| The intestinal tract is usually immature during weaning and abnormal mutations in microbial structure provide the possibility for colonization by foreign pathogens.Disturbed intestinal microbial structure is an important cause of enteritis,which leads to diarrhea,post-weaning anorexia and growth retardation,a phenomenon that is particularly prominent in meat rabbit production.Because of the many known beneficial functions of butyrate and its effectiveness in other animal production applications,we hypothesized that butyrate could alleviate the adverse effects caused with weaning by promoting intestinal homeostasis in young rabbits during weaning.Although butyrate and its derivatives are known to be beneficial for animal growth,their potential mechanism of action is not clear,especially in weanling rabbits.In the present study,we investigated the mechanism of dietary butyrate action during weaning of young rabbits by constructing in vivo and in vitro experimental models.At the animal level,we investigated the mechanism of action of dietary butyrate on the structure of intestinal microflora,development and maintenance of intestinal barrier,organismal inflammation occurrence and organismal metabolism during weaning of young rabbits by combining 16 S r RNA sequencing and LC-MS untargeted metabolomics.At the cellular level,the mechanism of the effect of butyrate on the action of harmful bacteria-induced apoptosis,inflammatory response and appetite protein expression was investigated.The main contents were as follows:1.Effect of dietary butyrate on the growth and development of weanling rabbits.The rabbits in the control group were fed a basal diet and the rabbits in the butyrate group were fed a basal diet containing 0.3% sodium butyrate.The main results were that dietary butyrate significantly increased the mean daily feed intake and mean daily weight gain of weanling rabbits(P < 0.05).At 30-60 days of age,the diarrhea rate was significantly reduced in the butyrate group(P < 0.05),while feed conversion and survival were improved to some extent(P > 0.05).Butyrate significantly increased small intestinal and cecum villi height and villi/crypt ratio(P < 0.05).Butyrate significantly upregulated the expression of intestinal epithelial barrier-related genes Claudin-1,Claudin-2,Occludin,and ZO-1(Zonula occludens 1)(P < 0.05).Also,butyrate significantly reduced the levels of IL-1β(Interleukin,IL)in the small intestine,cecum and plasma(P < 0.05).Butyrate significantly reduced the level of TNF-α(Tumor necrosis factor α)in the small intestine(P< 0.05),and IL-6 was also reduced but not significantly(P > 0.05)in the small intestine,cecum and plasma.CCK(Cholecystokinin)levels in the small intestine and plasma were significantly reduced by butyrate(P < 0.05),while PYY(Peptide YY)levels in the small intestine and cecum were significantly reduced by butyrate(P < 0.05).Butyrate significantly increased the levels of Ghrelin in the small intestine and plasma(P < 0.05),while there was no significant effect on the levels of GLP-1(Glucagon-like peptide 1)(P > 0.05).In brain microvascular endothelium,protein expression of ZO-1,Claudin-5and Occludin was significantly upregulated by butyrate(P < 0.05).In the hypothalamus,the protein expression of IL-6,IL-1β,and p-NF-κB p65(Nuclear factor-kappa B,NF-κB)was significantly downregulated by butyrate(P < 0.05).Butyrate decreased the expression of NF-κB p65 and TNF-α,but not significantly(P > 0.05).Meanwhile,butyrate significantly reduced the expression of POMC(Proopiomelanocortin),CART(Cocaine and amphetamine-regulated transcript)(P < 0.05).The expression of Ag RP(Agouti-related protein)and NPY(Neuropeptide Y)was significantly upregulated by butyrate(P < 0.05).The above results indicated that butyrate promoted the development of intestinal epithelial tight junctions and maintained intestinal barrier integrity as well as the permeability of the blood-brain barrier.Butyrate suppressed the inflammation and diarrhea caused by weaning,thus relieving and avoiding post-weaning anorexia and growth retardation.2.Effect of dietary butyrate on the intestinal microbial structure of weanling rabbits.Samples of the contents near the intestinal mucosa of the small intestine and cecum in Chapter 2 were taken for 16 S r RNA sequencing.The main results were that dietary butyrate had a significant effect on microbial β-diversity in the small intestine and cecum(P < 0.05).In the small intestine,the LEf Se multilevel species difference analysis showed that a total of eight species were enriched at the genus level: five in the control group and three in the butyrate group.Of