Study On The Mechanism Of Qingre Huoxue Prescription In Treating Rheumatoid Arthritis Based On Intestinal Flor

Rheumatoid arthritis（RA）is a chronic autoimmune disease of unknown etiology,with synovitis as the main pathological change.At present,the western medical treatment of RA is mainly represented by glucocorticoids,disease-modifying anti-rheumatic drugs（DMARDs）and biological agents,but there are problems such as large side effects,easy recurrence of drug discontinuation and high price.Chinese medicine has been shown to be effective in relieving joint symptoms and slowing down the progression of RA due to its multi-path and multi-target therapeutic advantages.Lu Zhizheng,a master of Chinese medicine,proposed the method of "Qing Re Huo Xue" and formulated his own formula to treat RA patients,which has proven to be effective,safe and reliable through more than 10 years of clinical practice.However,the specific mechanism of action of the Qing Re Huo Xue Fang in the treatment of RA is still unclear,and further experiments are needed to explore the mechanism of action of the Qing Re Huo Xue Fang in the treatment of RA,so as to provide the corresponding theoretical and practical basis for the prevention and treatment of RA in Chinese medicine.Objectives:1.To observe the difference of intestinal flora between RA patients and healthy individuals and its correlation with serum inflammatory indexes,immune indexes and quality of life by means of 16S rDNA sequencing;2.To observe the effect of Qing Re Huo Xue Fang on the intestinal flora of Collagen Induced Arthritis（CIA）mice to explore the pathogenesis of its treatment of RA3.To establish a pseudo-sterile mouse CIA model by using combined antibiotics,and transplant RA patients and healthy human fecal bacteria into the intestine of pseudo-sterile mice by fecal bacteria transplantation technique,and to observe the changes of intestinal flora of RA patients after transplantation by Qing Re Huo Xue Fang,and to explore the effects of Qing Re Huo Xue Fang on intestinal flora diversity and flora structure of RA patients.Methods:1.Twenty patients with RA were included in the clinical study,while 15 healthy individuals matched in gender and age with the RA patient group were included as the control group,and the differences in gender,age,and BMI between the two groups were not statistically significant.The above study subjects’ general data conditions,serum inflammatory indexes,serum immunological indexes,quality of life scores and other clinical data were collected,and stool samples were also retained.The intestinal flora of the stool samples from the two groups of subjects were sequenced and bioinformatically analyzed using 16S rDNA technology,and the changes in intestinal flora of RA patients and their correlation with serum inflammatory indexes,immunological indexes,and quality of life were analyzed;2.Twenty healthy,clean-grade 6-week-old male C57BL/6 mice were randomly divided into blank group,model group,clear heat and blood formula group,and methotrexate group,with 5 mice in each group.Except for the blank group,all mice were induced with chicken type II collagen-complete Freund’s adjuvant called CIA mouse model,followed by treatment with Qing Re Huo Xue Fang and methotrexate by gavage in the Qing Re Huo Xue Fang and methotrexate groups,respectively,for 21 days.The general condition,body weight,degree of joint swelling and changes in arthritis index of mice in each group were observed during the experiment,and fecal samples were collected at the end of the experiment for sequencing and bioinformatics analysis of the intestinal flora of mice in each group using 16S rDNA technology;3.Thirty healthy,clean grade 6-week-old male C57BL/6 mice were included and randomly divided into 2 groups:blank control group（n=5）and pseudo-sterile group（n=25）.The mice in the pseudo-sterile group were first gavaged with 1 mg/kg amphotericin B for three consecutive days,twice a day,during which they drank and ate normally;after that,1 g/L ampicillin,500 mg/L vancomycin,1 g/L neomycin and 1 g/L metronidazole were added to the quoted water,and the drinking water was changed once a week,during which they were gavaged with drinking water once a day for 2 weeks to construct a pseudo-sterile mouse model.The weight changes of pseudo-sterile mice were observed during the experiment,and the fecal flora of both groups were cultured and counted at the end of the modeling period,and the intestinal flora of both groups were sequenced and bioinformatically analyzed using 16S rDNA counting to assess the effect of pseudo-sterile modeling;4.Using the fecal bacteria transplantation technique,fecal flora from RA patients and healthy humans were transplanted into the intestine of pseudo-sterile CIA mice by gavage for 1 week.After the fecal bacteria transplantation,the pseudo-sterile CIA mice transplanted with fecal bacteria from RA patients were treated with the Qing Re Huo Xue Fang（or methotrexate）,and the general condition,body weight,degree of joint swelling and changes in arthritis index of each group were observed,and the intestinal flora of each group was sequenced and bioinformatically analyzed by 16S rDNA technology at the end of the experiment.Results:1.Correlation analysis between intestinal flora analysis and serum inflammatory indexes,serum immune indexes and quality of life in RA patients（1）The species richness and diversity of intestinal flora of RA patients were significantly lower than those of healthy individuals,and there were significant differences in the structure of flora between the two groups.