased plasma concentrations of 5-HT,decreased mRNA levels for TPH1,reduced protein abundances for claudin-1,ZO-1,and occludin and enhanced pro-inflammatory cytokines mRNA expression in jejunal tissues.L-tryptophan supplementation markedly attenuated CUMS-induced decrease in body weight(BW)and average daily gain(ADG),decreased the intestinal permeability and reversed plasma corticosterone and 5-HT concentration,mRNA levels for TPH1 and IDO,and protein abundances for claudin-1,ZO-1,and occludin and inflammatory cytokines expression.These results showed that CUMS exposure resulted in poor growth performance with impaired intestinal inflammatory response.These effects induced by CUMS were reversed by L-tryptophan supplementation.Above results were further tested in mice exposed in CUMS.The beneficial effects of L-tryptophan on maintaining intestinal function were associated with enhanced mRNA levels for L-tryptophan transporters and protein abundance for tight junction proteins,inhibited mRNA levels for pro-inflammatory cytokines and enhanced expression of anti-inflammatory cytokines in jejunal tissues.Exp.2 was conducted to investigate the mechanism whereby L-tryptophan regulates CUMS-induced impaired intestinal barrier integrity.Chicken primary intestinal epithelial cells were cultured with L-Trp in the presence or absence of dexamethasone(DEX),adrenaline(AD)and norepinephrine(NE).The results showed that stress hormones increased the mRNA level for inflammatory cytokines(IL-6,IL-8,IL-1β,TNF-α,NF-κB and IL-10).DEX treatment decreased the protein abundances for occludin and ZO-1.However,the pretreatment with L-Trp and specific antagonists for stress hormone receptors protected cell from stress hormones-induced high level of cytokines genes expression and lower protein abundances of occludin and ZO-1.Additionally,stress hormone influenced the Caco-2 cell TEER after 16 and 24 h incubation.Preincubation with L-Trp and specific antagonists for stress hormone receptors for 4 h attenuated the decrease of Caco-2 TEER.These results suggest that L-Trp protected intestinal barrier integrity through blocking the combination between stress hormones and their receptors to decrease the cytokines expression and increase tight junction protein expression.Exp.3 was conducted to investigate the hypothesis that L-tryptophan supplementation may regulate immune dysfunctions in chronic unpredictable mild stress(CUMS)exposed animals.Broilers were treated according to experiment 1.And mice were used to further test the results.Forty-eight 8-week-old mice were fed control diet(0.2%L-Trp)or L-Trp diet(0.4%L-Trp in total)in the presence or absence of CUMS for 8 weeks.The results showed that exposure of animals to CUMS resulted in stress-like behavior and induced a significant decrease in serum NDV level and proliferation of T and B lymphocytes.Additionally,broilers exposed to CUMS showed an increase in intestinal inflammatory response when challenged by LPS.The effect of CUMS was associated with increased in mRNA levels for stress hormones receptors in spleen.L-tryptophan supplementation markedly attenuated CUMS-induced stress-like behaviors,and lymphocytes proliferation were also up-regulated after L-Trp administration.Our data showed that CUMS exposure resulted in stress-like behaviors and impaired immune function.L-tryptophan improved CUMS-induced stress behavior and immune function,which are mediated partly by blocking the combination of stress hormones and their receptors.Exp.4 was conducted to investigate the mechanism whereby L-tryptophan regulates CUMS-induced immune dysfunction.T cells were cultured with or without L-Trp/5-HT in the presence or absence of dexamethasone(DEX),adrenaline(AD)and norepinephrine(NE).T-cell proliferation was determined.The results showed that stress hormones inhibited the proliferation of T lymphocytes.Our results confirmed that the blockade of stress hormone receptors by their specific antagonists protected T cells from stress hormones-induced proliferation inhibition,indicating that CUMS might stimulate the release of stress hormones and combination with their corresponding receptors,resulting in stress response.L-tryptophan and 5-HT treatments significantly attenuated the inhibitory action of stress hormones.These findings indicated that L-tryptophan could attenuate the CUMS-induced immunological dysfunction,partly through the serotonergic signals to block the combination between stress hormones and their receptors.In summary,L-tryptophan supplementation could attenuate stress response,protect intestinal epithelial barrier integrity and balance CUMS-induced immune dysfunction by blocking the combination between stress hormones and their receptors.