| Objective:In this experiment,the acute liver injury(ALI)rat model was established by Thioacetamide(TAA).The preventive and therapeutic effects of compound probiotic preparation(lived combined Bacillus subtilis and Enterococcus faecium enteric-coated capsules LCBE)on acute liver injury were observed.The changes of fecal intestinal flora were detected by 16 S rDNA and serum metabonomics were detected by LC-MS respectively,and analyzed the correlation,then analyzed the control mechanism to provide theoretical basis for its treatment of acute liver injury.Methods:The 36 male SD rats were randomly divided into three groups: normal control group(Nor),ALI group(TAA),intervention group(TAA+LCBE),with 12 rats in each group.The rats in ALI group and intervention group were intraperitoneally injected with TAA of 200 mg / kg body weight dose on the 6th and 7th day of the experiment respectively,and the rats in control group were intraperitoneally injected with saline of the same amount.At the same time,from the first day to the end of the experiment,the intervention group was given 200 mg / kg body weight dose of LCBE once a day for 8days,and the other two groups were given the same amount of normal saline for 8 days.On the 9th night after skipping meals,blood was collected from the abdominal aorta under anesthesia.ALT,ALB and TBIL were detected by ELISA to reflect liver function and D-lactic acid to reflect intestinal barrier.Serum metabolomics and KEGG enrichment were determined by liquid chromatography mass spectrometry.Rat liver was retained for HE staining.At the same time,fresh feces from the distal end of the colon were collected under aseptic operation for 16 S rDNA to detect changes in intestinal flora.Pearson's statistical method was used to analyze the correlation between different flora and different metabolites.Results:1.Changes in liver function in each group: compared with the control group,the ALT?TBIL were significantly increased and ALB was significantly decreased in the ALI group(P < 0.05).Compared with the ALI group,the ALT and TBIL in the intervention group tended to decrease,and albumin tended to increase,with no significant difference(P > 0.05).2.Changes in intestinal barrier function of rats in each group: D-lactic acid significantly increased in the ALI group compared with the control group(P < 0.05),and the difference was statistically significant.significantly decreased after LCBE treatment(P<0.05),the difference was statistically significant.3.Pathological results of liver HE staining in rats in each group: compared with the control group,the structure of liver plate in ALI group was destroyed,and some areas of liver cells showed balloon-like degeneration,punctate and eosinophilic necrosis,expansion of hepatic sinuses and infiltration of inflammatory cells.In the intervention group,there was a small amount of inflammatory cell infiltration in the hepatic lobule manifold area,a slight dilatation of the central vein and congestion,and a small amount of hepatocyte punctate necrosis around.4.Sequencing results of rat feces 16 S rDNA amplifiers:4.1 The analysis of alpha diversity index suggested that the treatment of LCBE couldsignificantly increase the diversity of intestinal flora and enrich the intestinal flora inrats with acute liver injury.4.2 Distribution of intestinal flora in rats:4.2.1 The relative abundance of species at the level of phylum suggested that the top5 intestinal dominant phylum of the three groups of rats were Firmicutes?Bacteroidetes?Proteobacteria?Verrucomicrobia and Actinobacteria,but the proportion in the intestinal flora of each group is still significantly different.4.2.2 The species analysis of phylum level difference indicated that compared with the control group,the Firmicutes increased in the ALI group and the Bacteroidetes decreased(P<0.05);after treatment,the Bacteroidetes abundance of the intervention group increased and the Firmicutes abundance decreased(P > 0.05).4.2.3 The analysis of species differences at genus level indicated that Compared with the control group,the abundance of Butyricimona?Bacteroides?Muribaculum?Phascolarctobacterium?Parabacteroides?Alistipes decreased significantly in ALI group,while that of Romboutsia and Lactobacillus increased significantly(P < 0.05).After LCBE treatment,the abundance of Butyricimona and Bacteroides increased significantly,while Romboutsia and Lactobacillus decreased significantly(P < 0.05).5.Results of liquid chromatography-mass spectrometry of serum metabonomics in rats:5.1 The results of quantitative analysis,PCA and PLS-DA showed that the expression of metabolites in the three sample groups had good homogeneity and significant difference among the groups.5.2 The analysis of differential metabolites suggested that LCBE could correct the significant increase of glycine goose deoxycholic acid ? glycine cholic acid ?diethanolamine concentration and the significant decrease of erucic acid ?calcitriol ?phenylacetic acid?canine quinolinic acid?1-methylhistidine and trimethylamine oxide concentration in ALI group,and the corrected level had no significant difference compared with the control group(P>0.05).5.3 The results of metabolic pathway analysis indicated that the metabolitesregulated by LCBE were mainly involved in lipid metabolism and amino acid metabolism.6.The multi-group association analysis suggested that the correction of1-methylhistidine concentration by LCBE in ALI rats was positively correlated with the increase of Butyricimonas and Bacteroides,and the correction of erucic acid and calcitriol was positively correlated with the increase of Butyricimonas.Conclusion:1.Acute liver injury induced by TAA in rats has obvious damage to liver tissue?function and intestinal barrier,while LCBE can play a role in prevention and treatment for ALI..2.The early intervention of LCBE can balance the intestinal microecology of acute liver injury,and regulate the diversity of intestinal flora,?the expression of beneficial bacteria?the function of intestinal barrier and inflammation.3.13 different metabolites,such as glycocholic acid and glycocholic acid,may be potential biomarkers of TAA induced acute liver injury in rats.4.LCBE has a positive regulatory effect on the key metabolites in serum of ALI rats,mainly including lipid metabolism and amino acid metabolism pathway related metabolites.5.LCBE can regulate part of intestinal flora to normalize the metabolomics abnormalities of acute liver injury. |
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