Study On The Effect Of Huayu Tongbi Recipe On Regulating Joint Inflammation In Mouse Rheumatoid Arthritis Model

Objective To investigate the effect and the actions of Huayu Tongbi prescription on the regulation of inflammatory cells and JAK/STAT signaling pathway,so as to provide the theoretical basis for the clinical application of Huayu Tongbi prescription in the treatment of rheumatoid arthritis.Methods DBA/1 mouse were used for molding.Huayu Tongbi prescription leflunomide and pure water were intragastric administration respectively 21 days after secondary immunization.Gavage continued for 32 consecutive days and the condition of paw swelling were measured and recorded each 7 days.Mouse were executed and the lymph nodes,synovial tissues of their limbs and joints,and their forelimbs were intact.The cells were extracted and Single cell suspension by cutting and grinding the lymph nodes and synovial tissues of mouse.The ratio of T helper cells in the lymph node cell suspension and the proportion of intrinsic typeⅡ lymphocytes(ILC2)in the synovial tissue suspension were detected by flow cytometry,and the effects of Huayu Tongbi prescription on T helper cells and ILC2 were investigated.After upper limb fixation,paraffin section was performed,and the distribution and number of osteoclasts,chondrocytes and blood vessels in the joints were observed by hematoxylin-eosin staining(HE staining),tartrate-resistant acid phosphatase staining(TRAP staining),and safranin O fast green staining,and the level of intraarticular inflammation and the effect of Huayu Tongbi prescription on inflammatory joints were evaluated.The expression levels of JAK/STAT signaling pathway related proteins in cells in synovial tissue of the joints were detected by Western-blot to study the mechanism of Huayu Tongbi prescription in regulating gene transcription and expression of related inflammatory cells by intravastal administration.Results The measurement of toe thickness of mice during intragastric administration showed that compared with blank group,model group,Huayu Tongbi prescription group and leflunomide group,toe thickness increased.Forelimb joint paraffin section of HE staining,observed in arthritic joints,model group,the blood from tong bi group,group and blank group has the obvious increase in the number of osteoclasts and bone defect,a few new small blood vessels,blood tong bi parties can adjust the inflammatory joint inflammation cells infiltration,reduce osteoclasts transformation,improve the inflammation of the environment.Flow cytometry results showed that after Huayu Tongbi prescription was administered intragastric,the Th17 ratio in lymph nodes of model group was significantly higher than that of blank group,huayu tongbi prescription group and leflunomide group(P<0.01,P<0.05,P<0.01).Lymph node memory T cells in model group were significantly higher than those in blank group,Huayu Tongbi prescription group and leflunomide group(P<0.05,P<0.01,P<0.05);The ratio of ILC2 in synovial tissues after treated with Huayu Tongbi prescription,compared with the model group,the ratio of ILC2 in leflunomide group and Huayu Tongbi prescription group was significantly higher(P<0.01,P<0.05).Western-blot results showed that compared with the model group,the expression levels of JAK2,p-JAK2,JAK3,p-JAK3,STAT6 and p-STAT6 in the leflunomide group and Huayu Tongbi prescription group decreased to some extent,and the decrease of p-JAK2,JAK3,p-JAK3 and p-STAT6 in Huayu Tongbi prescription group(P<0.01,P<0.05,P<0.01,P<0.05)was statistically significant.Conclusion By regulating the level of T helper cells,especially the proinflammatory T cells and ILC2 cells in the joints of CIA model mice,the joint inflammation can be partially alleviated and the normal cell population in the inflammatory joints can be restored.