Research On The Mechanism Of Xiaochaihu Decoction In The Treatment Of Rheumatoid Arthritis Based On The Theory Of "Shaoyang Main Bone"

Purpose:Rheumatoid arthritis belongs to the category of Bi syndrome of TCM.In this study,it was proposed that the dysfunction of "Shaoyang governing bone",the dysfunction of the pivot,would be one of the pathogenesis of RA,might be treated by Xiao Chaihu Tang which was on behalf of the prescriptions to treat shaoyang disease.While it is based on the theory of "Shaoyang governing bone",combined with the related factors of bone destruction in the pathogenesis of RA such as synovial inflammation(including inflammatory cytokines and NF-k B inflammatory signaling pathways)and angiogenesis cytokines(including VEGF,HIF1?)and bone metabolic pathway(including RANKL/RANK/OPG signal pathway)related indicators,to observe the effect of molecular biology with Xiao Chaihu Tang in RA classic animal model CFA.It will provide basic experimental basis for the treatment of RA with traditional Chinese medicine,and provide evidence for the theory of "Shaoyang governing bone" in the TCM pathogenesis of RA.Material and method:By carding the related ancient books in theory,we have analyzed the internal relation between the dysfunction of the "Shaoyang governing bone" and the pathogenesis of rheumatoid arthritis,analyzed the present situation of the treatment of rheumatoid arthritis with the treatment of its representative formula "Xiao Chaihu Tang",to put forward the research direction of the treatment of rheumatoid arthritis under the guidance of the theory of "Shaoyang governing bone",to provide necessary theoretical basis and experimental support for the selection of rheumatoid arthritis TCM pathogenesis and effective clinical treatment.In animal experiments,we applied SPF health adult female Wistar rats for the preparation of CFA animal model of rats with rheumatoid arthritis with freund's complete adjuvant and cattle ? collagen type.According to the random number table,the rats were randomly divided into the normal group,the model group,the XCHT low-dose group,the XCHT medium-dose group,the XCHT high-dose group,and the Tripterygium Glucosides group.Rats in each group were given the appropriate treatment for drug gavage,and the experimental drug intervention period was 28 d.During the experiment,we have observed the state of the rats in mental state,the situation of drinking and drinking water,hair color,behavior,body mass change,and the changes of arthritis index and joint swelling in rats.After the experiment,the radiological expression of each group would be observed and compared.We would kill rats with the method of taking blood from their abdominal aorta using 10% hydronium chloride intraperitoneal injection.After taking the materials,we would detect and analyze the changes of TNF-??IL-1??IL-6?M-CSF cytokines in rats serum in each group with ELISA method,observe and analyze the pathological changes and pathological grade of ankle joint specimens of rats in different groups,detect and analyze TARDD?IKK??IKB??NF-?B p65 protein expression of rats ankle tissue samples in the comparative experiment between groups with Western blot method,detect and analyze the expression of HIF-1??VEGF?RANKL?OPG m RNA in the ankle tissues of rats in each group with RT-PCR method,to explore the molecular biological mechanism of Xiao Chaihu Tang against rheumatoid arthritis.SPSS19.0 software was used for statistical analysis of experimental results,and one-way anova was used for parameter statistical analysis,and LSD test was used for comparison between groups,with p<0.05 was statistically significant.Results: 1.General state and arthritis state of rats(including joint morphology,arthritis index,and swelling degree)1.1 There were varying degrees of improvement the performances in the mental state,drinking and eating,color luster,behavior of the CFA rats with the increase of dose of Xiao Chaihu Tang.While the performances in the mental state,drinking and eating,color luster,behavior of the rats of tripterygium glycosides group were poor contrasting with Xiao Chaihu Tang group,and accompanied by a loose stool.1.2 We would observe the comparison of mass of rats at different time points in the experimental groups.Compared with the model group,the XCHT low-dose