| Background: Rheumatoid arthritis(RA)is a chronic autoimmune disease mainly manifested by multi-joint involvement.The prevalence of RA is about 0.32%-0.36% all the world,and the disability rate of RA patients is as high as 20% one year after the onset,which is one of the major diseases causing labor force loss.Currently,the commonly used therapeutic drugs in clinical practice mainly include glucocorticoid,non-steroidal anti-inflammatory drugs and biological agents,etc.For the treatment of RA,RA patients in China have achieved a low disease activity and a far lower clinical response rate than expected.Therefore,it is of great significance to develop safe and effective drugs with low toxicity.Chinese traditional medicine has a long history in the treatment of this disease.It can improve symptoms,assist western medicine to control the disease,and has less adverse reactions.In my teacher’s opinion,RA with a course of more than 5 years or glucocorticoid therapy is prone to syndrome manifestations of Qi and Yin deficiency,which cannot be explained by previous syndrome classification.In the guide for the Combination of Diseases and Syndromes of Rheumatoid Arthritis.My teacher is mainly responsible for the writing of the diagnosis and treatment of deficiency of both Qi and Yin.Based on the theory and clinical basis of supplementing Qi and nourishing Yin and combined with the tutor’s years of clinical experience,the prescription of Supplementing Qi and nourishing Yin and dredging collaterals formula is finally established as Sishen Decoction.Sishenjian is included in Bao Xiangzhi’s New Prescription of the Qing Dynasty,which has the effect of supplementing Qi and nourishing Yin and dredging collaterals and relieve pain,and can improve the symptoms of RA patients such as joint pain,morning stiffness,fatigue,dry mouth and eyes,etc.In a large number of clinical applications,the effect is remarkable.However,there is no study on the mechanism of rheumatoid arthritis treated by this method in foreign countries,and there are few animal experimental studies in China.Recent studies on RA mainly focus on the regulation of inflammatory signaling pathways.Many inflammatory cytokines are directly or indirectly involved in the inflammatory response of the joint,and the major ones are proinflammatory factors such asTNF-α,IL-1,IL-6,etc.The nuclear transcription factor IRAK1/IKK/NF-κB pathway and JAK2-STAT3 pathway are the major inflammatory pathways involved in the pathogenesis,which are also one of the research hotspots in recent years.The experiment research on the status quo as the starting point,verify the therapeutic effect of Sishen decoction,and provide an important basis for improving the inflammation of rheumatoid arthritis by tonifying qi,nourishing yin and dredging collaterals.Through the transcription factorIRAK1/IKK pathways,JAK2-STAT3 pathway research referred to in the function mechanism of the treatment of rheumatoid arthritis,aims to use the method of science reveals the unique advantage in the treatment of the disease and the system of Chinese traditional medicine,to combine traditional Chinese and western medicine treatment of RA to try some new method,provide the latest research data for future drug discovery.Objective:To observe the effect of Benefiting Qi,nourishing Yin and dredging collaterals on inflammatory indexes of SD rats with RA,and to explore the effect and mechanism of IRAK1/IKK/NF-κB and JAK2-STAT3 pathways.Method: 50 SPF SD male rats were randomly divided into 5 groups,and there were 10 rats in each group.In other words,noraml control group,model group,Sishen Decoction group,Sishen Decoction plus methotrexate group,methotrexate group,were used to model AIA rats with adjuvant arthritis method,and the CFA rats were continuously given corresponding drugs for 8 weeks.part I: Effects of the method of nourishing qi and nourishing yin on inflammatory expression in rats with RA.1.The degree of ankle swelling of SD rats in different groups was measured.To Observe the synovial histopathological changes of the ankle joints with light microscope.2.The histopathological changes of synovium of ankle joint in SD rats of different groups were observed by light microscopy.3.To observe the changes of arthritis index in SD rats of different groups.4.Levels of CRP,ESR,IL-1,IL-6 and TNF-αby serial serum test in SD rats of different groups were measured with the method of ELISA.part II:Effects of method of nourishing qi and nourishing yin and dredging collaterals on IRAK1/IKK/NF-κB signaling pathway in rats with RA.The protein expression levels of IRAK1,TRAF6,IKK and IRAK1/IKK/NF-κBin