Study On Mechanism Of Tongfeng Qingxiao Formula Regulating Intestinal Flora And Intestinal Mucosal Barrier In Gouty Arthritis With Damp-heat Syndrome

Objective:Study on the regulation mechanism of Tongfeng Qingxiao Formula on intestinal flora and intestinal mucosal barrier in Gouty arthritis（GA）model rats with damp-heat syndrome,and to provide a theoretical basis for the clinical application of Tongfeng Qingxiao formula in the prevention and treatment of damp-heat syndrome GA at the modern medical level.Methods:Forty male SD rats were randomly divided into normal group,model group,TFQXF group（Chinese medicine group）and diclofenac sodium group（positive group）,with 10 rats in each group.The rats in each group were modeled with damp-heat syndrome GA,and the normal group was not modeled.Based on the theory of traditional Chinese medicine,the animal model of combination of disease and syndrome was constructed by simulating the external environment and internal etiology of GA with damp-heat syndrome.With the help of artificial climate box,the temperature was set at 35°C and the relative humidity was 95%to simulate the external hot and humid environment,and the high-sugar,high-fat and high-alcohol diet was given to simulate the spleen-stomach damp-heat syndrome caused by improper diet,alcoholism and preference for fat and sweet taste.The MSU crystal suspension was prepared by improved method before the establishment of GA disease model,and 150μl MSU solution was injected into the ankle joint cavity of rats at a concentration of 65·mg^-1ml to induce acute GA.Gait analysis and ankle infrared thermal imaging were used as qualitative and quantitative indicators to evaluate the GA model of damp-heat syndrome in rats.The pathological changes of synovial tissue in model rats were observed by HE staining.The levels of CD4⁺and CD8⁺in peripheral blood of rats were detected,and the effect of TFQXF on intestinal mucosal immune barrier in rats was analyzed.The ultrastructure of rat intestinal epithelium was observed by transmission electron microscopy to analyze the effect of TFQXF on the mechanical barrier of rat intestinal mucosa.The effects of TFQXF on the diversity and abundance of intestinal flora in rats were analyzed based on 16S r RNA high-throughput sequencing.Results:（1）Rats with damp-heat syndrome showed typical signs of damp-heat after modeling,mainly manifested as body burnout,reduced feeding,fluffy hair with withered color,loose and drooping scrotum,loose or sticky stool,red tongue and fat tongue.After the GA disease model was established in rats,the peak of inflammation was characterized by obvious redness and swelling of the joints,disappearance of bone markers,and curling of the right lower foot claw and unwilling to contact the ground.Infrared thermal imaging showed that the temperature of the right ankle joint was significantly higher than that of the left ankle joint without GA modeling.The gait analyzer showed that the gait pressure value and standing phase duration of the right hind foot were significantly lower than those of the left foot,showing a three-foot gait.（2）After modeling,a large number of synovial cells proliferated in the ankle joint,and the synovial tissue was incompleted and broken.A large number of inflammatory cell infiltration and vasodilation were observed,which was showed typical inflammatory pathological changes.The intestinal mucosal immune barrier and mechanical barrier of rats were damaged,mainly manifested by the significant decrease of CD4⁺/CD8⁺level in peripheral blood（P<0.05）,the loss of intestinal microvilli and the destruction of epithelial cell membrane,the blurring of cell tight junctions and the appearance of naked nuclei,the overflow of organelles outside the cells,and the destruction of mitochondria in the cells.（3）After TFQXF intervention,the CD4⁺/CD8⁺levels in the Chinese medicine group returned to normal levels;synovial epithelium is relatively complete,synovial edema,synovial epithelial damage,shedding and other pathological phenomena,synovial cell proliferation and inflammatory cell infiltration were significantly reduced compared with the model group;there was no damage to the colonic epithelial cells.The microvilli were arranged normally and closely,and clear cell tight junctions were seen.There were gaps of different sizes between cells,and mitochondria were slightly vacuolated.After the intervention of diclofenac sodium,the level of CD4⁺/CD8⁺in the positive group also recovered,but lower than that in the Chinese medicine group.The synovial tissue remained intact,with mild synovial cell proliferation and a small amount of inflammatory cell infiltration.The colonic epithelial cells were slightly damaged,the microvilli were partially missing,the cell membrane was slightly damaged,lysosomes were visible,and the organelle structure was generally normal.（4）The results of 16S r RNA high-throughput sequencing showed that at the phylum level,Firmicutes（83.82%）,Bacteroidetes（13.16%）and Actinobacteria（1.47%）were the main components of the intestinal flora of rats in the normal group.After modeling and treatment intervention,the community abundance of Firmicutes decreased,while the community abundance of Bacteroidetes and Actinobacteria increased.The abundance of Bacteroidetes（42.49%）in the Chinese medicine group increased most significantly,and the abundance of Radiobacteres（3.45%）increased most significantly in the positive group.At the genus level,the abundance of Escherichia-Shigella in the model group and the traditional Chinese medicine group was significantly higher than that in the normal group.The abundance of Staphylococcus in the positive group was significantly higher than that in other groups.Tongfeng Qingxiao Decoction can increase the abundance of beneficial bacteria such as Bacteroidetes and unclassified＿f＿Lachnospiraceae in model rats,and reduce the abundance of harmful bacteria such as Firmicutes,Shigella,Staphylococcus and Escherichia.Conclusion:（1）TFQXF can improve the inflammatory response of ankle joint synovium in GA model rats with damp-heat syndrome;the abundance of Escherichia-Shigella and Staphylococcus increased significantly after GA modeling of damp-heat syndrome,suggesting that the intestinal flora of model rats was in a pathological imbalance state;TFQXF significantly increased the abundance of Bacteroidetes in model rats,making it more adaptable to various intestinal microecological environment changes,which may help rats to transform and utilize traditional Chinese medicine polysaccharides to fight against intestinal barrier damage and GA inflammation.（2）TFQXF can regulate the intestinal immunity and mechanical barrier of GA model rats with damp-heat syndrome,regulate the structure of intestinal flora,and increase the abundance of beneficial bacteria producing butyric acid,such as Roseburia.