| The existence of gut microbiota-brain axis in human and animals.The intestinal dysbiosis is often closely associated with the functional changes of nervous system in hosts.In contrast to small intestinal microbes,large intestinal microbes have larger quantity and are more diverse with stronger substrate fermentation abilities.Therefore,large intestinal microbiota may play an important role in the functional modulation of host nervous system.Antibiotic cocktail(ampicillin,gentamycin,and metronidazole)suppresses microbial carbohydrate metabolism,and promotes nitrogenous metabolism.On the contrary,corn starch promotes microbial carbohydrate metabolism,and suppresses nitrogenous metabolism.The effects of these two approaches on the function of nervous system are still unclear.Therefore,by using large intestinal microbiota targeted strategies,our research aimed to investigate the effects of large intestinal microbiota on the modulation of nervous system in host.In addition,the potential underlying mechanism was further analyzed using microbial in vitro culture,mouse model and in vitro neural cell model.1 Effects of distal ileal antibiotic infusion on the neurochemistry in the hypothalamus of pigletsIn the present study,twelve piglets(12.08 ± 0.28 kg)fitted with a T-cannula at the distal ileum were fed a standard diet and randomly assigned to two groups(n=6)for ileal infusion of either saline(control group)or antibiotics(ampicillin,gentamycin,and metronidazole;antibiotic group).After 25-days of infusion,ileal and fecal microbiota,serum amino acids and neurotransmitters,and hypothalamic transcriptomics were analyzed.Whilst the antibiotic infusion did not change the proximal ileal microbial composition,it markedly altered the fecal microbial composition and increased aromatic amino acid(AAAs)metabolism(P<0.05),suggesting the infusion specifically targeted large intestinal microbes.Concentrations of AAAs were likewise decreased in the blood and hypothalamus(P<0.05)by antibiotic infusion.Antibiotic infusion further decreased concentrations of hypothalamic BDNF,and neurotransmitters(5-HT and dopamine),in line with AAAs being their precursors(P<0.05).Furthermore,an upregulation in gene expressions of neurotransmitter transporters and synthetases was observed(q<0.001).In conclusion,the ileal-antibiotic infusion altered neurotransmitter expression in the porcine hypothalamus and this effect occurred simultaneously with changes in both the large intestinal microbiota,and AAAs in large intestine,blood and hypothalamus.These findings indirectly indicate that antibiotic-induced alterations in the large intestinal microbiota may decrease hypothalamic expressions of neurochemical factors by reducing circulating AAA availability.2 Effects of cecal corn starch infusion on the neurochemistry in the hypothalamus of pigletsThis study used a large intestinal microbiota-targeted strategy by increasing large intestinal carbohydrate availability in a cecal-cannulated piglet model.Sixteen piglets(12.08 ± 0.20 kg)with a cecal T-cannula were fed with a standard diet and randomly assigned to two groups(n=8)for cecal infusion of saline(control group)or corn starch(starch group),respectively.After 19-day experiment,ileal and fecal microbiota,concentrations of 5-HT,dopamine and their precursors AAAs,and hypothalamic gene expressions were analyzed.Cecal starch infusion led to a decrease in large intestinal AAA metabolism,an increase in systemic AAA availability,central AAA-derived neurotransmitters(5-HT,dopamine)and neurotrophic BDNF in piglets(P<0.05).These findings indirectly indicate that corn starch-induced alterations in the large intestinal microbiota may enhance hypothalamic expressions of neurochemical factors by increasing circulating AAA availability.3 Isolation and identification of aromatic amino acid utilizing bacteria in cannulated pigletsThe present study mainly investigated the metabolism of tryptophan,tyrosine and phenylalanine of Lactobacillus derived from large intestine of cannulated piglets in in vitro culture.Fecal samples in cannulated pigs were inoculated in vitro in MRS medium to isolate Lactobacillus bacteria.The isolated Lactobacillus bacteria were inoculated into a single AAA medium(10 mM)and then cultured for 24 h in vitro.After 24 h,the concentration of free AAA in each group was determined,and the disappearance rate of AAA was calculated.At the same time,major metabolites of bacteria,including lactic acid,ammonia