| Nonalcoholic fatty liver disease(NAFLD)is a pathological syndrome characterized by excessive lipid deposition and hepatic steatosis,which is independent of alcohol or other liver injury factors.The incidence of NAFLD increases year by year,and become the most popular chronic liver disease in China.Epidemiological investigation found that the increase of fructose intake was closely related to the occurrence of NAFLD.Animal experiments also showed that fructose could significantly induce hepatic lipid deposition.Gut microbiota system is the largest and most complex micro ecosystem in human and animals.In recent years,more and more attention has been paid to the role of gut microbiota in the occurrence and development of NAFLD.Intestinal dysbiosis has become a key factor causing NAFLD.As one of the main fermentation products of intestinal bacteria,butyric acid has a variety of biological effects.Previous studies have shown that sodium butyrate(NaB)can significantly alleviate NAFLD induced by high-fat diet,however,whether it can alleviate NAFLD induced by fructose has not been reported.Therefore,in this study,the NAFLD model of C57BL/6j mice was induced by fructose drinking water,the effects of intestinal microbiota on alleviating NAFLD were explored by fecal microbiota transplantation(FMT),and the potential effective microbial metabolites in hepatic portal vein blood were screened for further investigation in vitro.According to the results of in vitro and in vivo experiments,γ-Aminobutyric acid(GABA),the metabolic molecules from intestinal bacteria,was screened and applied to prevent highfat diet induced fatty liver in broilers.1.Effect of NaB on alleviating fructose induced NAFLD in micePrevious studies have reported that sodium butyrate(NaB)has preventive and therapeutic effect on high-fat induced NAFLD in mice,but whether it can alleviate fructose induced NAFLD has not been reported.Therefore,based on the establishment of NAFLD model in mice induced by fructose drinking water,this study explored its alleviating effect on hepatic steatosis and metabolic disorders by gavage of NaB.Sixty male mice were first divided into two groups: control group(C,20 mice)and fructose group(F,40 mice).C group drank tap water and F group drank water containing 30%fructose(w/v)for 8 weeks.After establishing the fatty liver model,F group mice were divided into two groups: sodium butyrate group(B)mice were gavaged with 200 mg NaB / kg BW,and F and C group mice were gavaged with equal volume of saline once a day for 6 weeks.Fructose drinking water decreased food intake and increased water intake(P < 0.05),but did not affect total energy intake and body weight(P > 0.05),NaB treatment had no significant effect on food intake,water intake,energy intake and body weight(P > 0.05).Oral glucose tolerance test(OGTT)showed that NaB can alleviate the decreased glucose tolerance caused by fructose(P < 0.05).The results of H&E,oil red O staining and TG content detection showed that after 8 weeks of fructose drinking,the hepatic lipid deposition and the plasma glucose level increased significantly(P < 0.05).After 6-week treatment,NaB significantly reduced the lipid deposition,steatosis and the content of TG in the liver(P < 0.05).The results of blood biochemical test showed that compared with F group,the concentrations of glucose and uric acid in plasma of group B mice decreased significantly(P < 0.05).The results of real-time q PCR showed that compared with C group,the expression abundance of genes related to fatty acid synthesis and transport in F group was significantly upregulated(P < 0.05),β-oxidation key gene CPT1α was significantly down-regulated(P< 0.05);Compared with F group,the expression of genes and proteins related to fatty acid oxidation(PPARα and CPT1α)in group B were significantly up-regulated(P <0.05).The results suggested that NaB can significantly alleviate increased hepatic lipid deposition and steatosis induced by fructose.This effect may be achieved by enhancedβ-oxidation in the liver.2 Effects of NaB on intestinal barrier,microbiota and portal vein metabolites in NAFLD miceThe liver and intestine originate from the endoderm,and they are closely related in anatomy and physiological function.This section was aimed to investigate whether NaB alleviated NAFLD mice was related to the intestinal mucosal structure,microbiota and metabolites in portal vein plasma.The results showed that there was no significant difference in the epithelial mucosa and gene expression of tight junction in ileum and