group and the XCHT medium-dose group at 21 d,the statistical results showed p<0.05,and the body mass increased significantly,and at the end of 28 d to the end of the experiment,the statistical results showed p<0.01,the body mass increases,the difference was very significant.While the statistical results of the XCHT high-dose group at the end of 14 d to the end of the experiment showed p<0.01,the difference of mass increase was very significant.Compared with the tripterygium glycosides group,the XCHT high-dose group showed p<0.01 at the end of 28 d to the end of the experiment,the body mass increases,the difference was very significant.1.3 The changes in AI scores at the end of the experiment.Compared with the model group,the AI statistical results of the XCHT low-dose group,XCHT medium-dose group,XCHT high-dose group,and the tripterygium glycosides group showed p<0.01,the difference was very significant.1.4 Comparison of the changes of the right hind foot swelling of rats at different time points.Compared with the model group,the XCHT low-dose group,the XCHT medium-dose group,the XCHT high-dose group and the tripterygium glycosides group,at 14 d up to end of the experiment,statistical results showed p<0.01,right rear foot swelling of rats reduced quite significantly.1.5 Comparison of the changes of the foot volume of rats at the end of the experiment.Compared with the model group,the statistical results of the XCHT medium-dose group,the XCHT high-dose group and the tripterygium glycosides group,statistical results showed p<0.01,the decrease of the foot volume was very significant.2.Radiological changes in the joint of rats and pathological changes of ankle arthroplasty in rats 2.1 Performance in radiology: In the model group,the soft tissue swelling around the ankle joint was obvious,the right rear ankle was heavy,the joint between the ankle and the metatarsal joint was not clear,and part of the joint was fused to explain the success of the model.While,with the increase of the dose of Xiao Chaihu Tang,the degree of soft tissue swelling and the improvement of joint space between the CFA joints were more obvious.2.2 In the light microscope,the histopathological analysis of the histopathological section of the right posterior ankle joint of the rats was analyzed:With the increase of Xiao Chaihu Tang dose,bone fibre organization arrangement,cartilage surface smoothness,synovial cell hyperplasia,pannus formation conditions,articular cavity,inflammatory cells have different degrees of improvement.Computing pathological grading,compared with model group,XCHT low-dose group,XCHT medium-dose group,XCHT high-dose group and tripterygium glycosides group,the statistical results showed p<0.01,the pathologic scores were reduced and the differences were very significant.With the increase of the dose of Xiao Chaihu Tang,the pathological score decreased,and the statistical results showed p<0.01,the difference was very significant.3.Changes in serum TNF-??IL-1??IL-6?M-CSF inflammatory cytokine concentrations in rats In the serum levels of inflammatory cytokine TNF-??IL-1??IL-6?M-CSF examination,with the increase of Xiao Chaihu Tang dose,their expression gradually decreased,compared with the model group,the XCHT medium-dose group and the XCHT high-dose group statistical results showed p<0.01,decrease quite significantly.4.The expression of TARDD?IKK??IKB??NF-?B p65 protein in the NF-k B signaling pathway in ankle tissues of rats In the expression of TARDD?IKK??IKB??NF-?B p65 key protein in the NF-k B signaling pathway in ankle tissues of rats,comparing with the model group,the XCHT low-dose group statistical results showed p<0.05,the protein expression was decreased and the difference was significant.While the XCHT medium-dose group,the XCHT high-dose group and the tripterygium glycosides group statistical results showed p<0.01,the protein expression to reduce,the differences are very significant.5.Expression of HIF-1??VEGF m RNA in ankle tissues of rats HIF1 and VEGF m RNA expression in the right posterior ankle tissue specimens,comparing with the model group,the expression of HIF1?and VEGF m RNA in the right posterior ankle tissues of the rats in the XCHT medium-dose group,the XCHT high-dose group and the tripterygium glycosides group statistical results showed