| There are large and diverse bacterial colonies in the gastrointestine of animals,which play an important role in regulating metabolism and maintaining intestinal health.In vitro studies showed that intestinal microorganisms could participate in the metabolism of amino acids,and there were differences in the metabolism of amino acids among different intestinal segments of chyme.However,the effect of intestinal microorganisms on host nitrogen metabolism is not clear.In addition,there are many kinds of microorganisms in the large intestine and their metabolic activity is the highest.However,the contribution of large intestinal microorganisms in regulating host metabolism and maintaining intestinal health is not clear.As one of the most effective intervention methods,antibiotics can effectively reduce the number of intestinal microorganisms.In addition,intestinal nutrient substrates can change the structure and metabolic types of intestinal microflora.Increasing the level of carbohydrates or nitrogen compounds in the intestine is another effective way to interfere with intestinal microorganisms.Thus,based on the above considerations,the growing pigs were used in this experiment with antibiotics feeding,ileal infusion of antibiotics or cecal infusion of corn starch and casein hydrolysate to intervene the intestinal microbiota,then to investigate the responses of gut microbiota,host metabolism and gut barrier function.1 Antibiotic feeding-induced alterations of the gut microbiota and microbial fermentation in protein parallel the changes in host nitrogen metabolism of growing pigsThis study was designed to investigate the effects of feeding a cocktail of antibiotics(AGM)(ampicillin,gentamycin and metronidazole)on intestinal microbiota,N utilization efficiency,and amino acid(AA)digestibility in cannulated pigs,with the aim of exploring the impact of gut microbiota on host N metabolism.In total,16 piglets(42 days old;weighing 12 ±0.5 kg)were surgically fitted with a simple distal ileal T-cannula and a jugular venous catheter(n=8).The pigs were fed a basal diet without antibiotics(control)or with antibiotics(antibiotics),for 2 weeks.The results showed that antibiotics feeding did not affect weight gain or digestive enzyme activity.The antibiotics increased the concentration of urea N(P<0.05).However.antibiotics feeding reduced N utilization,and the total tract apparent digestibility of isoleucine,methionine,valine,tyrosine and total AA(P<0.05).Furthermore,antibiotics feeding increased the terminal ileum apparent digestibility of CP,phenylalanine,valine,alanine,tyrosine and total AA(P<0.05).Antibiotics feeding markedly altered the composition of the microbiota in the ileum and feces,with a reduction in populations of Bifidobacterium,Lactobacillus and Ruminococcus,and an increase in the abundance of E.coli.Antibiotics feding also significantly increased the concentration of cadaverine and ammonia,both in ileal digesta and feces(P<0.05),suggesting a marked impact on N metabolism in the intestine.The analyses indicated that the alteration of gut microbiota was correlated with the apparent digestibility of CP and AA in the intestine.These findings suggest that the antibiotics feeding-induced alteration of gut microbiota may contribute to the change in intestinal N metabolism,and consequently,N excretion from the body.These results also suggest that antibiotics could have a significant effect on host N metabolism.The present study contributes to our understanding of the effects of antibiotics and provides a rational scientific basis for diet formulation during AGM use.2 Antibiotics infusion-induced alterations of large intestinal microbiota regulated host bile acid profile and lipid metabolism of growing pigsThis experiment was to investigate effects of antibiotics infusion induced microbiota changed in the large intestine on microbial bile acid metabolism,liver transcriptome,and metabolome of serum and urine using a growing pig model.Twelve growing pigs(45 days old;weighing 12.08 ± 0.28 kg)were fitted with a T-cannula at the terminal ileum and randomly assigned into two groups(n=6)and infusion with either sterile saline(control)or saline with mixed antibiotics AGM(antibiotic).After 25 days' treatment,blood,urine,proximal ileal digesta,proximal colonic digesta,and liver samples were collected.The bile acid profile in