rat synovial tissue were analyzed by Western blot,and the mRNA levels of IRAK1,TRAF6,IKK and IRAK1/IKK/NF-κB in rat synovial tissue were detected by real-time fluorescence quantitative PCR.part III: The effect of method of nourishing qi and nourishing yin and dredging collaterals on JAK2/STAT3 signaling pathway in rats with RA.1.Expression levels of JAK2,STAT3,p-JAK2 and p-STAT3 in rat synovial tissue were detected by Western blot,and expression levels of JAK2 and STAT3 in rat synovial tissue were detected by real-time fluorescence quantitative PCR.2.The expression level of SOCS3 protein in rat synovial tissue was detected by Western blot,and the expression level of SOCS3 mRNA in rat synovial tissue was detected by real-time fluorescence quantitative PCR.Result:1.Compared with the normal group,the degree of ankle swelling in the model group significantly increased,and and there were statistically significant differences between them(P<0.05).Compared with the model group,the degree of ankle swelling in the Sishen Decoction group significantly reduced,and the results were statistically significant differences between them(P< 0.05).Compared with the model group,the ankle swelling degree of methotrexate group,methotrexate + Sishen Decoction group significantly reduced(P<0.05).Compared with methotrexate group,the degree of ankle swelling in Sishen Decoction group was the same as that in methotrexate group,and there were no statistically significant differences between them(P>0.05).Compared with methotrexate group,the degree of ankle swelling in Sishen Decoction+methotrexate group was significantly lower than that in methotrexate group.(P<0.05).2.Compared with the normal group,the cartilage tissue structure of rats in the model group was destroyed,and the synovial cells were deformed,fibrotic and disordered,with a large number of inflammatory cells infiltrating and proliferating,and pannus formation in the synovial membrane.In the model group,the synovial tissue surface was thickened,inflammatory cell infiltration was observed,synovial cells were disorganized,pannus formation and a few cartilage destruction were observed.In Sishen Decoction group and methotrexate group,cartilage destruction and synovial tissue proliferation were alleviated,cell morphology was slightly disordered and pannus formation was alleviated;In the Sishen Decoction + methotrexate group,the rat cartilage tissue was smooth without damage,the synovial cell structure and morphology were relatively normal,and the cells were relatively orderly arranged.3.Compared with the normal group,the score of ankle arthritis index in the model group was significantly higher.(P<0.05)The score of ankle arthritis index in the Sishen Decoction group was significantly lower than the model group.(P<0.05),the score of arthritis index in the methotrexate group,the methotrexate + Sishen Decoction group was significantly lower,and the difference was statistically significant(P<0.05).Compared with methotrexate group,the score of ankle arthritis index in Sishen Decoction group was similar to that in methotrexate group,but there was no significant difference,(P>0.05);Compared with methotrexate group,the score of ankle arthritis index in Sishen Decoction + methotrexate group was significantly lower than that in methotrexate group(P<0.05).4.The levels of CRP and ESR significantly increased in the model group than the normal group(P<0.05).Compared with the model group,the CRP and ESR levels in the Sishen Decoction group significantly reduced,and there were statistically significant differences between them(P<0.05).Compared with the model group,CRP and ESR levels in methotrexate group,methotrexate+Sishen Decoction group significantly decreased,(P<0.05).Compared with methotrexate group,the CRP and ESR levels of Sishen Decoction group and methotrexate group were similar,and the results were no statistically significant differences between them(P>0.05).Compared with methotrexate group,the level of CRP and ESR in the sishen decoction+methotrexate group was significantly lower than that in the methotrexate group.(P<0.05).5.Compared with the normal group,the levels of TNF-α,IL-1 and IL-6 in the model group significantly increased(P<0.05).Compared with the model group,the levels of TNF-α,IL-1 and IL-6 in the Sishen Decoction group significantly decreased.