| Objective:To analyze and compare the differences,and research a possible mechanism controlling the changes of intestinal flora among patients with Primary Biliary Cholangitis(PBC)and Sj?gren’s syndrome(SS)through studying the mainly changed characteristics of intestinal flora among these patients.Methods:79 patients admitted to the Department of Rheumatology in the author’s hospital and 30 healthy controls were selected in the study.The patients were divided according to their clinical diagnoses into the PBC Group(n = 38)and the SS Group(n = 41),from which stool specimens were collected.To sequence the V3-V4 region of 16 S rDNA in those stool specimens,a special technology named high-throughput technology was used,and DNAs were extracted for PCR amplification.The products were purified,quantified and homogenized to form a sequencing library.On the illumina novaseq PE250 sequencing platform,the composition and diversity of intestinal flora were analyzed.LEfSe,an analysis method of the question from the view of microbial diversity,was used to compare the differences between the Case Group and the Control Group in the markers of intestinal flora.Combined with the clinical indicators of patients,the SS Group was divided into Active Group and Inactive Group,and the differences in intestinal flora of the two groups were compared.PBC Group was divided into Severe Cholestasis Group and Mild Cholestasis Group,and the changes in bacterial flora of the two groups were compared.Results:1.There was no discrepancy from the view of statistics found in gender,age or BMI of all groups(P > 0.05).2.A total of 6,473,536 high-quality sequences were obtained through sequencing analysis from all the three groups(averaging 58,850 sequences/specimens),among which2,211,304,2,733,525 and 1,528,707 high-quality sequences were obtained from the PBC Group,the SS Group and the Control Group respectively.Additionally,a total of 64,141OTUs(PBC Group,n = 18,243;SS Group,n=19,913;Control Group,n = 25,985)were obtained through cluster analysis with a consistency of 97%.Among them,11,315,12,819 and 17,965 OUTs were specific to the PBC Group,the SS Group and the Control Group respectively.3.Comparison in Alpha diversity of intestinal flora: Chao 1,the difference was so clear that we found that the species and Shannon indices in PBC and SS Group were remarkably lower than that of the Control Group,which was higher than that of the later one,showing statistically significant differences(P < 0.05).No significant difference was found in Chao 1,observed species,Shannon or Simpon indices between the PBC Group and the SS Group(P > 0.05).4.Comparison in beta diversity of the intestinal flora: based on principal coordinates analysis(PCo A),a comparison was made in intestinal microbial community structure,which showed the remarkable differences in intestinal microbial diversity of the above three groups(P < 0.05).5.To make an analysis and a comparison among the species annotation at the phylum level,all the 3 groups were mainly composed of Firmicutes,Bacteroidetes,Actinobacteria and Proteobacteria.At the genus level,they were mainly composed of 10 species.It is shown that the ratio of other two groups was higher than that of the Actinobacteria in SS Group in the pairwise comparison we made at the phylum level on intestinal flora,whereas the proportion of Proteobacteria in the PBC Group was higher than that of the other two groups,showing statistically significant differences(P < 0.05).The remarkable differences are shown to us that the ratio of Bifidobacteria in the SS and PBC Group was reduced in the comparison we made between the Control Group at the genus level(P<0.05).6.Comparisons based on LEfSe analysis and Heatmap: The dominant species in the PBC Group were p-Proteobacteria,c-Gamaproteobacteria,c-Bacilli,f-Enterobacteriaceae,o-Enterobacterales,Collinsella and Shigella,and those in the SS Group were f-Ruminococcaceae,g-Faecalibacterium,p-Gemmatimonadetes,f-ACK-M1 and Xanthomonadales.In the Control Group,Lachnospiraceae,g-Clostridium,Acorus,g-Mitsuokella and g-Hyphomicrobium were the dominant species.7.In Severe PBC Cholestasis Group,the proportion of probiotics(Bifidobacteria)significantly decreased,while that of pathogenic bacteria(Shigella,Corinella and Streptococcus)increased.8.Compared with the Active Group,the proportion of Bifidobacteria was lower in the SS Active Group,while that of Roseburia was higher.Conclusions:1.The intestinal flora diversities and structures among patients with PBC and SS were significantly different from those of the Healthy Control Group,which suggested that chronic inflammation and immune activation caused by disordered floral balance might be one of the pathogeneses associated with both diseases.2.The abundance and diversity of intestinal flora showed no significant difference between the PBC Group and the SS Group,indicating that changes in intestinal flora diversity of both groups were similar,together with comparable flora imbalance characteristics.3.There were many consistent variable signals caused by changes in intestinal flora among patients with PBC and SS.For example,probiotics,Bifidobacterium as well as Lachnospiraceae,Coprococcus and Clostridium can produce one unique acid and short-chain fatty acids in a strange way.Thus,it was reduced remarkably compared with the Control Group(P<0.05).While,it also caused an imbalance in disease-specific flora of the above-mentioned diseases.Actinobacteria among patients with SS increased significantly,while in the case of PBC,Proteobacteria was the one showing a significant increase.4.The dominant species in the PBC Group were more than pathogenic bacteria in the Control Group,and this phenomenon was also found in the Severe Cholestasis Group,suggesting that these bacteria might be important strains involved in the pathogenesis of PBC.5.The proportion of Bifidobacteria in the SS Active Group was lower than that in the Inactive Group,suggesting that the severity of the disproportion of Bifidobacteria might be related to the activity of the disease. |
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