the eight enriched species at the genus level,seven belonged to Firmicutes and one to Actinobacteria.We performed a univariate correlation network analysis of 208 genus-level microorganisms in the small intestine.The results showed a significant positive correlation between all the differentially dominant microorganism within the group(P < 0.05).There was a significant negative correlation between all the differentially dominant microorganism between the groups(P < 0.05).A significant positive correlation was found between the differentially dominant microorganism in the control group and Escherichia-Shigella(P < 0.05).There was a significant positive correlation between the differentially dominant microorganism in the butyrate group and Lactobacillus(P < 0.05).In the cecum,a total of six species were enriched at the genus level by LEf Se multilevel species difference discrimination analysis.The control and butyrate groups each included three,five of which belonged to Firmicutes and the other one to Proteobacteria.We performed a one-way correlation network analysis of 318 microorganisms at the genus level.There was a significant positive correlation between all the differentially dominant microorganism within the group(P <0.05).A significant negative correlation was found between all the differentially dominant microorganism in both groups(P < 0.05).We functionally annotated all OTUs and showed that butyrate significantly increased the fermentative capacity and chemoenergetic heterotrophic levels of intestinal microorganisms and reduced the risk of pathogenicity in weanling rabbits(P < 0.05).We correlated the differentially dominant microorganism in each gut with growth traits of weanling rabbits.The results showed that differentially dominant microorganism in the butyrate group promoted intestinal development,suppressed inflammation and promoted positive regulation of appetite during weaning in young rabbits(P < 0.05).These results indicated that dietary butyrate had a positive regulatory effect on the formation of intestinal microbial structure during weaning of young rabbits.With the action of butyrate,the abnormal and unfavorable changes of intestinal flora caused by weaning of young rabbits were avoided,and the occurrence of inflammation and anorexic behavior of young rabbits were alleviated.3.Effect of dietary butyrate on organismal metabolism in weanling rabbits.The contents of the small intestine and cecum near the intestinal mucosa and plasma samples in Chapter 2 were taken for LC-MS untargeted metabolomics assay.The main results were: in the small intestine,30 metabolites were increased and 7 metabolites were decreased in the butyrate group compared to the control group(P < 0.05).We performed KEGG pathway enrichment analysis of the small intestinal metabolome in the butyrate and control groups using a hypergeometric distribution algorithm.The results showed that the differentially dominant metabolites Cholestane-3,7,12,25-tetrol-3-glucuronide and Deoxycholic acid in the small intestine were enriched to the bile acid secretion pathway(P< 0.05)and Sphingosine was enriched to the apoptosis pathway(P < 0.05).In the bile acid secretion pathway,digestion and absorption of intestinal fat and biosynthesis of secondary bile acids were facilitated by the increased abundance of Deoxycholic acid,which improved the transport capacity of ileocytes and ultimately enhanced the potential for portal and systemic circulation of bile acids.In the apoptotic pathway,a decrease in the abundance of Sphingosine(regulated by cascades in this pathway)may lead to a decrease in the degradation of nuclear DNA fragments and a decrease in the level of apoptosis.In the cecum,a hierarchical analysis of the top 50 metabolites in terms of VIP value using the Euclidean distance algorithm showed that 36 metabolites were increased and 14 metabolites were decreased by the addition of dietary butyrate(P < 0.05).We found that the differential metabolites Deoxyinosine,Uric acid were enriched to the purine metabolic pathway and Deoxyinosine was enriched to the ABC transporter protein pathway by KEGG pathway enrichment analysis.In plasma,the top 50 metabolites in terms of VIP values were analyzed in a hierarchical clustering.The results showed that the control group contained 7 metabolites and the butyrate group contained 43 metabolites(P < 0.05).By KEGG pathway enrichment analysis,the alpha-linolenic acid metabolic pathway was enriched by Dodecanedioic acid(P < 0.05)and the production of Traumatic acid may be promoted by the increased abundance of Dodecanedioic acid,thus improving the anti-inflammatory