| Nonalcoholic fatty liver disease(NAFLD)refers to a condition characterized by excessive fat accumulation in the liver in the absence of certain of cause,such as alcohol consumption.In recent years,the NAFLD incidence of the word shows an explosive growth,and people still have the limitation in understanding of it due to the mechanism complexity and the difficulty of treatment.Until now,no approved drugs for NAFLD have been for marketing.The common clinical therapeutics include drugs for lipid-lowering,weight-loss and liver-protecting,but the effectiveness and safety are still lacking in clinical research.Previous studies have shown that the "live-gut axis" plays a key role in the onset and development of NAFLD,which may shed light on targeting intestinal flora to prevent NAFLD.In addition,many NAFLD related population-based cohort demonstrate that the significant changes in the diversity and composition of intestinal flora in subjects,especially characterized by abundance reduction of Faecalibacterium prausnitzii.It has been reported that the standard strain of F.prausnitzii(A2-165)can alleviate liver steatosis with unknown mechanism.So,we aim to optimize the screening method to obtain a certain number of F.prausnitzii,then based on their physiological,biochemical and genomic characteristics 11 strains were selected to investigate the mitigation effect on NAFLD;Finally,the mechanism of its effect was clarified by means of animal validation test,metabolome,transcriptome and molecular biology method.The main results and conclusions are as follows:(1)39 strains of F.prausnitzii was screened by the modified "negative screening" method from 17 human feces,which improved the isolation efficiency up to 75.43%.And the physiological and biochemical and comparative genomic analysis of isolates were carried out.It showed that F.prausnitzii can utilize fructose,cellobiose,maltose and inulin,but not arabinose,raffinose,ribose,mannitol,xylose and sorbose;The optimum p H of F.prausnitzii was 6.0～7.0,and its growth was significantly inhibited when the bile salt concentration was0.1%;The range of butyric acid production of F.prausnitzii is 0.43～9.52 m M,showing great difference in butyric acid production capacity.The pangenome and phylogenetic analysis of93 F.prausnitzii genomes(36 genomes obtained from this study and 57 genmoes downloaded form NCBI database)showed that the genomes of 93 F.prausnitzii had open pangenome,including 27849 pangenomes and 472 core genomes,mainly involving translation,ribosome structure and biosynthetic function;The phylogenetic tree of F.prausnitzii is mainly clustered in 6 branches(A,B,C,D,E and F),and the characteristics of these branches have no relation to the host region and disease status.Moreover,safety evaluation of F.prausnitzii showed that there were 407 prophages(including 73 complete prophages)annotated in 93 F.prausnitzii genomes,and the number was related to the disease status of the host,but not to the region upon the genome level;In addition,169 virulence factors and 23 antibiotic resistance gene entries were noted.Most gene entries were specific to some strains,and only a few were common.(2)Mice model was established with high-fat diet for 12 weeks.The body weight,pathological characteristics of liver and adipose tissue,lipid metabolism,insulin resistance and other indicators were investigated to evaluate the alleviating effect of 11 F.prausnitzii isolates and type strain A2-165 on NAFLD,and the changes of intestinal flora diversity and composition were analyzed.The results showed that the strains CCFM1204,LB8,ZF21,PL45,and A2-165 significantly reduced the body weight,liver index,leptin content,liver oxidative stress and inflammation level after intervention,and increased the adiponectin content,and improved liver steatosis,adipose tissue inflammation,blood lipid disorder,pancreatic insulin resistance,while the other strains had no significant mitigating effect on NAFLD,indicating that F.prausnitzii had strain heterogeneity in relieving NAFLD.Principal component analysis indicated that CCFM1204 had the best effect on NAFLD among the supplemented F.prausnitzii strains.Moreover,CCFM1204 was 58.82% and 17.62% higher than the standard strain A2-165,respectively,in liver pathological score and HOMA-IR index,showing a great potential to alleviate NAFLD;At the same time,it was found that strain HW29 had the worst response to NAFLD.CCFM1204 changes the intestinal flora of NAFLD mice β diversity,not α diversity,reducing the abundance of harmful strains Erysipelatoclostridium and Tyzzerella,and increasing the abundance of beneficial bacteria Lactobacillus and Dubosiella;It also regulates and restores the expression of metabolic pathways that are beneficial to the host,including glutathione metabolism,tryptophan metabolism,riboflavin metabolism,etc.(3)Using HW29 as a negative control,the possible mechanism of relieving NAFLD upon CCFM1204 was analyzed by means of metabonomics,transcriptomics and molecular biology.The analysis of untargeted metabolome in mice serum showed that CCFM1204 significantly enriched indoles,lysophosphatidylcholines,carnitines,bile acids and unsaturated fatty acid compounds in mice;With HW29 group as the control,indole-3-propanoic acid(IPA),indole-3-pyruvic acid,indole-3-lactic acid(ILA),linoleic acid and glycocholic acid were obtained as the potential material.The expression of ILA,indole-3-formaldehyde(I3C)and indole acrylic acid(IA)in feces of mice in all group had no significant difference,and CCFM1204 and HW29 could significantly increase the content of indole-3-acetic acid(IAA),but the significantly increased content of IPA is only found in CCFM1204,indicating that IPA might be a characteristic substance of CCFM1204;There was no significant difference in the ability producing IAA,ILA,I3 C and IA between two strains,but only CCFM1024 could produce IPA,which further confirmed that IPA was the substance to alleviate NAFLD.Transcriptome analysis show that CCFM1204 mainly enriched the lipid metabolism pathway and the pathway related to xenobiotics in liver;Protein interaction network analysis shows that constitutive androstane receptor(CAR)and liver X receptor α(LXRα),Fatty acid synthase(FASN),sterol regulatory element binding protein-1(SERBP1)and other genes related to lipid metabolism and response to xenobiotics are core genes.In summary,CCFM1204 may attenuate NAFLD by producing IPA activatting CAR receptors in the liver to inhibit hepatic lipogenesis and gluconeogenesis.(4)CINPA1,an inhibitor of CAR receptor,was used to interfere with NAFLD mice to verify whether CCFM1204 could alleviate NAFLD by activating CAR receptor in liver.The results show that IPA and CCFM1204 could significantly improve the phenotype of NAFLD mice,but strains HW29 and CINPA1 had no such effect.In addition,after the intervention of IPA and CCFM1204,the expression of CAR receptor in the liver was significantly up-regulated,and the proteins or genes related to lipid metabolism were significantly down regulated,including SREBP1,FASN,and acetyl CoA carboxylase α(ACC)and stearoyl CoA desaturase 1(SCD1),but there was no significant change in the expression of liver nuclear factor 4 α(HNF-4α),indicating that the activation of CAR may affect more adipogenesis in the liver than gluconeogenesis. |
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