| BackgroundThe intestinal flora is the most complex and important micro-ecological system in the human body,which provides nutrition and regulation for the human body.The intestinal epithelium develops and functions to promote the development and maturation of the immune system and to protect the host from attack by foreign pathogens.Intestinal flora can induce both innate and acquired immune responses.For example,maturation of intestinal lymphoid tissue must be mediated by bacterial and intestinal epithelial cells and dendritic cells,thereby regulating T cells and B cells.Systemic lupus erythematosus(SLE)is a multi-system involving autoimmune connective tissue disease.Intestinal microbes are associated with autoimmune diseases.Immune disorders in SLE patients and changes in the body's environment and intestinal barrier function lead to intestinal microecological disorders.However,previous studies mainly use reverse transcription-PCR(RT-PCR),denaturing gradient gel electrophoresis(DGGE)and other molecular biological methods and biochips to study intestinal flora.These methods have some disadvantages.They have a low detection of flux,can not accurately reflect the abundance of microorganisms,can not detect trace amounts of microbes,and can not explore unknown microorganisms.This study aimed to use the next-generation sequencing technology Illumina PE250/PE300 sequencing stereotyped bacterial 16S rRNA V3 region in the stool samples,while using quantitative PCR to analyze the dominant intestinal flora to fully elucidate the intestinal flora of SLE patients,therefore to reveal the relationship between SLE and intestinal microecology.MethodsIn this study,we analyzed the characteristics of the intestinal flora and its correlation with disease activity in patients with newly diagnosed and referral patients with systemic lupus erythematosus and healthy control individuals.The 16SrRNA-based Illumina PE250/PE300 sequencing and Fluorescent Quantitative PCR were used to analyze the characteristics of the patient's intestinal microflora,and the biomarkers of intestinal microflora in patients with systemic lupus erythematosus were searched.Results1.In the SLE patients enrolled in this study,90%were female patients aged 16-68 years old.They were all newly diagnosed patients in the active period of disease.The levels of complement C3 and C4 in SLE patients decreased significantly,and the newly diagnosed patients were significantly lower.In the referral patients,more than 50%of patients had strong ANA titers.2.There was no statistically significant difference in the intestinal flora between the newly diagnosed and the referral SLE patients.Overall,compared with the healthy controls,the intestinal beneficial bacteria Bifidobacterium and B/E values of SLE patients were significantly lower(P<0.05),while E.jejuni and the harmful bacterium Enterobacteriaceae were significantly increased(P<0.001).3.Enterococci in the intestine of SLE patients were positively correlated with the SLE disease activity score(SLEDAI)(R=0.41,P=0.0197).4.Proinflammatory cytokines IL-1?(interleukin-1?)and IL-lra(interleukin-1 receptor antagonist)in plasma of SLE were significantly increased compared with healthy controls(P<0.001);immunoregulatory factors IL-10(Interleukin-10)(P<0.05),IL-12(Interleukin-12)(P<0.01)levels were significantly higher than those of healthy controls;cytokines with multi-function and pleiotropic effects TNF-a(tumor necrosis factor a)was significantly higher in SLE patients than in healthy controls(P<0.05);however,IL-17(Interleukin-17),which plays an important role in SLE,was not significantly different between SLE patients and healthy controls(P>0.05).5.A total of 2,843,401 valid sequences were obtained in 5,58 samples(1,269,735 valid sequences were obtained for healthy control samples,with an average of 48,836 sequences per sample;1,573,666 valid sequences were obtained for SLE patient samples,averaging 49,177 sequences per sample).The sobs,chao,and ace indices reflecting the richness of the community were slightly higher in the SLE patients than in the healthy controls,but there was no significant difference between the two groups(P>0.05).The shannon and coverage indices reflecting the diversity of the communities were significantly greater in the SLE patients(P<0.05).The simpson index was significantly higher in the SLE patients than in the healthy controls(P<0.05).The diversity of intestinal flora in SLE patients was changed.6.At the OTU(operational taxonomic unit)classification level,the difference between the SLE patients and the healthy controls was significantly greater than the intra-group difference,with statistical significance(P=0.001).At the level of genus classification,the degree of dispersion between individuals in the SLE patients was significantly greater than that in the healthy controls,and there was a large difference between individuals in the intestinal flora.The classification of the intestinal microbiota shows that Bacteroidetes and Firmicutes account for more than 85%of the total sequence and is the absolute dominant flora in the gut microbiota.Compared to the healthy controls,the levels at the gate,the proportions of Bacteroidetes,Firmicutes,Proteobacteria,and Fusobacteria in the intestinal flora of SLE patients were not significantly different,and the proportion of actinomycetes was significantly increased(1.14%vs 0.15%,P=0.0497).At the department level,the rumen bacteria and desulfovibriobacteria in the intestinal flora of the SLE patients were significantly reduced,while the proportion of Enterococci was significantly increased(4.88%vs 0.0004%,P<0.01);at the genus level,the intestinal flora of the genus Sartiphaea and Plavococcus were significantly reduced,while the genus Veillonella(2.15%vs 0.16%,P<0.01)and Enterococcus(4.88%vs 0.0004%,P<0.01)were increased significantly.7.Based on the multi-level discriminant analysis,it was found that from the family level to the genus level,there were 41 species with significant differences in abundance in the SLE patients,and the genus Enterococcus and Veillonococcus were significantly different in the SLE patients(P<0.05),and the dominant influence on the abundance difference can be considered as the main difference between the SLE patients in this experiment.ConclusionsThe proportion of Enterobacteriaceae in the intestinal tract of SLE patients was significantly increased,but the proportion of Bifidobacterium in the beneficial bacteria was significantly reduced,resulting in a significant decrease in the B/E value of intestinal colonization resistance.These changes in the structure of the dominant intestinal flora led to intestinal tract.The imbalance of bacterial flora affects the intestinal mucosal immune function and leads to immune dysfunction,which may be related to the occurrence and development of SLE disease.The diversity of intestinal microflora in SLE patients had some changes;and the difference between the SLE patients and the healthy controls was significantly greater than the difference within the SLE patients,and the experimental reliability was higher.There were large differences in the intestinal flora between the SLE patients.The proportion of potential pathogenic bacteria such as Veillonella and Enterococcus in the intestinal tract of SLE patients was significantly increased,and the proportion of colonic resident Campylobacter was significantly reduced.Changes in the structure of these bacteria may be associated with complications in SLE patients.Concurrently related to the degree of damage to the gastrointestinal tract and the different conditions of kidney involvement.Enterococcus and Veilella have a greater influence on the abundance of different species and can be considered as the major differential group of SLE patients.At the same time,plasma pro-inflammatory and immunomodulatory cytokines were significantly increased in SLE patients,further demonstrating immune dysfunction in SLE patients.The above results provide some evidence for the future diagnosis of the SLE disease and the possible pathogenesis,intestinal micro-ecological therapy and cytokine therapy. |
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