| Chapter One Effect of TNF-α antagonist treatment on intestinal flora in patients with rheumatoid arthritisObjective:So far,most of the studies on the analysis of intestinal flora disorder in patients with rheumatoid arthritis(RA)are cross-sectional studies,and there are few literatures on the changes of intestinal flora in patients with RA after drug intervention.The purpose of this study was to explore the intestinal flora disorder in RA patients and the changes of intestinal flora after treatment with tumor necrosis factor(TNF)-α antagonist.Materials and Methods:Thirty one RA patients and 10 healthy family members were enrolled in the outpatient department.Stool samples were collected before and after treatment with TNF-α antagonist.Clinical information such as disease activity and laboratory examination were recorded,and fecal samples of healthy family members were collected at the same time.A total of 62 stool samples of RA patients and 10 family samples were collected for 16S rRNA sequencing.OTU(operational taxonomic unit)clustering,species annotation,α-diversity analysis,β-diversity analysis and LEfSe analysis were used to study the differences of intestinal flora diversity,composition structure,species abundance and enriched flora in fecal samples of patients and their family members,as well as the correlation between species abundance and clinical indicators such as autoantibodies.Results:α-diversity analysis showed that after treatment with TNF-α antagonist,the intestinal flora richness and Chao index in stool samples of patients decreased,and the level in family members further decreased(P<0.05);Simpson index did not change significantly in RA patients before and after treatment,but it increased significantly in family members(P<0.05);Shannon index did not change significantly in RA patients before and after treatment and in family members.There was no correlation between α-diversity and rheumatoid factor(RF),anti-cyclic citrullinated peptide(CCP)antibody titer,duration and body mass index(BMI).β-diversity analysis showed that there were significant differences in the composition and distribution of flora among RA patients before and after treatment and healthy family members,and there was a significant positive correlation between the composition and distribution of flora and the richness index of intestinal flora.After treatment,Proteobacteria and Actinobacteria increased and Bacteroidetes decreased in the intestinal tract of RA patients,which was close to the level of family members.Enterobacteriaceae,Peptostreptococcaceae,Clostridiaceae and Bifidobacteriaceae increased,and the proportion was similar to that of healthy family members;Prevotellaceae,Streptococcaceae and Erysipelatoclostridiaceae decreased.At the species level,Enterococcus caccae and Holdemania filiformis DSM 12042 were significantly enriched before treatment,but decreased significantly after treatment,which was close to the family level.Conclusion:After treatment with TNF-α antagonist,the overall structure of intestinal flora of RA patients showed a gradual dynamic change,and the richness and structure of flora gradually approached the level of healthy family members.Enterococcus caccae and Holdemania filiformis DSM 12042 were enriched before treatment and decreased significantly after treatment,which may be potentially associated with RA status.Chapter Two Effect of TNF-α antagonist treatment on metabolites of intestinal flora in patients with rheumatoid arthritisObjective:At present,most of the research samples involving metabolites and RA are from the synovial fluid,serum and plasma of human or animal.There is almost no literature to directly detect the level of metabolites in fecal samples,and few literature to explore the changes of intestinal metabolites before and after drug intervention.Therefore,we designed this part of the experiment to study the difference of intestinal flora metabolites in fecal samples of RA patients and healthy people,and the changes of intestinal flora metabolites in RA patients after treatment with TNF-α antagonist.Materials and Methods:Thirty one RA patients and 10 healthy family members were enrolled in the outpatient department.Stool samples were collected before and after treatment with TNF-α antagonist.Clinical information such as disease activity and laboratory examination were recorded,and samples of healthy family members were collected at the same time(same as chapter one).After homogenization,centrifugation and drying,non-targeted hydrophilic metabolites,lipid metabolites and targeted short chain fatty acids were detected by liquid chromatography and mass spectrometry.The hydrophilic metabolites,lipid metabolites and short chain fatty acids were analyzed qualitatively and quantitatively.Principal component analysis(PCA),partial least squares discriminant analysis(PLS-DA)and orthogonal partial least squares discriminant analysis(OPLS-DA)were used to screen different hydrophilic metabolites,lipid metabolites and short chain fatty acids.HMDB database was used to identify metabolic markers of intestinal flora.Results:A total of 6477 metabolites were detected in all samples,including 4487 lipid metabolites and 1990 hydrophilic metabolites.After comparison with HMDB database,5535 metabolites could be