p<0.05,the expression decreased,and the difference was significant.6.Expression of RANKL and OPG m RNA in RANKL/RANK/OPG signaling pathway in rat ankle joint.6.1 RT-PCR technique was used to analyze RANKL m RNA expression in the right posterior ankle tissues of CFA rats.Compared with the normal group,the expression of RANKL m RNA in the right posterior ankle of the model group showed p<0.05,the expression increased and the difference was significant.Compared with the model group,the expression of RANKL m RNA expression in the right posterior ankle tissue specimens in the XCHT low-dose group,the XCHT medium-dose group,the XCHT high-dose group,the statistical results showed p<0.05,the expression decreased,and the difference was significant.6.2 RT-PCR technique was used to analyze OPG m RNA expression in the right posterior ankle tissues of CFA rats.Compared with the normal group,the expression of OPG m RNA in the right posterior ankle of the model group showed p<0.01,the expression decreased and the difference was very significant.Compared with the model group,the expression of OPG m RNA expression in the right posterior ankle tissue specimens in the XCHT low-dose group,the XCHT medium-dose group,the statistical results showed p<0.05,the expression increased,and the difference was significant.While in the XCHT high-dose group and the tripterygium glycosides group,the statistical results showed p<0.01,the expression increased,and the difference was very significant.Conclusion: 1.The dysfunction of "Shaoyang governing bone",the dysfunction of the pivot,would be one of the pathogenesis of RA.While that causes the disorder of qi and blood fluid running and metabolic disturbance,is the pathological basis for RA formation.So we could use Xiao Chaihu Tang which was on behalf of the prescriptions to treat shaoyang disease to treat rheumatoid arthritis under the condition of the dysfunction of "Shaoyang governing bone ",which would provide another effective prescription for clinical treatment of rheumatoid arthritis.2.This study would have shown that as the dose of Xiao Chaihu Tang increases,we could improve the general state of CFA rats(including mental state,drinking water condition,luster,behavior and body mass)and the state of arthritis(including joint shape,arthritis index,swelling degree).We could improve the histopathological performance of the ankle tissue specimens of the CFA rat and the radiological manifestations of bilaterally foot joints,and effectively inhibit bone destruction.Xiao Chaihu Tang might inhibit the expression of the key protein TARDD?IKK??IKB??NF-?B p65 in inflammatory signaling pathways important,NF-?B signaling pathway,by inhibiting the expression of inflammatory cytokines related to synovitis related to synovitis in the serum,such as TNF-??IL-1??IL-6 and inhibiting the expression of the important cytokines M-CSF in the activation of NF-?B signaling pathway,to control the inflammatory response of rheumatoid arthritis,thereby delaying bone destruction.It inhibits the related factors of bone destruction,the blood vessels formed the relevant cytokines HIF-1? and the downstream target gene VEGF,to reduce angiogenesis and hyperplasia of synovial membrane,and to control the occurrence and development of rheumatoid arthritis.It might inhibit RANKL and promotes OPG which are in the RANKL/RANK/OPG signaling pathway in bone destruction related factors,to inhibit osteoclast differentiation and prevent bone destruction.All that might be its mechanism for preventing rheumatoid arthritis.3.This study could conclude that Xiao Chaihu Tang might inhibit the activation of the NF-?B signaling pathway under the involvement of M-CSF cytokines,inhibit the activation and secretion of TNF-??IL-1??IL-6 and other inflammatory cytokines,block the RANKL/RANK/OPG signaling pathway,inhibit osteoclast differentiation,at the same time,inhibit HIF-1? and downstream target gene VEGF to reduce angiogenesis and hyperplasia of synovial membrane,to prevent bone destruction.That may be its mechanism for preventing rheumatoid arthritis.It would provide the basic experimental basis for the treatment of rheumatoid arthritis of traditional Chinese medicine,and provide the evidence for the theory of "Shaoyang governing bone" in the TCM pathogenesis of rheumatoid arthritis.