Features Of Omics Of Patients With Non-Alcoholic Steatohepatitis(NASH) And The Mechanism Of Action Of Probiotics Improving Health Of Mice With NASH

Non-alcoholic fatty liver disease（NAFLD）,as the hepatic manifestation of the metabolic syndrome,is a global health issue.With the improvement of people’s living standards and changes in dietary structure,the incidence of NAFLD has been rising rapidly in recent years.NAFLD refers to a wide range of diseases from steatosis to nonalcoholic steatohepatitis（NASH）,featuring as excess lipids accumulation in the liver.NASH is critical for the progression of NAFLD,linking to steatosis,hepatocyte damage,and inflammation infiltration.NASH renders the risk of scarring or fibrosis,and ultimately leads to cirrhosis and hepatocellular carcinoma.However,the pathogenesis of NASH remains unclear.Lacking effective treatments for NASH makes lifestyle interventions as a main approach to prevent the development of the disease.In this study,we investigated the features of omics of patients with non-alcoholic steatohepatitis（NASH）and the mechanism of action of probiotics improving health of mice with NASH using the combination of gut microbiome and metabolome.Objectives:1.To elucidate the signature of differential metabolites and gut microbiome of patients with NASH.2.To investigate the consistency of the metabolome and microbiome of NASH model of mouse with those of patients with NASH.3.To clarify the safety and probiotic potential of Lactiplantibacillus plantarum LPJZ-658（CGMCC No.22908）improving health of mice with NASH,and investigate the mechanism of action of LPJZ-658.Methods:1.This study enrolled 52 patients with NASH and 32 healthy volunteers.Plasma and fecal samples were collected from those patients and healthy volunteers.The plasma samples were assayed using UHPLC-Q-Orbitrap/MS to obtain plasma metabolic profiles and related data.The signature of differential metabolites of patients with NASH were screened using principal component analysis（PCA）and orthogonal partial least-squares discrimination analysis（OPLS-DA）.The metabolic pathways involved in differential metabolites was analyzed using KEGG pathway enrichment.2.The differences of intestinal flora between those patients and healthy volunteers were investigated using 16 S r RNA gene high-throughput sequencing,thereby revealing intestinal flora relevant to the improvement of health of patients with NASH.3.The safety of Lactiplantibacillus plantarum LPJZ-658 was evaluated using genome sequencing,bioinformatics analysis,hemolytic activity assay,antibiotic susceptibility assay,and in vivo acute oral toxicity test.In addition,the probiotic properties of Lactiplantibacillus plantarum LPJZ-658 were further investigated using acid and bile salt tolerance tests,hydrophobicity and self-aggregation tests,and antimicrobial tests.4.The NASH model of mouse was induced using the combination of Western diet combined with low-dose carbon tetrachloride（WD/CCl₄）treatment for 12 weeks.LPJZ-658 was administrated to those mice by gavage at 1×109CFU/mouse/day for the last 4 weeks.The indices of lipid metabolism and inflammation damage in serum and liver tissues were detected by ELISA.The pathological features of liver tissue were analyzed using by H&E and Sirius Red staining.The gene expression levels of lipid metabolism,inflammatory and fibrosis were analyzed using RT-PCR and immunoblot.Differential metabolites and gut microbiome of those mice were analyzed using LC-MS and 16 S r RNA gene high-throughput sequencing Results:1.A total of 128 differential metabolites and several metabolic pathways relevant to NASH,such as primary bile acid biosynthesis,sphingolipid metabolism,and arginine biosynthesis,were obtained in the plasma of patients with NASH and healthy volunteers.2.NASH induced dysbiosis of the intestinal flora in the fecal from the patients with NASH: the species richness and diversity were significantly reduced in these fecal samples generally.In terms of phylum,compared with healthy volunteers,the patients with NASH had the increased abundance of Proteobacteria,Bacteroidota,and Fusobacteriota and the decreased abundance that of Firmicutes.In the light of genus,compared with healthy volunteers,the patients with NASH had genera with increased abundance（Escherichia-Shigella,Bacteroides,Klebsiella,Fusobacterium,Veillonella,Streptococcus,and Enterobacter）and those with decreased abundance（Faecalibacterium,Blautia,Eubacterium＿coprostanoligenes＿group,Dorea,UCG-002,Ruminococcus,Butyricicoccus,Coprococcus,NK4A214＿group,Lachnospiraceae＿NK4A136＿group,Incertae＿Sedis,Eubacterium＿eligens＿group,and UCG-005）.The function predicted using KEGG database showed the top five enriched metabolism-related pathways（membrane transport,carbohydrate metabolism,biosynthesis of other secondary metabolisms,tissue repair,and protein translation）.3.Lactiplantibacillus plantarum LPJZ-658 was characterized,with 3.26 Mbp of whole genome length,44.83% of GC content,3,254 ORFs with biological functions,a bile salt hydrolase（BSH）with 70.4% identity,and a secondary metabolite gene cluster consisting of 51 genes.Lactiplantibacillus plantarum LPJZ-658 exhibited non-toxic and non-hemolytic activity and was sensitive to a wide range of antibiotics tested.Lactiplantibacillus plantarum LPJZ-658 also exhibited tolerance to acid and bile salts,high hydrophobicity and self-aggregation,as well as excellent antimicrobial activity against gastrointestinal pathogens.4.Lactiplantibacillus plantarum LPJZ-658 significantly alleviated liver injury,steatosis,fibrosis,and inflammatory damage in the NASH model of mouse.NASH was associated with metabolism pathways revealed by serum metabolomics,purine metabolism,glycerophospholipid metabolism,linoleic acid metabolism,and primary bile acid biosynthesis.Lactiplantibacillus plantarum LPJZ-658 significantly regulated the levels of bile acid metabolism-related products in the serum.Moreover,LPJZ-658 significantly ameliorated NASH-induced intestinal flora dysbiosis,significantly increasing the abundance of Firmicutes and Actinomycete at the phylum level and decreasing the abundance of Oscillibacter and Mucispirillum at the genus level.Conclusions:1.NASH affects multiple metabolic pathways,including primary bile acid biosynthesis,sphingolipid metabolism,and arginine biosynthesis;2.The gut microbiome of NASH patients is disturbed;3.The NASH model of mouse and patients with NASH have the consistency and reproducibility of bile acid metabolism and gut flora;4.Lactiplantibacillus plantarum LPJZ-658 possesses the safety and probiotic properties;5.Lactiplantibacillus plantarum LPJZ-658 ameliorates non-alcoholic steatohepatitis in the NASH model of mouse by modulating bile acid metabolism and intestinal flora disorders.