Effect Of Lactulose On The Intestinal Flora And Serum Metabolites In Rats With Acute Liver Injury

Objective:The purpose of this experiment is to establish a rat model of acute liver injury by thioacetamide(TAA),observe the changes of intestinal flora and serum metabolites of the model,and then explore the effect of lactulose on the above observation indexes of the model,so as to provide a new idea for the clinical treatment of acute liver injury.Methods:Thirty six healthy and clean male SD rats were randomly divided into three groups:normal control group(DZ group),model group(ALI group)and lactulose intervention group(RG group),12 rats in each group were fed with common feed,RG Group A was diluted with lactulose warm boiled water at a dose of 7 ml / kg,and group DZ and group Ali were given the same amount of normal saline.Lactulose intervention was given throughout the experiment for 7 days.On the 6th and 7th day of the experiment,rats in RG group and ALI group were injected with TAA(200 mg/kg).On the eighth day of the experiment,the rats in the overnight fasting group were taken blood from the abdominal aorta under anesthesia for the detection of alt,TBIL,ALB,D-lactate and serum metabolites.After the blood was taken,the liver tissue samples were cut for HE staining to observe the pathological changes.The rats were collected by aseptic operation In fresh feces,16 S r DNA sequencing technology was used to study the changes of intestinal microflora structure.Finally,LC-MS was used to analyze the metabonomics of the metabolites in the blood of rats in each group and identify the different metabolites among each group.Results:1.Results of liver function in rats: The level of ALT and TBIL in the ALI group was significantly higher than that in the DZ group(P < 0.05);the level of ALT and TBIL in the RG group was significantly lower than that in the ALI group(P < 0.05);the above indexes in the RG group were significantly higher than those in the nor group(P < 0.05)There were also differences in the two groups(P < 0.05);in the ALI group,ALB was significantly lower than that in the DZ group(P < 0.05);in the RG group,ALB was higher than that in the ALI group,but there was no statistical difference(P > 0.05);in the RG group,there was also a statistical difference(P < 0.05).2.Changes of intestinal barrier function in rats: compared with ALI group,the serum D-lactate level in RG group was significantly lower(P < 0.05);there was no significant difference between RG group and DZ group(P > 0.05).3.Liver pathological results: in DZ group,the structure of liver lobule was complete,there was no congestion in the central vein,the hepatic cord arranged radially around the central vein,and the structure of hepatic sinuses was clear.ALI group: the structure of liver lobule is complete,blood stasis can be seen in central vein,punctate necrosis,focal necrosis and bridging necrosis can be seen in liver lobule,and a large number of inflammatory cell infiltration can be seen in portal area;RG group: balloon like change can be seen in liver cells,punctate necrosis can be seen around some liver lobules,and no focal necrosis can be seen,and the degree of inflammatory cell infiltration is light.There was no fibrosis and obvious hepatocyte regeneration in the three groups.4.HE staining of ileum: in DZ group,the structure of ileum mucosa was clear and complete,the villi were arranged closely and orderly;in ALI group,the ileum mucosa became atrophic and thickened,the villi were arranged sparsely and disorderly,some cells were exfoliated,accompanied with inflammatory cell infiltration.Compared with ALI group,the structure of ileum mucosa in RG group was basically complete,the arrangement of villi was slightly sparse,and the villi was slightly edematous.5.Scanning electron microscopy of ileum: in DZ group,the villi of ileum were broad in shape,compact and regular in arrangement.ALI Group : the villi of ileum were flat,loose and irregular,and some of them were collapsed and damaged.Compared with ALI group,the villi in RG group were plump,dense and regular.6.16 S high-throughput sequencing of fecal flora6.1 Diversity analysis of intestinal flora in experimental rats The results of alpha diversity index analysis: compared with DZ group,the ace index of ALI group decreased,the difference was statistically significant(P < 0.05);compared with ALI group,the ace index of RG group increased,but the difference was not statistically significant(P > 0.05),suggesting that the intestinal microbial richness of rats with acute liver injury decreased,while lactulose could make the intestinal microbial richness of rats with acute liver injury tend to recover.Compared with DZ group,the simpson index of ALI group decreased(P < 0.05).RG compared with the ALI group: the simpson index increased,but the difference was not statistically significant(P > 0.05),indicating that the intestinal microbial diversity of rats with acute liver injury decreased,while lactilose could restore the intestinal microbial diversity of rats with acute liver injury.6.2 distribution of intestinal flora In this experiment,the changes of intestinal microflora in the three groups of rats were mainly concentrated in the following phylum: Firmicutes ? Bacteroidetes ?proteobacteria?Verrucomicrobia and actinobacteria.Group DZ: the intestinal flora was mainly composed of phylum,Bacteroides,accounting for 70% and 18% respectively,followed by verruca,actinomycetes and Proteus;group ALI: the flora was mainly composed of phylum,Bacteroidetes,accounting for 31% and 45% respectively,followed by Proteus and Proteus;RG intestinal flora was also composed of phylum,Bacteroidetes,The abundance of flora was about 38% and 32% respectively,followed by actinomycetes and verruca microflora.The abundance of flora was about 16% and 12% respectively.6.3 analysis of species differences between groups6.3.1 Species analysis of phylum level differences: ALI group compared with DZ group:the proportion of sclerecta in the intestinal flora of rats decreased,while that of Bacteroides increased,the difference was statistically significant(P < 0.05),in addition,the proportion of actinomycete also decreased,but there was no statistical difference(P >0.05);RG group compared with ALI group: the proportion of Actinomycete in the intestinal flora of rats increased(P < 0.05),while sclerecta increased(P < 0.05)The proportion of Bacteroides was higher than that of Bacteroides,but there was no significant difference(P > 0.05).6.3.2 species analysis of genus level differences: comparison of ALI group and DZ group:in ALI group,the abundance of Bacteroides in Bacteroides increased,while that of bifidobacteria in actinomycetes and lactobacilli in pachytenella decreased.Except for bifidobacteria,the above differences were statistically significant(P < 0.05);RG Compared with ALI group,the abundance of bifidobacteria increased(P < 0.05),lactobacilli increased and Bacteroides decreased(P > 0.05).7.Analysis of serum metabonomics Compared with DZ group,the decreased metabolite in ALI group was kynurenic acid,and the increased metabolite was Glycochenodeoxycholic acid(P < 0.05).Compared with ALI group,the change of the metabolites in RG group was contrary to the above results,the increased metabolites were kynurenic acid,the decreased metabolites were Glycochenodeoxycholic acid,the difference was statistically significant(P < 0.05).Conclusion:1.TAA can induce rat model of acute liver injury,lead to liver function damage,pathological changes of liver,decrease of diversity and abundance of intestinal flora,increase of opportunistic bacteria Bacteroides,decrease of beneficial bacteria bifidobacteria and Lactobacillus.2.Lactulose can reduce the degree of liver necrosis,protect liver function,and decrease ALT and TBIL in blood.3.Lactulose can restore the diversity and abundance of intestinal flora in rats with acute liver injury,enrich Bifidobacterium and Lactobacillus,and reduce Bacteroides.4.There are cholestasis and tryptophan metabolism abnormalities in rats with acute liver injury,which are manifested as the increase of conjugated bile acid-Glycochenodeoxycholic acid content,and the decrease of tryptophan metabolite-kynurenic acid content.The above abnormalities may be related to the disorder of intestinal flora.Lactulose may improve the above abnormalities by regulating intestinal flora,reducing Glycochenodeoxycholic acid and increasing the level of kynurenic acid content,so as to improve the liver and intestinal disease.