and antioxidant capacity of the organism.The above results showed that dietary butyrate exerted a significant positive effect on the metabolism of the organism during weaning of young rabbits.Butyrate may have facilitated intestinal fat digestion and absorption and bile acid circulation in weanling rabbits by altering the bile acid secretion pathway.Butyrate enhanced the potential of the body’s anti-inflammatory and antioxidant functions through the alpha-linolenic acid metabolic pathway.Butyrate may have reduced the chance of apoptosis through the apoptotic pathway.4.Effects of butyrate-mediated in vitro Escherichia coli(E.coli)infestation on apoptosis,inflammation,and appetite regulation.We used E.coli infested cells in vitro and treated hypothalamic neurons with IL-1β.The main results were: in enterocyte,brain microvascular endothelial cells,astrocytes and hypothalamic neurons,the level of apoptosis was significantly lower in the-/B and-/-groups than in the E/-and E/B groups(P < 0.05),and the level of apoptosis was significantly higher in the E/-group than in the E/B group(P < 0.05).In the transepithelial electrical resistance of the in vitro blood-brain barrier composed of brain microvascular endothelial cells and astrocytes,the-/B group was the largest,followed by the-/-group,then the E/B group,and the E/-group was the smallest,and the difference between the groups was significant(P < 0.05).In enterocyte,the expression of NF-κB p65,TNF-α,IL-1β,p-NF-κB p65,and IL-6 was significantly lower in the-/B and-/-groups than in the E/-and E/B groups(P < 0.05),and the expression of TNF-α,IL-1β,p-NF-κB p65,and IL-6 was significantly lower in the E/B group than in the E/-group(P < 0.05).In hypothalamic neurons,NF-κB p65 and IL-6expression was significantly lower in the-/B and-/-groups than in the E/-and E/B groups(P < 0.05).The protein expression of TNF-α was significantly higher in the E/-group than in the-/B,-/-and E/B groups(P < 0.05).The expression of IL-1β and p-NF-κB p65 was significantly lower in the-/B and-/-groups than in the E/-and E/B groups(P < 0.05),and the expression of IL-1β and p-NF-κB p65 was significantly lower in the E/B group than in the E/-group(P < 0.05).The expression of POMC and CART was lowest in the-/B group,followed by the-/-group,then the E/B group,and highest in the E/-group,and the difference between the groups was significant(P < 0.05).Ag RP and NPY had the highest protein expression in the-/B group,followed by the-/-group,then the E/B group,and the lowest was the E/-group,and the difference between the groups was significant(P <0.05).Treatment of hypothalamic neurons with IL-1β showed that IL-1β significantly increased the level of apoptosis in hypothalamic neurons(P < 0.05).Butyrate increased the protein expression of NF-κB p65 to some extent(P > 0.05),while the protein expression of p-NF-κB p65 was significantly upregulated(P < 0.05).Also,POMC and CART protein expressions were significantly up-regulated by butyrate(P < 0.05)and Ag RP and NPY protein expressions were significantly down-regulated by butyrate(P <0.05).The above results indicated that the occurrence of apoptosis and inflammation caused by E.coli was significantly reduced mediated by butyrate.Butyrate increased the transepithelial electrical resistance of the blood-brain barrier in vitro and achieved positive regulation of appetite promotion by hypothalamic neurons.And the cytokine IL-1β may be a key target involved in regulation after oral administration of butyrate in weanling rabbits.In conclusion,dietary butyrate had a positive regulatory effect on the construction of the intestinal microbial structure of young rabbits during weaning.Butyrate reduced the colonization of harmful intestinal bacteria and the production of pro-inflammatory factors in the body.Butyrate promoted the development of intestinal epithelial tight junctions and maintained intestinal barrier integrity and blood-brain barrier permeability.Butyrate promoted beneficial metabolic processes in the organism and increased the production of beneficial metabolites.Butyrate may increase the body’s digestion and absorption of nutrients through the bile acid secretion pathway,enhance anti-inflammatory and antioxidant capacity through the alpha-linolenic acid metabolism pathway,and reduce the occurrence of apoptosis through the apoptosis pathway.Butyrate inhibited the transmission of inflammatory and appetite suppression signals between the gut-brain axis,thereby alleviating and avoiding anorexic behavior and growth retardation in young rabbits after weaning. |
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