colon,moreover,the levels of lipopolysaccharide and diamine oxidase in portal vein plasma did not show significant difference(P > 0.05).It suggested that fructose and NaB did not affect the intestinal mucosal structure and permeability in NAFLD mice.Compared with group F,the portal vein blood fructose content in C and B groups decreased significantly(P < 0.05),but there was no significant change in the expression of key genes involved in fructose metabolism and transport in small intestinal mucosa(P > 0.05),suggesting that the effect of NaB on reducing fructose may occur in the intestinal cavity.16 S r RNA gene sequencing showed that fructose treatment significantly changed the microbial composition in cecal content.Ace index increased significantly in F and B groups,but Shannon index increased significantly only in B group(P < 0.05).At the phylum level,the average Bacteroidete abundance in F group was lower than that in C and B groups(P < 0.05);At the genus level,NaB treatment alleviated the reduction of Lactobacillus,Bifidobacterium and Desulfovibrio abundance caused by fructose(P < 0.05),and NaB significantly increased the abundance of Roseburia(P < 0.05).Metabolomic analysis of portal vein plasma showed that the plasma differential metabolites between group F and group B were mainly amino acids,including L-glutamate,L-arginine and γ-Aminobutyric acid(GABA),etc.The pathway impact analysis of differential metabolites showed that the disturbed metabolic pathways were involved in amino acids metabolism.These results suggested that NaB can improve the intestinal dysbiosis caused by fructose and increase the abundance of beneficial bacteria,the intestinal microbiota remodeled by NaB may have a positive effect on hepatic TG metabolism by affecting amino acid metabolism.3.Effect of fecal microbiota transplantation(FMT)on liver lipid metabolism in NAFLD mice and screening of key microbial metabolitesIn order to further explore whether NaB can alleviate NAFLD through microbiotaliver axis,we performed FMT experiment.The remaining mice in C,F and B groups were used as donor mice for providing stools as transplant materials.Fifty 6-week-old C57BL/6J male mice were randomly divided into 5 groups as recipient mice.The grouping is as follows: CC: tap water + feces from group C,FC: fructose + feces from group C,FF: fructose + feces from group F,FB: fructose + feces from group B,FM:fructose + sterilized feces from group B.FMT was performed by gavage once a day for6 weeks.H&E,oil red O staining and TG content detection showed that hepatic lipid deposition and steatosis in FB group were significantly lower than those in FF and FM groups(P < 0.05).Compared with FF and FM groups,the levels of plasma fructose and uric acid in FB group were significantly lower(P < 0.05),the blood glucose concentration tended to decrease(0.05 < P < 0.1).RT-q PCR results showed that compared with FF and FM groups,the gene expression of SCD1,CD36 and FATP4 in FB group were significantly down-regulated,while gene and protein expression of PPARα and CPT1α were up-regulated(P < 0.05).The detection of short chain fatty acids in cecal contents showed that the butyric acid level decreased in fructose-exposed mice,but compared to the other furctose-exposed mice the butyric acid level significantly increased in FB group(P < 0.05).16 S r RNA gene sequencing showed that FMT significantly affected the composition of gut microbiota in each group.Compared with CC group,ACE index in FC,FF and FM groups significantly increased,Shannon index in FB,FC and FF groups significantly increased(P < 0.05).At the phylum level,the average abundance of Bacteroidetes in FF and FM groups decreased compared with CC and FB groups.Compared the differential bacteria between donor and recipient groups,we found that both NaB and stools of NaB-treated mice could significantly improve the abundance of Roseburia and Lactobacillus in fructose-exposed mice(P <0.05).Metabolic analysis portal vein plasma showed that there were great differences between FF and FB groups,and most of the disturbed pathways were still involed in amino acids metabolisms.NaB and stools of NaB-treated mice significantly decreased the levels of glutamate,arginine,uric acid,and increased GABA levels in fructoseexposed mice(P < 0.05).Correlation analysis showed that Lactobacillus abundance was negatively correlated with plasma uric acid,glutamate,fructose,hepatic TG contents,and positively correlated with plasma GABA contents(P < 0.05).Gene abundance analysis of cecal contents showed