| Non-alcoholic fatty liver(NAFLD)is currently considered one of the most common diseases worldwide.The accumulation of lipids in liver cells is associated with a variety of toxins,drugs,and diseases.However,the most common liver steatosis in recent years has been associated with high-fat diets,dysbacteriosis,and hyperlipidemia,but the exact cause is not fully understood.Non-alcoholic fatty liver and alcoholic fatty liver are extremely similar,and only if there is a long-term drinking history can they be distinguished.Because patients with NAFLD produce more endogenous ethanol than healthy people,some researchers suspect that the similarity between the two is not accidental.For this reason,we speculate that endogenous ethanol may be one of the main causes of NAFLD.However,in patients with NAFLD,the concentration of endogenous ethanol produced by the gut bacteria is almost undetectable in the blood.To explain these conflicting results,the ethanol high-yield Klebsiella pneumoniae strains isolated from patients with ABS/NASH was given intragastrically to induce NAFLD,which explained the important role of endogenous ethanol in the formation of NAFLD.It has been initially explored that high-fat-induced and ethanol high-yield strains-fed mice will promote the formation of fatty liver and further produce significant hepatic fibrosis,and study the changes in fecal metabolites.1.Biological characteristics of Klebsiella pneumoniae high-yield strains(1)In this study,we collected stool samples from different periods of the patients and performed sequencing analysis of the 16 S rDNA amplicon.We found that the abundance of Klebsiella bacteria in the proteobacteria of the feces was approximately 900 times higher than that of the healthy controls,and the proteobacteria in the host intestine is closely related to the fluctuation of its blood alcohol content.In addition,yeast content in fecal samples is negative,excluding yeast interference.This indicates that the elevated blood alcohol content of the patient is closely related to the klebsiella.85 alcohol-resistant colonies were isolated from the feces.They were identified as including Klebsiella pneumoniae,Providencia alcalifaciens,and Escherichia coli.22 strains of ethanol-producing strains with an ethanol production capacity of 20 mmol/L were selected.Combining the results of amplicon sequencing analysis of fecal microbiota,two Klebsiella pneumoniae strains W14 and TH1 with high ethanol production were selected,which can be cultured in YPD medium containing 2% glucose for 8 hours,with an ethanol production capacity of 47.73 and 43.47 mmol./L(aerobic conditions),22.7 and 25.2 mmol / L(anaerobic conditions),far higher than the K.pneumoniae standard strain ATCC2146(15.6 mmol/L,5.4 mmol/L).(2)W14,TH1 strain was stained with 2% phosphotungstic acid and observed under transmission electron microscopy(TEM).Their shapes are short rods with thicker capsule.And they can grow on MacConkey culture medium,and a bubbling circle of bile salt precipitates around the colony,indicating that it can be fermented with lactose;and the smooth surface of the colonies can be observed on the BHI medium.Compared with the standard strain ATCC2146,W14 and TH1 grew faster and could enter a stable phase after 6 hours.The average growth rate was about 1.5 times that of the standard strain ATCC2146,and the biofilm could be observed in approximately 16 hours by laser scanning confocal microscope.(3)Proteomic analysis revealed that the differential proteins are closely related to the mixed acid pathway(2,3-butanediol pathway).Klebsiella pneumoniae oxidizes glucose and other substances through the mixed acid pathway.The final metabolite is 2,3-butanediol,with ethanol,acetic acid,and lactic acid being bypass products.The differential proteins including alcohol dehydrogenase,acetolactate decarboxylase,diacetyl reductase and acetoin reductase,and other key enzymes may increase the yield of ethanol,and the strains grow rapidly and may have overflow metabolism phenomenon.This can further explain the high ethanol production capacity of W14 and TH1.2.Establishment and study of non-alcoholic fatty liver model induced by Klebner pneumonia in mice(1)Mice fed with W14,TH1 strain stimulated by glucose solution,detected the presence of blood ethanol at 2 hours and peaked at 4 hours(W14 111.16 mg / L,TH1 75.65 mg / L).Histopathology showed that hepatic lobule structure was disordered in W14,TH1 and ethanol-fed mice,degeneration of hepatic adipocytes,and fatty degeneration gradually increased from 4 weeks to 8 weeks,with partial granule degeneration and focal necrosis,infiltration of inflammatory cells in portal area and its surrounding area.After 8 weeks of gavage,the serum AST and ALT contents and the liver MDA and TG contents in the experimental group were significantly higher than those in the control group.This indicates that lipid peroxidation in the liver is higher,which reflects a large degree of hepatocyte damage and a higher content of fat deposits in the liver,consistent with histopathological results.Compared with ethanol-fed mice,FLD(fatty liver)mice induced by these strains showed the same histopathological findings,suggesting that the endogenous ethanol produced by the strain may lead to liver disease similar to long-term ethanol intake(Fatty liver).