The Impact Of BMI On The Gut Microbiota Characteristics In Non-alcoholic Fatty Liver Disease Based On Metagenomics

BackgroundNon-alcoholic fatty liver disease(NAFLD)refers to a group of diseases characterized by fatty degeneration of hepatocytes without heavy drinking[1].NAFLD includes two pathological features with different prognosis:simple steatosis without inflammation or fibrosis and non-alcoholic steatohepatitis(NASH),the latter of which may progress to fibrosis,cirrhosis and hepatocellular carcinoma(HCC)[2].NAFLD is one of the important causes of chronic liver disease.It has become an important public health issue,affecting 20%-30%of the global population[3],and is on the rise worldwideThe pathogenesis of NAFLD is not completely clear.It is thought to involve in complex connections between genetic susceptibility variation,dietary and environmental factors,insulin resistance and changes in the gut microbiota[4].The interaction between these factors leads to dyslipidemia and excessive lipid accumulation in hepatocytes,which ultimately leads to the development of NAFLD.More and more evidences showed that the human gut microbiota is related to a variety of diseases,such as inflammatory bowel disease,colorectal cancer,Parkinson’s disease,uipolar disorder,and so on[5].The liver and gut are closely related in anatomical position and function.Intestinal microorganisms play an important role in liver inflammation,injury,chronic fibrosis,and tumor development through gut-liver circulation[6].Studies have shown that the imbalance of gut microbiota will lead to intestinal mucosal barrier destruction,increased intestinal permeability,thereby promoting bacterial translocation and endotoxemia.The lipopolysaccharide(LPS)produced by the gut microbiota activates the TLR4/NLRP3 inflammatory pathway,thereby increasing the content of liver fat and inflammation[7].A multi-country collaborative study by Hoyles and his colleagues comprehensively analyzed the multiomics characteristics of non-diabetic obese women with fatty liver[8],putting forward that the richness of patient’s gut microbiota were decreased.Besides,the lipid metabolism,aromatic and branched-chain amino acid biosynthesis capabilities were enhanced,and the expression of genes such as liver immune inflammation is enhanced,and fecal microbiota transplantation experiments have shown that gut microbiota can cause fatty liver.The incidence of NAFLD usually parallels with the prevalence of obesity,type Ⅱdiabetes and metabolic syndrome[9].However,it is obvious that not all obese people will develop NAFLD.More importantly,NAFLD also exists in nonobese individuals,and its histopathological manifestations are similar to those of obese NAFLD patients[10].Globally,the reported prevalence of nonobese NAFLD varies widely,ranging from 3%to 30%[11].This difference may be attributed to differences in the choice of research subjects,diagnosis methods,and the lifestyle and eating habits of specific populations.The prevalence data of nonobese NAFLD in the East and the West are not directly comparable,at least partly because the cut-off value of the body mass index(BMI)that defines obesity is different.For Asians,it is recommended that a BMI greater than 25kg/m2 is defined as obesity,while it is recommended that a BMI greater than 30kg/m2 be defined as obesity for western people[12].Compared with obese NAFLD,what are the characteristics of gut microbiota in nonobese NAFLD population,this question remains largely unexplored.ObjectiveTo understands the impact of BMI on gut microbiota in NAFLD population,this research aimed to study the gut microbial community structure of nonobese NAFLD and obese NAFLD populations based on the metagenomic sequencing data of gut microbiota,and explore the important species related to nonobese NAFLD through bioinformatics methods.Methods1.A total of 183 NAFLD patients were selected from Chinese Healthy Integrative-omics Project(CHIP)of the First Affiliated Hospital of Zhengzhou University and their fresh stool samples were collected.The selected NAFLD subjects were in compliance with the 2010 "Non-alcoholic Fatty Liver Disease Diagnostic Criteria" revised by the Fatty Liver and Alcoholic Liver Disease Group of the Chinese Medical Association Hepatology Branch.Exclude viral hepatitis,alcoholic liver disease,autoimmune liver disease and other special cases that can cause fatty liver,and exclude patients with other serious diseases.The subjects were divided into two groups with BMI=25kg/m2 as the cut-off value.Among them,there were 52 nonobese NAFLD patients with BMI<25kg/m2,and 131 obese NAFLD patients with BMI≥25kg/m2.In addition,31 obese healthy controls(nonobese HC)and 20 obese healthy controls(obese HC)were randomly selected from the CHIP project cohort and stool samples were collected.2.The demographic characteristics,laboratory examination results,and imaging examination reports of the research subjects were collected from the hospital’s electronic medical record system.The past history,medication history,and drinking history of the research subjects were obtained through the questionnaires.3.After collecting a fresh stool sample from the research subject,quickly