（2）At the phylum level,Proteobacteria（Aspergillus phylum）and Actinobacteria（Actinobacteria phylum）were significantly increased in the RA group compared to the healthy human group,while Bacteroidetes（Bacteroidetes phylum）was significantly decreased compared to the healthy human.At the genus level,Bacteroides,Faecalibacterium,Prevotella₉,Paraprevotella,Prevotellaceae_NK3B31_group,Clostridium_sensu_stricto₁ in the intestinal flora of RA patients showed a significant increase in relative abundance increased significantly compared to the healthy group,while the relative abundance of Roseburia,Blautia,Parabacteroides,Bifidobacterium,Escherichia-Shigella decreased significantly compared to the healthy group.（3）Combining the results of correlation analysis with those of LEfSe analysis,Roseburia was found to be possibly negatively correlated with CRP,ESR,IL-6,IL-8,RF,anti-CCP,IgA,IgM and IgG,while positively correlated with the scores of 9 dimensions of the SF-36 scale and negatively correlated with the scores of the BRAF-MDQ scale;Faecalibacterium spp.and Prevotella₉ spp.may be positively correlated with CRP,ESR,IL-6,IL-8,RF,anti-CCP,IgA,IgM and IgG,while negatively correlated with SF-36 scale scores on 9 dimensions and positively correlated with BRAF-MDQ scale scores.2.Effects of Qing Re Huo Xue Fang on the joints and intestinal flora of CIA mice（1）Qing Re Huo Xue Fang can effectively alleviate the degree of joint swelling,reduce the arthritis index score and improve joint function in CIA mice.（2）In terms of intestinal flora,the alpha diversity of mice in the CIA model group was significantly lower than that of the blank group,and the richness and uniformity of intestinal flora were significantly improved after treatment with the Qing Re Huo Xue Fang In terms ofβ-diversity,the differences between the Qing Re Huo Xue Fang group and the blank group were smaller and more similar.At the phylum level,Qing Re Huo Xue Fang could down-regulate the relative abundance of mouse Thick-walled Bacteria（Firmicutes）and Proteobacteria（Proteobacteria）and up-regulate the relative abundance of Bacteroidota;at the genus level,it could up-regulate the relative abundance of Muribaculaceae,Lachnospiraceae_NK4A136_group,Ruminococcaceae_UCG009,Alistipes,Parabacteroides,Roseburia,Colidextribacter,and down-regulate the relative abundance of Bacteroides,Lactobacillus,Prevotella.3.Establishment of pseudo-sterile mouse model（1）After drinking and gavage with mixed antibiotics,the body weight of the pseudo-sterile mice group decreased significantly compared with normal,and the number of fecal flora after culturing was significantly reduced compared with the normal group.（2）The abundance and diversity of intestinal flora in the pseudo-sterile mice were significantly decreased compared with the blank group,and the difference of intestinal flora between the two groups was large.The mice in the pseudo-sterile group were significantly different from the mice in the blank group in each taxonomic level.4.Effect of Qing Re Huo Xue Fang on the joint and intestinal flora of pseudo-sterile CIA mice after fecal transplantation（1）Mice in the pseudo-sterile CIA model group had lower joint swelling and arthritis scores compared to other mice that had undergone fecal transplantation.（2）After fecal bacteria transplantation,the change in the ratio of flora in the intestine of mice was similar to that in the human intestine,but there were still differences in some phyla of bacteria.（3）The degree of joint swelling and arthritis scores were significantly higher in mice transplanted with RA patient flora compared to healthy human fecal bacteria transplantation group.（4）Qing Re Huo Xue Fang can effectively improve the species abundance and diversity of fecal bacteria colonized in the intestine of mice with RA,adjust the relative abundance of Muribaculaceae,Lachnospiraceae_NK4A136_group,Lachnoclostridium,Bacteroides,Prevotella and other intestinal flora,and regulate the imbalance of intestinal flora.Conclusion:1.The diversity and abundance of intestinal flora in RA patients were significantly decreased compared to healthy individuals,and the structure of flora was significantly different from healthy individuals.Among them,the genus Roseburia was inversely associated with the severity of serum inflammatory indexes and serum immune indexes and positively associated with quality of life,while the genus Faecalibacterium and Prevotella₉ were positively associated with the severity of serum inflammatory indexes and serum immune indexes and inversely associated with quality of life.2.Qing Re Huo Xue Fang can effectively relieve joint swelling of CIA mice,reduce arthritis score,increase the abundance and diversity of intestinal flora of CIA mice,and up-regulate the relative abundance of Lachnospiraceae_NK4A136_group,etc.Down regulating the relative abundance of Lactobacillus,Prevotella and so on,making the bacterial structure tend to normal mouse flora.3.Fecal bacteria in RA patients can significantly aggravate joint swelling degree and increase arthritis score of pseudo-sterile CIA mice,while Qing Re Huo Xue Fang can inhibit joint inflammation of CIA mice by improving intestinal flora disorder in colonized RA patients.