nitrogen and AAA metabolites were elevated.The results showed that,after 24 h in vitro culture,the Lactobacillus bacteria significantly increased the rate of AAA elimination(P<0.05),and the concentrations of lactic acid,ammonia nitrogen and AAA metabolites significantly increased(P<0.05).Six strains including Trp 1,Trp 2,Tyr 1,Tyr 2,Phe 1 and Phe 2 were isolated and purified,respectively to explore their AAA metabolic capacity and to identify their similarities.The results showed that all 6 strains could metabolize single AAA.The 16S rRNA identification showed that all six strains were belonging to Lactobacillus.The phylogenetic tree analysis showed that Trp 1,Tyr 1 and Phe are in the same branch with Lactobacillus reuteri strain DSM,with the 99%similarity;Trp 2 and Lactobacillus taiwanensis strain BCRC are in the same branch,with the 99%similarity;Tyr 2 and Phe 2 are in the same branch with Lactobacillus salivary strain JCM,with the 99%similarity.Therefore,this research confirms that some bacteria in Lactobacillus from large intestine of cannulated piglets have AAA metabolic capacity.4 Time-course effects of intraperitoneal injection of aromatic amino acids on the expression of central neurotransmitters and BDNF in miceThis study verified the role of circulation AAA on the expression of brain neurotransmitters(5-HT and dopamine)and BDNF using mouse model.In this study,mice were randomly assigned to four groups(n=30)for intraperitoneal(i.p.)injection of saline,or single AAA(tryptophan,tyrosine,or phenylalanine),respectively.At a given time point(0,1,2,4,and 8 hours),six mice per group were decapitated and samples of brain and blood were collected for amino acid profiling,neurotransmitter productions and gene expressions determination.The concentrations of AAA in the circulation and brain were increased after 1 hour of single AAA i.p.injection,in parallel with the decrease in concentrations of other neutral amino acids(P<0.05).The concentrations of 5-HT and BDNF were increased in brain after 2 hours of single tryptophan i.p.injection(P<0.05).The concentrations of dopamine and BDNF were increased in brain after 1 hour of single tyrosine i.p.injection(P<0.05),but not phenylalanine i.p.injection.In conclusion,our findings reveal that the up-regulation of circulating tryptophan or tyrosine,but not phenylalanine promotes the biosynthesis of neurotransmitters(5-HT and dopamine)and BDNF in mice brain in a time-course manner.5 Mechanistic insights into the role of aromatic amino acids on the expression of neurotransmitters and BDNF in mouse neuronal cells in vitroIn this study,the underlying mechanism by which AAA regulates the expression of neurotransmitters and BDNF in the host was further studied using mouse neuronal cell in vitro culture.Mouse neuronal cells were incubated with L-tryptophan(0.80 mM),L-tyrosine(4.40 mM),or L-phenylalanine(4.20 mM)for 24 h,the supernatants and cells were sampled for the analysis of AAA levels,cell signaling activation,and neurotransmitter concentrations.Results showed that compared with the control group,after 24 h of treatment with tryptophan and tyrosine,the concentrations of 5-HT and dopamine were significantly increased,and the concentration of BDNF was significantly increased(P<0.05).However,phenylalanine treatment had no significant effect on the expression of neurotransmitters and BDNF(P>0.05).The expressions of cAMP-PKA-CREB signaling pathway molecules were significantly increased in tryptophan and tyrosine treatment groups(P<0.05).After the expressions of neurotransmitter synthase(TPH2 and TH),neurotransmitter receptor(5-HT1A and DRD1)and downstream signal regulator CREB were inhibited by specific interfering RNA,the concentrations of elevated neurotransmitters(5-HT and dopamine)and BDNF induced by AAA returned to normal levels.The above results indicate that tryptophan and tyrosine can activate the cell 5-HT 1A/DRD1-CREB signaling pathway to promote the expression of BDNF through the synthesis of neurotransmitters(5-HT and dopamine).Therefore,this study reveals the mechanism by which AAA regulates the expression of neurotransmitters and BDNF in the host.In conclusion,by using large intestinal microbiota-targeted strategies,the interaction between large intestinal microbiota and central neurochemistry has been revealed.Tryptophan and tyrosine can act as mediators along the large intestinal microbiota-brain axis. |
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