serum,liver,ileum,colon and the main bacteria in colonic digesta and ileal digesta were investigated.The metabolites in serum and urine were investigated by metabolome.Liver transcriptomics were investigated by RNA-sequencing.Results showed that antibiotics had no effect on main bacteria in ileum,whereas could specifically change the main bacteria both in proximal colonic digesta by decreasing the populations of Firmicutes,Actinobacteria,Bifidobacterium,Clostridiun cluster ?,and Clostridium cluster ??a,and increasing the populations of Bacteroidetes,Bacteroides spp.,Lactobacillus and E.coli(P<0.05).Antibiotics infusion also reduced the populations of the bacteria which encoding bile salt 7?-dehydroxylase gene(baiJ)and decreased the secondary bile acid transformation ability of bacteria in colon.In addition,antibiotics infusion also changed the secondary BA profiles(deoxycholic acid,and lithocholic acid)in colonic digesta and this change was paralleled with the changes of these bile acid in serum and liver(P<0.05).Further correlation analysis showed that the markedly changed gut bacteria in colon were strongly correlated with the changes in bile acid profile.Moreover,antibiotics infusion markedly affected host fatty acid biosynthesis revealed by the results of both metabolomics in serum and urine and transcriptome in liver.These results indicate that antibiotics infusion-induced alterations of main bacteria in the large intestine can change host bile acid metabolism and lipid metabolism.These findings may provide a new insight into that the contributions of microbiota in the large intestine to gut bile acid metabolism and host metabolism.3 Colon transcriptome response to antibiotics-induced alterations of microbiota in the large intestine of growing pigsThis experiment was to investigate the effects of large intestinal microbiota alterations by antibiotics infusion on colon transcription of growing pigs.After 25 days' treatment,colon tissue were collected.Colon transcriptomics were investigated by RNA-sequencing.Results showed that the concentration of D-lactate and the activity of diamine oxidase were significantly decreased after antibiotics infusion.In addition,antibiotics infusion significantly changes the colon transcription,the genes related to immune response such as proinflammatory cytokines(IL-18 and IL-33)and cytokines&chemokines(CCL2,CCL5,CCL23 and CXCL13)were up-regulated(P<0.001).Genes related to oxidative stress such as GPX8 and CAT were also up-regulated after antibiotics infusion(P<0.001),suggesting there may be an increase in the immune response.However,genes related to gut barrier function such as mucin protein related(MUC4,MUC20,GALNT18 and ST3GAL1),tight junction protein(TJP3,CLDN3 and CLDN23)were down-regulated after antibiotics infusion(P<0.001),suggesting colonic barrier function was impaired.Antibiotics infusion also decreased the capacity of transport by down-regulated expression of genes involved in ABC transporters(ABCB8,ABCB9 and ABCA7)and vitamin digestion and absorption(SLC19A1 and ABCC1).In addition,antibiotics infusion also upregulated the genes related energy metabolism(NDUFAF2,NDUFA5 and NDUFA12),these results may caused by the decreased level of SCFAs(butyrate)in colon after antibiotics infusion.Collectively,these results suggest that antibiotics infusion-induced alterations of large intestinal microbiota can regulate the gut function in colon.These findings may provide a new sight into the cross-talk between large intestinal microbiota and host.4 Increasing carbohydrate or nitrogenous compound concentrations in the large intestine by cecal infusion of corn starch or casein hydrolysate lead to opposite modulating influence in colonic microbiota and microbial metabolites of growing pigsThis study was to investgate the colonic microbiota and microbial metabolites responses to an increase in carbohydrate source or nitrogenous compounds in the large intestine.To study this,twenty-four cecum cannulated-piglets(40 days old;weighing 12.08 ± 0.28 kg)were employed(n=8),and sterile saline(Control group),carbohydrate source(corn starch,50g/d,Starch group)and nitrogenous compounds(casein hydrolysates,50g/d,Casein group)were infused into cecum.After 19 days infusion,colonic digesta and mucosa were collected for analysis of microbiota and microbial metabolites.Compared with control group,starch