that the expression of glutamate decarboxylase(gad B)in FB group was significantly higher than that in FF group(P <0.05).In conclusion,the gut microbiome remodeled by NaB increased the hepatic fatty acid oxidation and intestinal butyric acid level in fructose-exposed mice,decreased portal vein blood fructose level,hepatic lipid deposition,and increased gad B in intestinal microbitoa.It suggested that microbial metabolite GABA may have positive effect on preventing hepatic lipid deposition.4 Mechanism of GABA alleviating fructose-induced lipid deposition in mouse hepatocytesIn order to explore the direct effect of GABA on fructose-induced lipid deposition in hepatocytes,we used mouse normal hepatocyte cell line AML12 to perform in vitro studies.10 m M fructose treatment for 12 or 24 h did not affect the cell viability(P >0.05),but significantly promoted lipid deposition(P < 0.05);62.5 μM – 250 μM GABA could significantly inhibit fructose-induced TG deposition in cells(P < 0.05).Oil red O staining showed that 125 μM GABA can significantly reduce the lipid deposition caused by fructose.Compared with the Con group,fructose treatment significantly down-regulated gene expression of CPT1α and PPARα,while the gene and protein expression of CPT1α and PPARα were increased in FRU + GABA group(P < 0.05).Compared with Con group,fructose treatment significantly decreased the protein expression of GABAB receptor in hepatocytes,while the addition of GABA could significantly increase the expression of GABAB,and the addition of GABAB receptor agonist baclofen could alleviate the lipid deposition of hepatocytes caused by fructose(P < 0.05).These results suggested that GABA can significantly inhibit the fructose induced lipid deposition on hepatocyte,which may be achieved by activating GABAB receptor and fatty acid oxidation.5 Effects of GABA on fatty liver and intestinal microbitoa in broilersFatty liver is a common metabolic disease in poultry,which has become one of the main causes of non infectious death in cage chickens.In order to explore whether GABA can prevent fatty liver in broilers,90 1-day-old white feather broilers were randomly divided into three groups: low-fat diet group(Con),high-fat diet group(HF)and high-fat diet + GABA group(FG,100 mg / kg diet).The experimental period was40 days.The results showed that compared with Con group,at 40 day the body weight of broilers in HF and FG groups increased significantly,moreover,the abdominal fat weight and serum TG content significantly increased in HF group compared with Con and FG groups(P < 0.05).H&E staining of liver tissue sections showed that there were a large number of lipid droplets and obvious steatosis in HF group,while the staining results of FG group were similar to that of Con group.The content of hepatic TG in HF group was significantly higher than that in Con and FG group(P < 0.05).RT-q PCR showed that GABA treatment significantly up-regulated hepatic PPARα and CPT1αgene expression compared with Con and HF groups(P < 0.05).High-fat diet did not affect the short chain fatty acids(SCFAs)concentrations in cecal contents,but the levels of propionic acid and butyric acid in FG group were significantly higher than that in HF group,moreover,GABA supplementation alleviated hepatic oxidative stress induced by high-fat diet,decreasing MDA content and increasing the activity of GSHPx and CAT in the liver(P < 0.05).NMDS analysis of intestinal microbiota showed that the intestinal microbial communities of FG and Con groups were highly coincident,but separated from HF group,and the abundance of Bacteroidetes increased significantly(P < 0.05).At genus level,compared with HF group,the abundance of Romboutsia and Ruminococcus torques group decreased while the abundance of Barnesiella increased in Con and FG groups(P < 0.05).Spearman correlation analysis showed that hepatic TG content negatively correlated with the relative abundance of Barnesiella,Romboutsia and Ruminococcus torques group,abdominal fat weight positively correlated with the abundance of Ruminococcus torques group.In conclusion,GABA can significantly reduce serum and hepatic TG content caused by high-fat diets,and increase the SCFAs concentrations in cecal contents.GABA can promote the expression of β-oxidation genes in liver and significantly alleviate the intestinal dysbiosis in broilers fed high-fat diet.The increase of the abundance of beneficial bacteria Barnesiella is related to the decreased hepatic TG content. |
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