(P<0.05).Compared with the model group,the levels of TNF-α,IL-1 and IL-6 in the methotrexate group and the methotrexate+Sishen Decoction group significantly decreased,with statistically significant differences(P <0.05).The levels of TNF-α,IL-1 and IL-6 in Sishen Decoction group and methotrexate group were the same as methotrexate group(P>0.05).The levels of TNF-α,IL-1 and IL-6 in the Sishen Decoction+methotrexate group were significantly lower than those in the methotrexate group(P<0.05).6.Compared with the normal group,the levels of IRAK1,TRAF6,IKK and NF-κBp65 protein significantly increased in the model group with statistically significant differences(P<0.05).The protein levels of IRAK1,TRAF6,IKK and NF-κBp65 in the Sishen Decoction group were significantly lower than the model group(P<0.05).Compared with the model group,IRAK1,TRAF6,IKK and NF-κBp65 protein levels in methotrexate group and methotrexate + Sishen Decoction group significantly decreased,with statistically significant differences(P<0.05).IRAK1,TRAF6,IKK and NF-κBp65 protein levels in Sishen Decoction group and methotrexate group were as similar as methotrexate group(P>0.05).The protein levels of IRAK1,TRAF6,IKK and NF-κBp65 protein in Sishen decoction+ methotrexate group were significantly lower than those in methotrexate group(P<0.05).7.Compared with the normal group,IRAK1,TRAF6,IKK and NF-κBp65 mRNA levels significantly increased in the model group,with statistically significant differences(P<0.05).Compared with the model group,IRAK1,TRAF6,IKK and NF-κBp65mRNA levels in the sishen decoction group significantly decrased(P<0.05).Compared with the model group,the levels of IRAK1,TRAF6,IKK and NF-κBp65mRNA in methotrexate group and methotrexate+sishen decoction group significantly decreased,with statistically significant differences(P<0.05).IRAK1,TRAF6 IKK and NF-κ Bp65 mRNA levels of sishen decoction group and methotrexate group were as similar as methotrexate group(P>0.05).Compared with methotrexate group,the mRNA levels of IRAK1,TRAF6,IKK and NF-κBp65mRNA in sishen decoction+methotrexate group were significantly lower than those in methotrexate group(P<0.05).8.The protein levels of p-JAK2,p-STAT3,SOCS3 in the model group were significantly higher than those in the normal group(P<0.05).The protein levels of p-JAK2,p-STAT3 and SOCS3 in the sishen decoction group significantly decreased compared with the model group(P<0.05).Compared with the model group,the protein levels of p-JAK2,p-STAT3 and SOCS3 in methotrexate group and methotrexate+sishen decoction group significantly decreased,with statistically significant differences(P<0.05).The protein levels of p-JAK2,p-STAT3 and SOCS3 in the sishen decoction group and the methotrexate group were as similar as the methotrexate group(P>0.05).The protein levels of p-JAK2,p-STAT3 and SOCS3 in the sishen decoction+methotrexate group were significantly lower than those in the methotrexate group,and there have statistically significant differences.(P<0.05).9.Compared with the normal group,the JAK2,STAT3 and SOCS3 mRNA levels of the model group significantly increased(P<0.05).The levels of JAK2,STAT3 and SOCS3 mRNA in the sishen decoction group were significantly lower than those in the model group(P<0.05).Compared with the model group,the JAK2,STAT3 and SOCS3 mRNA levels in methotrexate group and methotrexate+sishen decoction group significantly decreased,with statistically significant differences(P<0.05).The levels of JAK2,STAT3 and SOCS3 mRNA in the sishen decoction group and the methotrexate group were similar as the methotrexate group(P>0.05).Compared with methotrexate group,the levels of JAK2,STAT3 and SOCS3 mRNA in the sishen decoction+methotrexate group were significantly lower than those in the methotrexate group(P<0.05).Conclusion:1.It is concluded that Sishen Decoction,a traditional Chinese medicine compound,can improve inflammation in rats with rheumatoid arthritis,and Yiqi Yangyin Tongluo Decoction is an effective method for treating rheumatoid arthritis.Sishen Decoction plus methotrexate is better than methotrexate alone in the treatment of rheumatoid arthritis rats;Sishen decoction has the same effect as methotrexate in the treatment of rheumatoid arthritis rats;Sishen Decoction plus methotrexate is better than methotrexate alone in the treatment of rheumatoid arthritis rats;Sishen decoction can be improved by inhibiting the levels of TNF-a,IL-1 and IL-6 factors.Inflammation in rats with rheumatoid arthritis.2.It is suggested that the inhibition of IRAK1/IKK/NF-kappa B signaling pathway by Sishen decoction may be one of the anti-inflammatory mechanisms of rheumatoid arthritis.3.It is suggested that the inhibition of JAK2-STAT3 signaling pathway and the expression of SOCS3,a negative feedback factor,may be one of the anti-inflammatory mechanisms of rheumatoid arthritis. |
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