Study On The Changes Of Intestinal Flora In Patients With Chronic Hepatitis B Syndrome Of Spleen And Stomach Dampness-Heat And Liver Depression And Spleen Deficiency And Their Association With Methylation-Related Proteins

Objective:To observe the changes of intestinal flora in patients with chronic hepatitis B spleen-stomach damp-heat syndrome and liver-stagnation and spleen-deficiency syndrome,and to explore the relationship between intestinal differential flora and chronic hepatitis B methylation-related proteins.Methods:Using the case-control study scheme,12 chronic hepatitis b patients were recruited from the outpatient department of pixian traditional Chinese medicine hospital in chengdu,including 5 cases of spleen-stomach damp-heat syndrome,7cases of liver-stagnation and spleen-deficiency syndrome,and the control group consisted of 6 healthy subjects;16SrDNA high-throughput sequencing technology was used to observe the changes in the diversity and abundance of the three groups of samples,and to compare the structural characteristics of each group.The automatic biochemical analyzer was used to detect liver function indexes.16 SrDNA high-throughput sequencing technology was used to observe the changes in the diversity and abundance of the three groups of samples,and to compare the structural characteristics of each group.Liver function index was detected by automatic biochemical analyzer.Elisa method was used to detect the concentrations of methylation-related proteins DNMT1,MECP2,E-cad and P53.The Pearson correlation coefficient was used to analyze the relationship between intestinal differential flora and DNMT1,MECP2,E-cad and P53.Results:1.A total of 1980988 high-quality Clean reads were detected from the three groups of 18 samples in this study,and 8352 OUTs were obtained.These OTUs belonged to 11 phylum,19 classes,25 orders,45 families,89 species,and 146 genus.Among them,healthy control group（3399）> spleen and stomach damp heat group（2654）> liver stagnation and spleen deficiency group（2299）.There were 263 crossover OTUs in the three groups.469 crossover OTUs between the healthy control group with the spleen and stomach dampness group,and 445 crossover OTUs between the the healthy control group with the liver and spleen deficiency groups.There were a total of 419 OTU in the spleen and stomach damp-heat group and the liver depression and spleen deficiency group.At the same time,the number of specific OTU in each group was 2748 in the healthy control group,1674 in the spleen and stomach damp-heat group,and 2053 in the liver and stomach damp-heat group.2.On the level of the phylum: the three groups of samples were dominated by Bacteroides and Firmicutes.Compared with the healthy control group,the abundance of Bacteroides in the spleen-stomach dampness group and the liver-stagnation and spleen-deficiency group decreased,The abundance of Firmicutes,Proteobacteria and Fusobacterium increased,and the B/E value decreased.Compared with the healthy control group,the chronic hepatitis B syndrome group Plevotobacter,Sutterella,Dorea,Lachnospiraceae_UCG-008,Ruminiclostridium₉,Allisonella,Anaeroglobus increased in abundance,and the Victivallis abundance decreased.Catenibacterium and Comamonas were detected in the spleen-stomach damp-heat group,and Tyzzerella was detected in the liver-stagnation and spleen-deficiency group.Compared with the spleen and stomach dampness group,Actinobacillus was detected in the liver stagnation and spleen deficiency group,while Catenibacterium and Intestinibacter disappeared.According to LEfSe analysis,the spleen-stomach damp-heat group and the liver-stagnation and spleen-deficiency group were significantly different on the Proteobacteria.3.Compared with the healthy control group,the ALT,AST,GGT,TBIL,DBIL and TBA of chronic hepatitis B spleen and stomach dampness syndrome and liver stagnation and spleen deficiency were significantly increased,and the HBV-DNA copy number was higher than the lower limit of detection（>1.0E+3）.（P<0.05）;Compared with the spleen-stomach damp-heat group,AST and GGT were decreased in the liver-stagnation and spleen-deficiency group（P<0.05）.4.Compared with the healthy control group,the concentrations of DNMT1,E-cad,P53 and MeCp2 in the chronic hepatitis B spleen and stomach dampness group and the liver stagnation and spleen deficiency group were significantly increased（P<0.05）.Compared with the spleen and stomach dampness group,the liver stagnation and spleen deficiency group E-cad,P53,DNMT1 and MECP2 increased,and the difference was not significant（P>0.05）.5.DNMT1 was strongly associated with B/E in the spleen-stomach damp-heat group（r=0.772）,E-cad,P53,MeCp2 and B/E were not significantly associated（r<0.3,P>0.05）,and E-cad in the liver-stagnation and spleen-deficiency group B/E showed a weak correlation（r=0.601）,and there was no correlation between DNMT1,P53,MeCp2 and B/E（r<0.4,P<0.05）.6.There was a weak correlation between DNMT1 and Proteobacteria in the spleen-stomach damp-heat group（r=-0.589）,E-cad,P53,MeCp2 and Proteobacteria were not correlated.There was no correlation between E-cad,P53,MeCp2 and Proteobacteria in the liver-stagnation and spleen-deficiency group（r<0.5,P>0.05）Conclusion:1.There is a significant difference in intestinal microbes between patients with chronic hepatitis B and healthy people;2.Chronic hepatitis B spleen and stomach dampness heat syndrome and liver stagnation and spleen deficiency syndrome patients have their own dominant genus;3.The progress of chronic hepatitis B is related to DNA methylation.DNMT1,E-cad,MeCp2 and P53 may also participate in the process of chronic hepatitis B while affecting host methylation;4.There is immune dysfunction in the body of chronic hepatitis B,intestinal micro-ecological disorders and methylation modification,B/E value,significantly different bacteria Proteobacteria and methylated proteins DNMT1,E-cad,MeCp2,P53 are related,It is very likely that the intestinal flora interferes with the expression of certain genes by methylation modification,and thus plays a role in chronic hepatitis B,but its specific mechanism needs further study.