froze the sample and stored it in a-80℃ refrigerator.Through DNA extraction,sequencing library construction,metagenomic sequencing,and sequencing data processing,the information of gut microbiota was obtained.4.The,"vegan" package of R 4.0.2 software(http://www.R-project.org/)was used to analyze the alpha diversity of the gut microbiota and presented it with the following parameters:Observed species,Shannon index,Simpson index,Gini index.The R 4.0.2 "phyloseq" package was used for principal coordinate analysis(PCoA)to calculate the β diversity of the gut microbiota,that is,the distance between samples.5.The R 4.0.2 software was used to analyze statistical data.The Wilcoxon test was used to compare the continuous variables between the two groups,and the Fisher exact test was used to compare the categorical variables between the two groups.P<0.05 was regarded as statistically significant.Results1.A total of 183 non-alcoholic fatty liver patients were included in this study,including 52 nonobese NAFLD patients and 131 obese NAFLD patients.There was no statistical difference in gender and age between the nonobese NAFLD group and the obese NAFLD group(P>0.05).The diastolic pressure,waist circumference,hip circumference of the nonobese NAFLD group were significantly lower than the obese NAFLD group,and high-density lipoprotein was significantly higher than the obese NAFLD group,the difference was statistically significant(P<0.05).Compared with the nonobese NAFLD group,leukocytes and uric acid in the obese NAFLD group were significantly increased(P<0.05).In addition,there were no statistical differences in fat attenuation parameter and liver stiffness measurement detected by Fibrotouch between the two groups(P>0.05).2.The Shannon index and Simpson index of the gut microbiota in the nonobese NAFLD group were significantly higher than the obese NAFLD group(P=0.0096,P=0.0071),and the Gini index was significantly lower than the obese NAFLD group(P=0.029),the difference was statistically significant.The nonobese NAFLD group and the obese NAFLD group were consistent in the number of observed species and there was no statistical difference.The results showed that there were differences in species diversity between the nonobese NAFLD group and the obese NAFLD group,and the species diversity of the nonobese NAFLD group is higher than that of the obese NAFLD group.3.Based on Hellinger Distance(HD),Jense-Shannon Divergence(JSD),Bray-Curtis Distance and Pearson-Coefficient Distance,at the level of the first principal component,the nonobese NAFLD group and the obese NAFLD group can be completely separated(P<0.05).Combining the four types of distances between samples,it was suggested that the nonobese NAFLD group and the obese NAFLD group have differences in the composition of major gut microbiotas.4.Through the analysis of species composition,the results suggested that the relative abundance of 28 species including Bacteroides ovatus,Ruminococcus bromii,Bacteroides uniformis,Roseburia inulinivorans,Bacteroides fragilis,Bacteroides thetaiotaomicron were significantly increased in the nonobese NAFLD group.The relative abundance of 8 species including Haemophilus parainfluenzae decreased significantly.The 28 species enriched in the nonobese NAFLD group were from Firmicutes,Bacteroidetes,Actinobacteria and Proteobacteria.The eight species enriched in the obese NAFLD group were from Firmicutes,Bacteroides and Proteobacteria.5.Through the correlation analysis of gut microbiota and clinical indicators,the results suggested that 14 species including Bacteroides cellulosilyticus enriched in nonobese NAFLD were significantly negatively correlated with BMI(P<0.05),and 10 species including Clostridium asparagiforme were significantly positively correlated with HDL.In addition,the abundance of Ruminococcus callidus,a species enriched in obese NAFLD,was significantly positively correlated with fasting blood glucose(rho=0.2457,P=0.0008)and fat attenuation parameter FAP(rho=0.1467,P=0.048).6.Further analysis of gut microbiota characteristics between nonobese healthy control and obese healthy control groups showed no significant difference in a diversity between the two groups.By comparing the characteristic gut microbiota between NAFLD and healthy population groups,it was suggested that the influence of BMI on gut microbiota of the body was heterogeneous.ConclusionThis study used the metagenomic sequencing method to conduct a comprehensive analysis of the gut microbiota structure of the non-alcoholic fatty liver disease population.The study found that BMI has a significant impact on the characteristics of the gut microbiota of NAFLD.Nonobese NAFLD and obese NAFLD have significant differences in the composition,richness and diversity of the flora.Bacteroides ovatus,Ruminococcus bromii,Bacteroides uniformis,Roseburia inulinivorans,Bacteroides fragilis,Bacteroides thetaiotaomicron and other species are significantly enriched in nonobese NAFLD.The changes in gut microbiota and metabolism products may be involved in preventing the progression of nonobese NAFLD.Our research results provide a theoretical basis for the treatment of NAFLD by regulating the gut microbiota.