inferred as class,5478 metabolites could be compared to subclass,1175 metabolites could be accurately compared to secondary map,and 252 metabolites were obtained by differential analysis.There were different metabolite enrichment in fecal samples of RA patients before and after treatment,and RA patients and healthy family members,respectively.Compared with the fecal samples after treatment and healthy family members,fatty acylthiolipids were more abundant before treatment;triglyceride,cholic acid,ethanol and their derivatives were higher before treatment than after treatment.Phosphatidyl ethanolamine,phosphatidyl serine,phosphatidylglycerol,(O-acyl)1-hydroxy fatty acid,phosphatidylcholine,ceramide,sphingomyelin and glucosinolates increased in fecal samples of after treatment and family members;monoglyceride,lysophosphatidylethanolamine and lysophosphatidylglycerol increased only in fecal s of after treatment;yeast sterol,dimethylphosphatidylethanolamine,glycerophosphatidylethanolamine and stigmasterol ester increased only in the family.In the short chain fatty acids,acetic acid,propionic acid decreased after treatment,close to the level of family members;butyric acid,formic acid,isobutyric acid,isovaleric acid and valeric acid did not change significantly.The metabolites enriched before treatment correspond to higher disease activity.Enterococcus caccae,Streptococcus lactis,Holdemania filiformis DSM 12042 and Lachnospiraceae bacterium had the strongest correlation with metabolite levels and clinical indicators.Conclusion:After TNF-α antagonists were used in RA patients,the levels of lipids,hydrophilic compounds and short chain fatty acids were significantly changed.Also,we found that Enterococcus caccae and several other heterobacteria in fecal samples were significantly positively correlated with the levels of metabolites and clinical indicators,suggesting that TNF-α antagonist may exert their drug efficacy by changing the intestinal flora and its metabolites..Chapter Three Effect of Enterococcus caccae on intestinal epithelial cell junctionsObjective:In previous study,we found that Enterococcus caccae(E.caccae)in fecal samples of RA patients had significant difference before and after treatment,and the level after treatment was close to that of healthy family members.At the same time,E.caccae had a strong correlation with the level of metabolites and clinical indicators.Thus,we speculate that E.caccae may be closely related to the disease state of RA,and may affect the intestinal barrier function and mucosal immunity.Therefore,we designed this part of experiment to study the effect of E.caccae on the intestinal epithelial model cell line Caco-2 cell junction through co-culture,so as to preliminarily explore the effect of E.caccae on the intestinal epithelial barrier function.Materials and Methods:The monolayer adherent Caco-2 cells were divided into five groups and co cultured with four concentrations of E.caccae(109/ml,108/ml,107/ml and 106/ml)and non co-cultured respectively.Western blot,RT-qPCR and immunofluorescence were used to detect the location,relative content and mRNA expression of Occludin,ZO-1 and E-cadherin in Caco-2 cells.Results:Western blot analysis showed that the relative contents of Occludin and E-cadherin decreased after E.caccae and Caco-2 cells were co-cultured for 1 hour and washed off E.caccae for another 24-hour incubation.And with the increase of bacterial concentration,Occludin and E-cadherin further decreased.Occludin and E-cadherin also decreased after 1 hour of co-culture and another 48-hour incubation.However,with the increase of bacterial concentration,the further decline of Occludin and E-cadherin contents was not as obvious as that of cultured cells after 24 hours.RT-qPCR mRNA analysis showed that the mRNA levels of Occludin,E-cadherin and ZO-1 did not decrease significantly after co-cultured with E.caccae and a followed 6-hour incubation after E.caccae washed off;after 12 hours of incubation,the expression of E-cadherin mRNA decreased when the bacterial concentration was 106/ml,107/ml,and 109/ml(P<0.05),but Occludin mRNA did not change significantly.ZO-1 mRNA decreased at the bacterial concentration of 106/ml(P<0.05);after 24 hours of culture,the mRNA expression of E-cadherin and Occludin decreased at all bacterial concentrations(P<0.05).ZO-1 mRNA decreased when the bacterial concentration was 106/ml(P<0.05).The results of immunofluorescence showed that the fluorescence intensity of E-cadherin,Occludin and ZO-1 in Caco-2 cells decreased after co-culture with E.caccae.The average fluorescence intensity of Occludin and ZO-1 decreased(P<0.05),and there was no significant difference in the change of E-cadherin.Conclusion:E.caccae reduces the contents of Occludin,ZO-1 and E-cadherin,interferes their expression and localization,and down-regulates the mRNA transcription levels of Occludin,ZO-1 and E-cadherin in Caco-2 cells.Therefore,E.caccae may damage the tight cell junctions,leading to the decrease of intestinal mucosal barrier function and the increase of intestinal permeability,and then may lead to the intrusion of pathogenic microorganisms and their secretions and metabolites into the tissues to activate local or even systemic immunity. |
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