Reseach On Molecular Mechanism Of Qingre Huoxue Decoction In Regulating Intestinal Microecology And Th/Treg Of CIA Rat Model

ObjectiveTo observe the effects of Qingre Huoxue recipe on intestinal microecologyand Th17/Treg balance in collagen-induced arthritis?CIA?rats model,to explore the mechanism of its treatment of rheumatoid arthritis?RA?.And provide scientific basis for the clinical application of Qingre Huoxue prescription.MethodsThirty rats were randomly divided into normal group,model group,methotrexate?MTX?group,Qingre Huoxue prescription group,probiotics group,6 rats in each group.The other rats were immunized 7 days after subcutaneous injection of collagen II and the mixed emulsifier of complete Freund adjuvantCIA model.MTXgroup 1.0 mg/kg weekly,Qingre Huoxue prescription group and probiotics group 20 g/kg Qingre Huoxue recipe and 350 mg/kg probiotics every morning,normal group and model group were given the same amount of normal saline water.During the course of administration,the degree of joint swelling was observed,the weight and degree of joint swelling were measured,the clinical score was recorded for 4 weeks,and the ankle joint and synovium of the rats were taken out to make pathological sections.The expression of CD31 and VEGF in synovial membrane was detected by.immunohistochemical method,and the effect of 16s rDNA on intestinal feces of rats was studied.The fecal flora was sequenced and analyzed by bioinformatics.and the percentage of helper cell subsets Th 17?CD4+IL-17+?cells andregulatory T cell Treg?CD4+CD25+Foxp3+?in splenic were detected by flow cytometry.The contents of IL-10 and IL-17 in rat serum were determined with ELISA method.ResultsPart one:Effect of Qingre Huoxue recipe on angiogenesis of synovium in CIA rats1 Effect of Qingre Huoxue recipe on weight,Joint swelling degree and Clinical score of CIA Rat modelCompared with the normal group,the weight of CIA model group increased slowly,and there was statistical difference from the 14th day after immunization?P<0.05?.Compared with the model group,the weight growth of Qingre Huoxue prescription group was stable and the weight of MTX group increased significantly from the 19th day,and the growth of probiotics group was slow Weight gain?P<0.05?.On the 10th day of immunization,the hind limbs of the rats in each model group developed redness and swelling reached the maximum on 27 days.Compared with the normal group,the swelling degree of the model group increased significantly?P<0.01?.The swelling degree of joint in Qingre Huoxue prescription group was significantly reduced on the 22 day,and the swelling degree of MTX group and probiotic group was also reduced compared with the model group.On the 7th day of immunization,the clinical score of the rats was 0.From the 10th day of immunization,some rats in the model group showed hind foot swelling,and then there were different degrees of redness of the forefoot,compared with the normal group,the clinical score of the model group increased significantly from the second week?p<0.001?;compared with the model group,Qingre Huoxue prescription group decreased significantly from the third week?P<0.05?,and the difference was statistically significant between the MTX group and the probiotics group?P<0.05?.2 Effect of Qingre Huoxue recipe on the Pathology of ankle and synovium in CIA Rat modelAnkle pathology:Compared with the normal group,no inflammatory exudates were found in the articular lumen of the model group,but some articular cartilage were damaged moderately to severely,and the fibrous tissue was obviously proliferated on the surface.The articular cavity disappeared.There were moderate to severe inflammatory cell infiltration in the surrounding soft tissue.Moderate destruction of articular cartilage in Qingre Huoxue prescription group,mild and moderate inflammation cell infiltration of surrounding soft tissue.Mild destruction of cartilage in MTX group;moderate and severe bone destruction in probiotics group.The inflammatory infiltration of surrounding soft tissue was not obvious.Synovial pathology:Compared with the normal group,the synovial cells proliferated obviously,a large number of inflammatory cells infiltrated,the number of pannus increased,and the structure of adipocytes was abnormal in the model group,compared with the model group,the infiltration of inflammatory cells in the Qingre Huoxue prescription group was significantly reduced compared with the model group.The neovascularization and pannus decreased obviously,most of synovial cells were disordered and the structure was abnormal,but some of the peripheral adipocytes were not infiltrated and deformed,but the probiotics group showed moderate inflammatory infiltration and more neovascularization.3 Effect of Qingre Huoxue recipe on CD31 and VEGF in synovial membrane of CIA Rat modelCompared with the normal group,the positive expression of VEGF in the synovial membrane of the model group was significantly increased,the expression area was increased,and the number of microvessels was significantly increased?p<0.01?.Compared with the model group,the expression of CD31 and VEGF and microvessel density withtreatment of Qingre Huoxue recipe was higher than that in the model group?p<0.01?.The microvessel density decreased significantly in MTX treatment group and probiotics group?p<0.05?.Part two:Effect and Mechanism of Qingre Huoxue recipe on intestinal Microecology in CIA Rat model1 Effect of Qingre Huoxue recipe on intestinal microflora ? Analysis