Analysis Of The Intestinal Microbiome From Patients With Rheumatoid Arthritis And Screening For Pathogenic Bacteria

Background Rheumatoid arthritis(RA)is a systemic autoimmune disease predominantly characterized by chronic,progressive,and aggressive arthritis.With the gradual development of the disease,if not effectively treated,the patient will eventually have joint deformity,loss of function,and even disability.Rheumatoid arthritis is distributed throughout the world with tens of millions of patients,the incidence racial differences,all ages can be onset,the peak age of 30-50 years old,with more women than men.Studies have shown that the occurrence of RA are closely associated with genetic factors and environmental factors,but the pathogenesis has not been determined.In recent years,more and more attention has gradually shifted to the correlation between RA and intestinal microbiome that is one of the environmental factors.Objectives This research was focus on analysis the distribution characteristics of intestinal microbiome in patients with RA,isolating and culturing the specific gut bacteria from the fecal of RA patients and identification the bacteria strain.The rat model of arthritis was established and the screened characteristic bacteria were used for gavage to rat.Observe the effect of the characteristic bacteria on the arthritis in rat and determine the pathogenicity of the selected bacteria.In order to further study the intestinal microbiome affect the mechanism of rheumatoid arthritis lay the foundation.Methods 1.The feces of patients with rheumatoid arthritis and healthy human were collected.The PCR-DGGE(Denaturing Gradient Gel Electrophoresis)was utilized to analysis the main bacteria composition of patients with rheumatoid arthritis.To identify the bacteria strains by sequencing the 16 S r RNA V3 region,identify the characteristic bacteria.2.Using the selective culture medium,the main characteristic bacteria were isolated from RA patients’ feces,and proliferated in right liquid medium.To identify the bacteria strains by sequencing the 16 S r RNA V3 region.3.Rats were treated with complete Freund’s adjuvant to establish an arthritic-sensitive animal model.The characteristic bacteria isolated from the stool of RA patients were intragastrically administered to rats.According to the multiple indexes,confirm the onset of RA disease was induced by the characteristic bacteria.The detection indexes include: the ankle joint morphological changes of the rats were observed by HE staining;the contents of TNF-α and IL-1β in the serum were detected by ELISA.4.The gut flora of RA rats was compared with the gut flora of RA patients to screen out more intestinal pathogenic bacteria associated with RA disease.Results 1.DGGE result showed that the diversity of intestinal microbiota in RA patients was lower than that in health people,and the distribution of bacteria was specific.At the same time,three kinds of potentially pathogenic characteristic bacteria were identified: Enterococcus faecium,Escherichia coli and Lactobacillus bervis.2.The bacteria in feces of RA patients were cultured in vitro by using selective culture medium.Through the 16 S r RNA V3 region sequencing comparison and analysis,the three kind of characteristic bacteria were successfully isolated from the cultured bacteria..3.The three kinds of bacteria were intragastrically administered to rats respectively.Then rats intragastrically administered with Enterococcus faecium and Lactobacillus berivis had joint deformities and even walking disorders.The ankle joint damage was observed by HE staining.The contents of TNF-α and IL-1β in both groups were significantly increased by ELISA,while the changes in the third group were not significant.4.Similar to the gut flora of RA patients,there is an increase of Lactobacillus bacteria in the gut flora.Prevotella genus was detected in the intestinal flora of RA rats.Conclusion Intestinal microbiome in patients with rheumatoid arthritis and healthy group compared with significant differences.Animal experiment confirmed that isolated Enterococcusa faecium and Lactobacillus berivis can induce arthritis in rats.Animal Lactobacillus was detected in the intestinal flora of RA rats,suggesting that the correlation between Lactobacillus with rheumatoid arthritis.