| Background and purpose:Rheumatoid arthritis(RA)is an autoimmune disease.Insect traditional Chinese medicine(TCM)has shown a unique effect in the treatment of RA.Soufeng Sanjie formula(SF)is composed of scorpions,scolopendras,black bean coat,and astragalus.SF has been widely applied in the clinical treatment of middle and late stages of RA,Jiangsu Province Hospital of Integrated Traditional Chinese and Western Medicine.It has definite curative effect and good clinical research basis,but the mechanism need to be clarified.This study further confirmed the efficacy of SF in the treatment of RA from animal experiments,and cell experiments,and clarified the potential mechanism of SF in the treatment of RA.Methods:Pharmacodynamics and mechanism study:(1)Analyze the chemical components in SF with the fingerprinting technique.(2)DBA/1J mice were used to establish the Collagen-induced arthritis(CIA)model.To explore the efficacy of SF in the treatment of arthritis by detecting the incidence of arthritis,arthritis score,joint swelling,joint pathology,joint Micro-CT,and inflammatory cytokines in CIA mice.(3)Flow cytometry was used to detect the level of CD4+T cells in CIA mice.(4)The expression level of Th17-related transcription cytokines in the spleen of CIA mice after administration of SF was detected by immunohistochemistry.(5)Establishing an in vitro Th17 polarization model to explore the effect of SF on Th17 cell differentiation.Toxicity experiments:To explore the toxicity of SF,ICR mice were given a single dose according to the maximum dose.Besides,1 and 3 times the dose of SF taken by clinical people was converted into the dose for mice,and the ICR mice were continuously administered for 30 days and 90 days.Results:(1)Preparation and composition analysis of SF:13 chemical components including arginine,proline,palmitic acid,leucine,and calycosin were analyzed in SF.The average contents of protocatechuic acid and calycosin in SF was higher.(2)Pharmacodynamic study in vivo:Compared with CIA model group,SF delayed arthritis,decreased arthritis score and reduced joint swelling in CIA mice;SF reduced inflammatory infiltration of ankle synovium,and delayed joint bone erosion in CIA mice.Moreover,SF decreased the levels of IL-17,IL-6 and TNF-α in serum and joint of CIA mice,and increased the level of IL-10.(3)Mechanism study:SF significantly decreased the level of Th17 cells and restored the imbalance of Th17/Treg ratio in CIA mice,and reduced the expression of retinoic acid-associated autism receptor gamma t(RORyt)and phosphorylated STAT3 in the spleen of CIA mice.In vitro,SF inhibited the differentiation of Th17 cells and the production of IL-17A.Meanwhile,SF reduced the mRNA levels of retinoic acid-associated autism receptor gamma t(RORyt)and interferon regulatory factor 4(IRF4).(4)Toxicity experiments:The maximum dose of a single dose showed that the body weight,liver and kidney function indexes,and main organ pathology of ICR mice had no significant changes compared with normal mice after administration.After 30 days and 90 days of continuous administration of ICR mice,the indexes of liver and kidney function and pathology of main organs in the treatment group had no significant change compared with normal mice,and there were no significant side effects compared with normal mice.Conclusion and significance:This study further confirmed the efficacy of SF in the treatment of RA from animal experiments and cell experiments.And it is also determined that SF can reduce the level of Th17 cells and restore the imbalance of Th17/Treg to alleviate joint inflammation and bone erosion in CIA mice.Moreover,SF was continuously administered to ICR mice for 90 d,and no significant side effects were found.The results of the study not only provide a theoretical basis for the clinical use of SF in the treatment of RA,but also provide a theoretical basis and new ideas for the clinical application of traditional Chinese medicine insect drugs and new drug research and development. |
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