Study On The Association Of Intestinal Flora And Trimethylamine Oxide With Primary Liver Cancer

ObjectiveThe aim of this study was to investigate the changes of intestinal flora and TMAO levels in patients with primary liver cancer by comparing the characteristics of intestinal microecological expression profiles and the levels of trimethylamine N-oxide(TMAO)in patients with hepatitis B cirrhosis combined with primary liver cancer with those in normal subjects,patients with chronic hepatitis B and patients with hepatitis B cirrhosis,and to further explore the flora closely related to TMAO levels,so as to provide new ideas for the pathogenesis,diagnosis or treatment of primary liver cancer.MethodsA total of forty study subjects were selected for this study,including ten patients with chronic hepatitis B(hepatitis group,CHB),ten patients with hepatitis B cirrhosis(cirrhosis group,LC),and ten patients with primary liver cancer complicated with primary hepatitis B cirrhosis at first diagnosis(liver cancer group,HCC),and the healthy control group(HC)was the healthy population of the physical examination center at the same time.Stool and plasma samples as well as clinical data were collected from all study participants.The abundance and composition of gut microbiota in stool samples were detected by macrogenome sequencing,and the plasma TMAO levels were measured using high-performance liquid chromatography-tandem mass spectrometry(LC-MS/MS).Alpha diversity was calculated to evaluate the diversity of gut microbiota within the samples and Beta diversity was used to measure the similarity of gut microbial composition between samples.LEfSe(Linear discriminant analysis(LDA)Effect Size)analysis was used to screen for gut microbiota with significant differences between groups.The difference in TMAO levels between the groups was analyzed using one-way ANOVA.Correlations between plasma TMAO levels and different intestinal flora were analyzed using Spearman’s rank correlation method.Statistical analysis of the data was performed using SPSS 24.0 and R(4.2.2)software.Analytical results were visualized using R(4.2.2)software.Results1.The baseline clinical characteristics of each group,such as age,gender,BMI,family history of hepatitis B,smoking history,and alcohol consumption history,showed no statistically significant differences(P>0.05).With regards to clinical examination indicators,the differences between the four groups were statistically significant(P<0.05)for the ALBI grade,HBV DNA,platelet count,albumin,alanine transaminase,and gamma-glutamyl transferase,while other testing indicators showed no statistically significant differences(P>0.05).2.Intestinal flora diversity analysis:(1)The results of Alpha diversity analysis revealed that the Shannon index was significantly lower in the cirrhosis group compared to the healthy control group(P=0.043).Compared to the healthy control group,the liver cancer group exhibited a significant decrease in both the Shannon index and Chao 1 index(P=0.032,P=0.014).The results of Alpha diversity analysis indicated that the intestinal microbial diversity was reduced in the cirrhosis and liver cancer groups.(2)The beta diversity analysis indicated a minimal overlap among the groups of healthy control,hepatitis,cirrhosis,and liver cancer,with further examination using Anosim(Analysis of similarities)confirming notable dissimilarities in the gut microbial structure among the four groups(P=0.028).3.Analysis of the species composition of the intestinal flora:(1)At the phylum level,the dominant organism(relative abundance>1%)in the four groups were mainly Bacteroidetes,Firmicutes),Proteobacteria,and Verrucomicrobia.(2)At the family level,Bacteroidaceae,Enterobacteriaceae,Lachnospiraceae,and Tannerellaceae were the dominant organism in the four groups.Veillonellaceae was the dominant organism in the liver cancer group.(3)At the genus level,Bacteroides,Faecalibacterium,and Megamonas were the dominant organism in the four groups.Pseudomonas was the dominant organism in the liver cancer group.(4)At the species level,Bacteroidesvulgatus,Bacteroides fragilis,Faecalibacterium Prausnitzii,Bacteroides ovatus,Megamonasfuniformis,Bacteroides uniformis,Bacteroides dorei,and Bacteroides thetaiotaomicron were the dominant organism in the four groups.4.A total of 22 significantly different species were identified by LEfSe analysis in the four groups(P<0.05,LDA>2).Among them,Veillonellales was significantly enriched in patients in the liver cancer group.Klebsiella pneumoniae was significantly enriched in the cirrhosis group.Lachnospira eligens,Lachnospira,and Bacteroides caccae were significantly enriched in the hepatitis group.Twelve microorganisms,including Firmicutes,Lachnospiraceae,Ruthenibacterium lactatiformans,and Ruthenibacterium were significantly enriched in the healthy control group.5.Analysis of TMAO levels:TMAO levels were significantly lower in patients in the hepatitis and cirrhosis groups compared to healthy control group(P=0.001,P=0.007);Patients in the liver cancer group demonstrated significantly elevated TMAO levels in comparison to those in the hepatitis group(P=0.032).6.Analysis of the association between intestinal flora and TMAO:The analysis showed that TMAO was positively correlated with Firmicutes(r=0.45,P=0.004),Ruthenibacterium lactatiformans(r=0.44,P=0.004),Lachnospiraceae(r=0.41,P=0.009),and Collinsella(r=0.38,P=0.016).7.Analysis of the association between TMAO and clinical indicators:the results showed that there was a correlation between TMAO and albumin,alanine aminotransferase,aspartate aminotransferase,total bilirubin,direct bilirubin,urea,creatinine,uric acid and platelets(P<0.05).Conclusions1.The intestinal microbial diversity and structure in patients with primary liver cancer exhibited a decrease when compared to the healthy population.This was reflected by a reduction in the levels of beneficial bacteria such as Lachnospiraceae,Collinsella,and Anaerobutyricum hallii.2.Compared to healthy populations,patients with chronic hepatitis B and hepatitis B cirrhosis exhibited a reduction in their TMAO levels.And Patients with primary liver cancer showed an increase in their TMAO levels as compared to those with chronic hepatitis B,possibly due to alterations in their intestinal flora.