(2)Fecal metabolomics studies revealed that six major metabolites,some of which were present in ethanol-fed mice.These metabolites were mainly associated with ethanol metabolism,fat metabolism,nitrogen metabolism,the original bile acid biosynthesis,butyrate metabolism,and the up-regulation of key pathways related to reverse endogenous cannabinoid signaling and ethanol production.Ethanol high-yield Klebsiella strains produce a large amount of endogenous ethanol and 2,3-butanediol,and these alcohols are difficult to completely convert into acetaldehyde and acetic acid,or gut-liver axis produces fatty acid glycerides by non-enzymatic metabolic reactions and leads to subsequent liver tissue damage and hepatic steatosis.Therefore,2,3-butanediol fermentation pathway is considered to be the main molecular mechanism for producing endogenous ethanol and increasing subsequent inflammation.3.Using gene expression microarray to screen genes related to non-alcoholic fatty liver diseaseTo further explore the progression of hepatic steatosis induced by high-yield strains of ethanol,we used gene expression microarray to construct liver transcriptome data of strain-fed(W14,TH1),ethanol-fed,and negative control-fed mice.And through enrichment analysis of differentially expressed genes,we found that genes involved in fatty acid metabolism,ethanol metabolism,immunity and inflammatory factors,cancer-related factors,and Oflr gene family were upregulated,including the hsd,ugt,and cyp gene families involved in fatty acid metabolism.By studying differential gene expression profiles in the liver,we found that steroid hormone biosynthesis,unsaturated fatty acid biosynthesis,PPARα,retinol metabolism,arachidonic acid metabolism and other pathways increase free fatty acid production and cause mitochondrial dysfunction,this promotes increased production of ROS in the liver to reflect liver damage caused by endogenous ethanol and ROS production.At the same time,we also found that genes associated with ethanol catabolism are up-regulated and enriched,suggesting that excess ethanol is cleared in the liver to resist its accumulation in the body,which is consistent with the results of AFLD disease progression.4.A preliminary study of high-fat diet synergistically induced non-alcoholic fatty liver and fecal metabonomics in miceBased on the ethanol-producing strain(W14)-induced nonalcoholic fatty liver disease in mice,a high-fat diet may lead to dysbacteriosis,further aggravating liver damage,and the liver cannot effectively decompose the intestinal-derived compounds resulting from the disordered flora.As a result,it accumulates in the body and induces further progression of NAFLD to hepatic fibrosis.At the same time,the fecal metabolites of mice in W14 group and control group were analyzed comprehensively.A total of 216 metabolites were detected,of which 35 metabolites were significantly different between the W14 group and the control group.Compared with the control group,there were 24 kinds of metabolites in the feces of W14 mice,mainly short-chain fatty acids,amino acids,and unsaturated fatty acids.And these elevated differential metabolites were analyzed by KEGG enrichment and a total of 16 metabolic pathways were found to be significantly up-regulated,including(1)aminoacyl-tRNA biosynthesis;(2)biosynthesis of ornithine-derived alkaloids;(3)propionate metabolism;(4)butyric acid metabolism;(6)Biosynthesis of tropine,piperidine,and pyridine alkaloids;(7)thioglycoll biosynthesis;(8)arginine and valine metabolism;(9)β-alanine metabolism;Glutathione metabolism;(11)phenylalanine metabolism;(12)biosynthesis of alkaloids derived from the shikimate pathway;(13)phenylpropanoid biosynthesis;(14)biosynthesis of secondary metabolites;(15)Purine metabolism;(16)Biosynthesis of terpenes and steroids.Combining modern diets with high-fat and sedentary lifestyles can lead to abnormal metabolism of the intestinal flora and accumulation of endogenous ethanol from the gut can increase liver burden and induce nonalcoholic fatty liver risk,Through the fine measurement of the overall structure and function of the intestinal flora,regulating the recovery of the intestinal microenvironment to a normal state is a key measure to prevent disease,and it is also a key area for future research. |
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