group increased total carbohydrate concentration and decreased total nitrogenous compounds concentration in colon,while on the contrary in casein group concentrations of total carbohydrate and total nitrogenous compounds decreased(P<0.05).Compared with control group,both starch and casein groups markedly altered microbial compositions both in colonic digeata and mucosa,but in different patterns.In colonic digesta,starch infusion significantly increased the relative abundances of Bacteroidales S24-7 group,Bifidobacterium,Megasphaera,and decreased the abundances of Anaerovibrio,Campylobacter,Veillonella,whereas casein infusion significantly increased Streptococcus,Desulfovibrio and Mogibacterium,and decreased the abundances of Coprococcus,Ruminococcus.In colonic mucosa,Starch group mainly increased Prevotella,Bacteroides and Bifidobacterium.whereas Lactobacillus was decreased(P<0.05).However,casein group mainly increased the abundance of Campylobacter and decreased the abundance of Lactobacillus and Coprococcus.These results indicate that differences in nutrient preference of gut microbes.In addition,the concentrations of acetate,propionate,and butyrate were increased,whereas the concentrations of cadaverine,putrescine,tyrosamine,deoxycholic acid and lithocholic acid were decreased in starch group.However,the casein group showed contrary effects(P<0.05).Therefore,these results suggest that higher level of carbohydrate in the large intestine could regulate the microbiota and increased microbial beneficial metabolites,while higher level of protein increased the potential pathogen and harmful metabolites.The findings may provide a new sight into the different response of gut microbiota to the pattern of nutrient substrates in the large intestine.5 Increasing carbohydrate or nitrogenous compound concentrations in the large intestine by cecal infusion of corn starch or casein hydrolysate lead to opposite modulating influence in host metabolism of growing pigsThe aim of this study was to investigate the response of host metabolism to increasing nutrient substrates availability(carbohydrates or nitrogenous compounds)in the large intestine.After 19 days of infusion,serum and liver samples were collected and serum metabolome and gene expression of liver were investigated.Compared with control group,starch infusion increasing the concentration of SCFAs,whereas casein infusion decreasing the concentration of SCFAs in liver(P<0.05).Starch infusion increased host metabolites associate with the TCA cycle(Fumaric acid,L-malic acid and oxaloacetic acid),and lipogenesis(stearic acid and palmitic acid),whereas casein infusion increased host metabolites related to glycolysis(lactic acid and glucose-6-phosphate)and lipolysis(glycerol and 3-hydroxybutyric acid).In addition,star-ch group upregulated the gene expression of FAS and ACC,and downregulated the gene expression of PPAR-? and CPT-1?,whereas casein group upregulated gene expression of PPAR-?,ACOX-1 and CPT-1?(P<0.05).these results indicated that increasing the carbohydrate level in large intestine could enhance the energy metabolism and fatty acid synthesis,while increasing the nitrogenous compounds level in large intestine could enhance the fatty acid oxidation and glycolysis.Moreover,starch group increased the SCFAs level in liver,whereas casein group drcreased the SCFAs level in liver(P<0.05).Further correlation analysis showed that the changed gut microbiota significantly correlation with parts of serum metabolites.Therefore,these results may indicate that large intestinal bacteria are involved in regulating host metabolism,and their metabolites(SCFAs)may mediate the gut-liver metabolic axis.These findings may provide new insights into the cross-talk between the large intestinal microbiota and host metabolism.6 Increasing carbohydrate or nitrogenous compound concentrations in the large intestine by cecal infusion of corn starch or casein hydrolysate lead to opposite modulating influence in mucosal immunity and barrier function of growing pigsThe aim of this study was to investigate the colon health to an increase in levels of carbohydrate source or nitrogenous compounds in the large intestine.After 19 days infusion,colonic tissue and mucosa were collected for analysis of immunohistochemical and mucosal gene expression related to gut barrier function and immunity.Compared