Research On The Correlation Between Different TCM Syndromes Of Rheumatoid Arthritis And Changes In Intestinal Flora

ObjectiveRheumatoid arthritis（RA）is a chronic systemic autoimmune disease with high disability rate.The pathogenesis of RA is the result of the interaction among gene predisposition,environmental factors and autoimmunity.Gut microbiota,as an important environmental agent,is closely related to the occurrence and development of RA.At present,Western medicine in the treatment of RA has side effects and cannot cure the disease.Traditional Chinese medicine（TCM）has played a unique advantage in the diagnosis and treatment of RA.However the TCM syndrome of RA are complex and lack of objective research.Therefore,this study intends to analyze the change of intestinal flora structure in RA patients with different TCM syndrome types by 16SrDNA high-throughput sequencing technology,comparing with the healthy subjects,so as to provide objective basis for the diagnosis of TCM syndrome of RA,and provide a new idea for TCM diagnosis and treatment of RA.MethodsIn this study,we collected 48 RA patients,including 15 cases of damp-heat impeding syndrome,15 cases of phlegm-blood stasis syndrome,18 cases of liver and kidney deficiency syndrome,and 12 healthy controls.The fecal samples of RA patients and healthy controls were collected.The total DNA in the samples was extracted using a DNA extraction kit,amplified by PCR,and sequenced by IlluminaMiSeq.The results were analyzed by bioinformatics correlation analysis to compare the differences of intestinal flora between RA patients with different syndrome types and healthy controls.Results1.There was no significant difference in sex,age and BMI between the RA patients with different syndrome types and controls.2.A total of 2220185 valid tags were obtained from all samples.The average valid tags were 36995 in RA patients and 44448 in controls.The average valid tag length was 406.61-425.78 bp.A total of 3198 OTU were obtained by OTU cluster analysis.3.Alpha diversity analysis showed that the sequencing depth of this study was reasonable,the sample size was sufficient,and the species richness was high.Observed index reflecting species richness was significantly lower in RA patients than those in controls.While shannon and simpson index reflecting species diversity were slightly lower in RA patients than those in controls,but the difference was no statistically significant.The richness of intestinal flora in RA patients was changed.4.In Beta diversity analysis,PCoA analysis showed that there were significant differences in the intestinal flora structure among individuals of RA patients with different syndrome types.UPGMA analysis showed that there were significant differences in the intestinal flora structure between RA patients and controls.ANOSIM and Adonis analysis showed that the difference between the RA patients and controls was significantly greater than the difference within the RA groups,and the experimental reliability was higher（P=0.001）.5.The results of different classification levels are as follows.At the phylum level,the abundance of Tenericutes in the phlegm-blood stasis syndrome group was significantly lower than that in controls（P<0.05）,and the abundance of Fusobacteria in the phlegm-blood stasis syndrome group was significantly higher than that in the liver-kidney deficiency syndrome group（P<0.05）.At the class level,the abundance of Acidimicrobiia in the damp-heat impeding syndrome group was higher than that in the controls（P<0.05）,the abundance of Mollicutes in the phlegm-blood stasis syndrome group was significantly lower than that in the controls（P<0.05）,and the abundance of Fusobacteriia in the phlegm-blood stasis syndrome group was significantly higher than that in the liver and kidney deficiency syndrome group（P<0.05）.At the order level,the abundance of Cellvibrionales in the phlegm-blood stasis syndrome group was significantly lower than that in the other syndrome groups and the controls（P<0.05）,while the abundance of Fusobacteriales in the phlegm-blood stasis syndrome group was significantly higher than that in the liver and kidney deficiency syndrome group（P<0.05）.At the family level,the abundance of Methylophilaceae in the damp-heat impeding syndrome group was significantly higher than that in other syndrome groups（P<0.05）,the abundance of Microtrichaceae in the phlegm-blood stasis syndrome group was significantly higher than that in other syndrome groups（P<0.05）,and the abundance of Fusobacteriaceae in phlegm-blood stasis syndrome group was significantly higher than that in liver and kidney deficiency syndrome group（P<0.05）.At the genus level,the abundance of Cupriavidus and Methylotenera in the damp-heat impeding syndrome group was significantly higher than that in other syndrome groups and controls（P<0.05）,the abundance of Pseudarthrobacter in damp-heat impeding syndrome group was lower than that in other syndrome groups and controls（P<0.05）,and the abundance of Fusobacterium in phlegm-blood stasis syndrome group was significantly higher than that in liver and kidney deficiency syndrome group（P<0.05）.At the species level,the abundance of Burkholderiales_bacterium_YL45 in damp-heat impeding syndrome and phlegm-blood stasis syndrome groups was significantly lower than that in controls（P<0.05）,the abundance of Lactococcus lactis,Lactobacillus mucosa,Lachnospiraceae bacterium 615 in liver and kidney deficiency syndrome group was lower than that in controls（P<0.05）.6.LEfSe analysis showed that there were 17 biomarkers with significant differences in abundance between different syndrome groups of RA and the healthy controls.The biomarkers of damp-heat impeding syndrome group were distributed in Proteobacteria,among which the most important biomarker was Cupriavidus.The biomarkers of phlegm-blood stasis syndrome group were distributed in Firmicutes,among which the most important biomarker was Anaerosporobacter.The most important biomarker of liver-kidney deficiency syndrome was prevotella₉.Conclusion1.There was no significant difference in sex,age and BMI between the RA patients with different syndrome types and controls.2.Compared with the controls,the richness of intestinal flora in different syndrome types of RA decreased,and the structure of intestinal flora in different syndrome types of RA changed.3.There were different biomarkers among different syndromes of RA.The most influential biomarker is the Cupriavidus in the damp-heat impeding syndrome group,Anaerosporobacter in the phlegm-blood stasis syndrome group,prevotella 9 in the liver and kidney deficiency syndrome group.