| Introduction: Rheumatoid arthritis(RA)is a chronic,symmetric facet joint involved systemic autoimmune disease,which is the second leading cause of disability in China.Although methotrexate and other anti-rheumatic drugs can alleviate conditions,the disease cannot cure.In addition,such drugs have serious side effects.At present,RA has become one of the major diseases endangering human health,and its etiology is not clear.RA related to bacterial infection with unknown mechanism.In July 2015,the data of metagenomics completed by researchers from Peking Union Medical College,Beijing Genomics Institution etc.revealed that abnormal microbial flora in oral and intestinal tract was one important part in RA pathophysiology and disease control.On March 11,2020,researchers at the university of California,San Diego,school of medicine developed a novel way to identify who is a cancer and what kind of cancer it is by simply analyzing the patterns of microbial DNA present in the blood,published in the journal ?nature?.The presence of microbial DNA in RA blood and synovial fluid is unclear.However,what kind of role does microbial flora play in the environment of in situ focus are still unclear.Our previous studies confirmed that Porphyromonas gingivalis,Prevotella,and Bacteroides were significantly highly expressed in RA.However,how these bacteria colonized in synovial tissue and fluid,and how they participated in the pathogenesis of RA were remaining unknown.To further our study about mechanism of intestinal microbial flora in RA pathogenesis,we selected fibroblast-like synoviocytes(FLS)in RA and established the model of collagen induced arthritis(CIA).Objective:We used Next Generation Sequencing to identify the differences of microbial flora between RA,OA(Osteoarthritis)and Health groups.In addition,to find out,in the meantime,prove the existence of the key microorganism in RA.Then,we established quantitative analysis of Bacteroides,Phorphyomonas gingivalis and Prevotella in blood,feces and synovial fluid of RA,analyzed accurate quantitative of each microbial flora at different sites,in order to provided experimental basis for RA prior diagnosis.For intervention experiments,we mixed three probiotics to assess whether they can improve or alleviate RA disease and to find out the mechanism.It is urgent to identify the mechanism of microorganism in RA and to investigate the efficacy of probiotics as an auxiliary treatment of RA.(1)Analysis of the characteristics of microbial flora in RAMethod: In the first part of this study,85 cases of RA,osteoarthritis(n=21)and health groups(n=43)were selected;the male to female ratio was 31: 152,and the age was in the range of 20-80 years old.Metagenomic sequencing was for study of characteristics of bacteria in fecal sample.16 S ribosomal RNA(16S r RNA)V1-V2 domain sequence was for study of characteristics of bacteria in RA and OA synovial samples,screening out potential biomarkers;correlation analysis of microbiome markers and clinical immune factors(IL-1?,IL-1 ?,TNF-?,LI-17 and IL-6);prediction of the functional genetic constitution of microbial flora.Non-targeted metabolomics screening of microbial metabolites,and The DNA of Prevotella in RA patients was analyzed by real-time fluorescence quantitative PCR,with OA and healthy group as control.Result: Intestinal and synovial sequencing analysis used to identify the imbalance of flora in RA.27 identifiable biomarkers(LDA score > 2,? < 0.05)were identified by LEf Se,the relative abundance in rheumatoid arthritis was significant different.Prevotella copri enriched in RA,while Prevotella copri DNA enriched in SFRA.Short-chain fatty acids,bile acid were involved in the lesions of RA.PCR validation and quantification of Prevotella copri is 1.98ng/?l.Bacteroides: 0.06ng/?l.Conclusion: This study focused on the intestinal tract and synovial fluid to screen the existence of microbial flora and identify those specific bacterial biomarkers Prevotella copri participated in the onset and development of RA.The result of absolute quantification of Prevotella and Bacteroides in different sites revealed that Prevotella acted as pathogenic bacteria in the process of RA.Bacteroides existed in samples from healthy control as a probiotic.The decrease of it in RA samples may due to changes of bacteria structure resulting in gut dysbiosis.These bacteria used as an auxiliary treatment of RA prior diagnosis since they detected in RA samples.