with control group,starch group decreased D-lactate concentration and the activity of diamine oxidase in serum,whereas casein group increased D-lactate concentration and the activity of diamine oxidase(P<0.05).In addition,starch group up-regulated the gene expression of mucin(MUC1),tight junction proteins(CLDN2,OCLD and ZO-1)(P<0.05),whereas casein group downregulated the gene expression of ZO-1,OCLD and CLDN1(P<0.05).Protein expression analysis further validated the gene quantification results.Compared with the control group,both the starch and casein group markedly modulated the gene expression of colonic mucosa immune function,but in different ways.Starch group up-regulated the gene expression of pro-inflammatory genes(IL-8 and TNF-?),anti-inflammatory genes(IL-10 and TGF-?),NFkB,MyD88 and Toll-like receptors genes(TLR2,TLR4 and TLR5)(P<0.05).Casein group significantly up-regulated the gene expression of pro-inflammatory genes(IL-1?,IL-8,IL-18 and TNF-?).The concentration of cytokines in colonic mucosa was consistent with the results of gene expression.Further cytokine network analysis showed that cecal infusion of casein hydrolysates enhanced inflammatory response compared with the control,whereas cecal infusion of corn starch inhibited inflammatory response compared with the control.In addition,compared with control group,the expressions of EGFR and TGR-5 genes were significantly down-regulated,while the expression of GPR41,GPR43,MCT-1 and HDAC1 genes were up-regulated in starch group(P<0.05).However,compared with control group,the gene expressions of GPR41,GPR43 and HDAC1 were down-regulated and the gene expression of EGFR was up-regulated in the casein group(P<0.05).Therefore,these results suggest that higher level of carbohydrate in the large intestine could improve colonic barrier function and enhance innate immunity development,while higher level of protein seemed detrimental to colon health by impairing barrier function and increasing proinflammatory response.The findings may provide a new sight into the different response of gut microbiota to the pattern of nutrient substrates in the large intestine and the cross-talk between gut microbiota and host.7 The mechanism of microbial metabolites secondary bile acids(DCA and LCA)on gut barrier function using Caco-2 cells modelDCA and LCA are the main secondary bile acid could regulate intestinal barrier function.However,the mechanism of DCA and LCA on gut barrier function still unclear.In this study,we use Caco-2 cell line as a model to investigate the mechanism of DCA and LCA on gut barrier function.(1)Primary bile acid(CA),secondary bile acid(DCA and LCA)were selected to treat Caco-2 cells at concentrations of 25,100,and 250 ?M for 24 hours.Cell viability,gene expression of ZO-1 and Occludin mRNA,TEER and cell permeability were investigated after treatment.The results showed that DCA and LCA at different concentrations could decrease the cell barrier function to a certain extent.The gene expression of ZO-1 and Occludin,TEER and cell permeability were decreased(P<0.05),but CA had no significant effect on cell activity and barrier function(P>0.05).(2)Cells were treated with DCA and LCA at a concentration of 100 ?M for 24 hours.The gene expression of barrier function genes and bile acid-related receptor gene were further measured.The results showed that DCA and LCA treatment could significantly downregulate the gene expression of ZO-1 and up-regulate the gene expression of EGFR receptor and Src gene.EGFR receptor is one of the bile acid receptors.Previous studies have shown that EGFR can participate in the regulation of intestinal barrier function.Src,as downstream pathway,can be regulated by the EGFR receptor,can participate in regulating cell oxidative stress and intestinal barrier function.These results indicated that DCA and LCA may regulate cell barrier function through EGFR-Src pathway.(3)EGFR inhibitors(AG-1478)and SRC inhibitors(PP2)were selected to treat the cells respectively.The results showed that both AG-1478 and PP2 could alleviate the effects of DCA and LCA on cell barrier function,suggesting that DCA and LCA decreased the cell barrier function through EGFR-Src pathway.In conclusion,both DCA and LCA can impaired the gut barrier function and may be regulated via EGFR-Src pathway. |
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