in CIA Rat modelCompared with the normal group,model group,Chao index,observed index,Shannon index,Simposon index decreased significantly?p<0.05,p<0.001?.Compared with the model group,Qingre Huoxue group increased significantly in three index?p<0.05,p<0.001?,raise of MTX group and probiotics group showed different degree,especially Shannon index,observed index was significantly increased?p<0.05,p<0.01?.Compared with the model group,the treatment group Simposon index rose,but there was no statistical difference.2 Effect of Qingre Huoxue recipe on intestinal microflora ? Analysis in CIA Rat modelThe distribution of intestinal flora in the model group and the normal group was intuitively reflected by the dendrogram,PCoA and MNDS.Compared with the model group,the differencewas obvious in the Qingre Huoxue prescription group,and the difference between the two groups was small but the similarity was high.The similarity between the two groups was higher than that of the normal group.?ANOSIM R=0.4012,P=0.001,Number of permutations=999?3 Effect of Qingre Huoxue recipe on intestinal flora in CIA Rat modelA total of 21 phylum were detected in the intestinal microflora of rats in each group,and 5 phylum were detected in the five groups,including Firmicutes,and Actinobacteriae.Compared with the normal group,in the model group,the proportion of phylum sclerotidis increased significantly?P<0.001?,while the other four decreased significantly?P<0.05?,showing the imbalance state.Compared with the model group,each treatment group detected bacteria species were increased,the Qingre Huoxue Formula groups of flora categories increased,includiong Fusobacteria,Deferribacteres.Gemmatimonadetes,Acidobacteria.Cyanobacteria,Gracilibacteria,Spirochaetae,Chloroflexi improved well?p<0.05?.The rate of Fusobacteria and Cyanobacteria in MTXgroupincreased?p<0.01?,the proportion of Firmicutes in each treatment group decreased,and Qingre Huoxue prescription group decreased the most?p<0.05?.4 Effect of Qingre Huoxue recipe on intestinal flora of CIA Rat modelThe samples of each group were analyzed in the first 30 genera with the highest proportion of genera.In the normal group,the species were abundant a nd evenly distributed,but the distribution of Prevotellal Fusicatenibacter was v ery small,but there was an increase in both the model group and the treatmen t group.Compared with the normal group,the distribution of Prevotellal Fusicat enibacter was very small.In the model group,there were fewer and uneven L actobacillus species increased significantly?P<0.001?,Up to 90%.the proport ion of other 29 species like Prevotella₉.Escherichia_Shigella?Romboutsia,Bac teroides?Lachnospiraceae_NK4Al 3 6_group decreased significantly?P<0.05?.C ompared withthe model group,the number of Lactobacillus in each treatment g roup significantly increased?P<0.001?.The Qingre Huoxue recipe group was rich in species and balanced,and the other 29 species all up-regulated.Thediff eren-ce ofPrevotella₉?Lachnospiraceae_NK4A136_group?Alistipes?Fusicatenib acter?Lachnoclostridium?Parasutterella was statistically significant?P<0.05?.The percentage of Prevotella9 in the MTX group was significantly higher tha n that in the control group?P<0.01?.Part three:Effect of Qingre Huoxue recipe on Th/Treg of CIA Rats and its Molecular Mechanism1 Effect of Qingre Huoxue recipe on Th/Treg Cell level in spleen of CIA Rat modelCompared with the normal group,the level of Th17 in the model group was increased and the level of Treg was decreased?p<0.01,p<0.05?.Compared with the model group,the level of Thl7 in Qingre Huoxue Fang group decreased and the level of Treg increased?p<0.05?.The level of Thl7 was higher than that in the group of probiotics?p<0.05?,but the down-regulation of the level of Th17 was not significant?P>0.05?.2 Effect of Qingre Huoxue recipe on IL-10 and IL-17 in Serum of CIA Rat modelCompared with the normal group,the serum level of IL-10 in the model group decreased significantly?P>0.05?and the level of IL-17 increased significantly?P<0.07?.Compared with the model group,Qingre Huoxue recipe could up-regulate the expression of IL-10 and the level of serum IL-10.There was no significant difference between the two groups?P>0.05?.The down-regulation level of IL-17?p<0.05?,MTX group and probiotics group increased the level of IL-10?P<0.01?and decreased the level of IL-17?P<0.05?.Conclusions1 Qingre Huoxue decoction can effectively alleviate the degree of joint swelling and clinical score in CIA rats.It can inhibit synovitis and joint destruction,reduce the expression of CD31 and VEGF,and inhibit the speed of progress of angiogenesis.2 The treatment of Qingre Huoxue decoction in CIA rats could increase the abundance?the species?evenness and reduce the species differences of intestinal flora of CIA rats,which tend to the intestinal flora of normal rats.In the CIA model group,Lactobacillus is the main dysbacteriosis group.3 Qingre Huoxue decoction upregulated the proportion of Treg cells in spleen,and down-regulated the proportion of Th17 cells of CIA rats,which regulated the steady-state of Th/Treg,By up-regulating IL-10 and down-regulating immune effectors such as IL-17,Qingre Huoxue decoction regulat the body immunity,so as to control synovitis and angiogenesis,and alleviate the clinical symptoms of CIA rats.4 Qingre Huoxue decoction regulates the imbalance of Th/Treg by regulating the imbalance of intestinal flora,which is inhibit the progress of arthritis and angiogenesis in CIA rats.