(2)Changes in biological activity of RA fibroblast-like synoviocytes(RA FLS)intervened by probioticsMethod: Three probiotics purchased from company,including Lactobacillus and Bifidobacterium.These probiotics mixed into probiotic preparation.To discuss the mechanism,probiotic preparation used to intervene RA FLS,including primary culture of synovial cells,and subculture of synovial cells;cell reproductive capacity tested by MTS,cell capacity tested by Wound healing,Transwell tested cell invasion ability,and expression difference of inflammatory factors tested by enzyme-linked immunosorbent assay(ELISA)kit.Result: Through the intervention with probiotic preparation by gradient dilution method,the best concentration was 800 pg/?l.Follow-up experiments conducted after three generations of primary culture of synovial fibroblast.It found that proliferation ability of synovial fibroblasts influenced after 24 hours of direct stimulation of synovial fibroblasts by probiotics(tested by MTS).The migration capacity of synovial fibroblasts was impeded;the number of synovial fibroblasts was significantly different;the expression of IL-17 was significantly different(P<0.05)(the expression of IL-17,IL-6 and IL-1 was tested by ELISA).Conclusion: Probiotics could offer protection to chronic inflammation of RA FLS.Probiotic preparation could significantly reduce the proliferation,migration and invasion of RA FLS,which provided a new way for clinical use of probiotics to alleviate RA symptom.(3)Study on intestinal flora characteristics and mechanism of mice with collagen-induced arthritis intervened by probioticsMethod: Lactobacillus and Bifidobacterium mixed in vitro for gavage to DBA/1 mice for 6 weeks from 2 weeks before the primary immunization.We recorded the arthritis score.In addition,we analyzed the changes of intestinal flora in collagen intervention group,control group,collagen group and collagen resistance group.Computed tomography(CT)used to test bone erosion in mice model group.Hematoxylin-eosin staining(HE)was used to observe the changes of synovial spatial structure in mice.ELISA used to test the changes of the level of serum inflammatory factors IL-17,IL-6 and IL-1 in mice.To explain the mechanism of microbial flora in RA,this part of study focused on whether probiotics intervention can alleviate RA disease.Result: Intragastric administration of probiotics applied to the collagen intervention group;the control group divided into three groups: complete control group,collagen group,and collagen resistance group with six mice in each group.CT showed that bone erosion was not severe in collagen intervene group,which treated with probiotics.No obvious structural change in synovial tissue of joint of mice observed by H E staining.ELISA tested the expression of IL-17,IL-6 and IL-1 in serum of mice,and it revealed that collagen intervention group had a tendency to alleviate the level of inflammatory factors,but there was no significant difference.However,the changes of intestinal flora in mice were significant.Conclusion: At present,the research on microbial flora provided a very good entry point for the study of many diseases.However,the study of microorganisms involved in the pathogenesis of RA and OA was not deep enough,which impeded the exploration of more effective and specific therapeutic methods.With the continuous development of molecular biology technology and means,we can have a deeper understanding of the relations between microorganisms and RA,to provide a broad prospect for the treatment of RA.Based on the above scientific suppositions and previous studies,the possible mechanism of microorganism to stimulate the occurrence,and development of RA includes that bacteria could regulate the differentiation of immune T cells through molecular simulation,induce the production of IL-17 and other inflammatory cells and factors to attack synovial and cartilage tissue,which changed the permeability of mucosa,destroyed mucosal barrier and immunity,and leaded to RA autoimmune disease.Based on “bacteria-intestine-joint”,we hypothesized that gut microbial flora,its metabolites first caused mucosal symbiosis,impeded autoimmune tolerance,and autoantibodies then led to the imbalance of T cells subset,and the release of inflammatory cytokines,finally induced the occurrence of rheumatoid arthritis by waterfall effect of cascade amplification. |
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