Study On The Mechanism Of Yiqi Yangyin Tongluo Prescription In Treating Rheumatoid Arthriti

Background:Rheumatoid arthritis(RA)is a typical chronic autoimmune disease,which is characterized by joint damage and joint dysfunction.The survey results show that the incidence of RA in China is about 0.32%-0.36%,and the disability rate of patients reaches 20% after one year,which leads to a large number of patients losing their ability to work every year.At present,the main drugs in clinical treatment of RA are hormone drugs,non-steroidal anti-inflammatory drugs,biological agents,immunosuppressants,small molecule targeted drugs and so on.Due to the patients’ fear of side effects of drugs,irregular medication,slow onset of commonly used drugs to control the disease and poor compliance,the clinical remission rate related to the treatment of rheumatoid arthritis in China is still at a low level,which is far from the expected level.Therefore,it is found that the drug for treating rheumatoid arthritis is safe,efficient and quick-acting,which is of great clinical significance.Traditional Chinese medicine has a very long history in the treatment of rheumatoid arthritis,and medication has the advantages of good effect,long lasting effect and less toxic and side effects.The teacher thinks that the long-term or irregular use of glucocorticoid during the treatment of rheumatoid arthritis or the long course of rheumatoid arthritis is prone to syndrome manifestations of deficiency of both qi and yin,which is difficult to follow the previous syndrome differentiation and dialectical treatment.In the Guide to Diagnosis and Treatment of Rheumatoid Arthritis by Combining Disease with Syndrome,a new dialectical syndrome type of deficiency of both qi and yin was added according to the teacher’s experience in diagnosis and treatment,and the prescription of benefiting qi,nourishing yin and dredging collaterals was established based on the composition of Sishen Decoction and the principle of benefiting qi and nourishing yin.This prescription is composed of Radix Astragali,Flos Lonicerae,Herba Dendrobii,Radix Cyathulae,and Radix Polygalae.Because of its therapeutic effects of invigorating qi and nourishing yin,promoting blood circulation and dredging collaterals,it can obviously improve the long-term and chronic accompanying symptoms of rheumatoid arthritis in RA patients,such as joint pain,fatigue,dry mouth and eyes,joint morning stiffness,etc.It is widely used in our hospital,and has played a great role in a wide range of patient applications,and the curative effect is extremely remarkable.However,at present,there is a lack of basic experimental research on the treatment of RA with Yiqi Yangyin Tongluo prescription at home and abroad.In recent years,the research on the basic mechanism of RA has paid more attention to the research on inflammatory signaling pathways that regulate diseases.The inflammatory reaction of joints is closely related to various inflammatory factors,among which the most important are pro-inflammatory factors such as TNF-α,IL-1β,IL-6 and IL-17.The nuclear transcription factor IL7A/IL17RA/MMP9 pathway is directly involved in the inflammatory pathway,while the Nrf2/Keap1/Ho1 pathway is mainly involved in the oxidation mechanism and the formation of cytokines,which indirectly causes the disease.These two pathways are also one of the research hotspots in recent years.In the course of this experimental study,the therapeutic target of Yiqi Yangyin Tongluo recipe was determined based on information analysis and network pharmacology,and then the therapeutic effect of Yiqi Yangyin Tongluo recipe was verified according to animal experiments,which provided an important basis for improving the inflammatory level and effect evaluation of RA by this method,and revealed some mechanisms of Yiqi Yangyin Tongluo recipe in treating RA through transcription factors IL7A/IL17RA/MMP9 pathway and Nrf2/Keap1/Ho1 pathway.The purpose is to determine the role and advantages of traditional Chinese medicine in the treatment of RA by drug targets and experimental methods,to provide support for the treatment of RA with integrated traditional Chinese and western medicine,and to play a reference role in the development of related new drugs.Purpose:The drug target of Yiqi Yangyin Tongluo recipe in the treatment of rheumatoid arthritis was clarified through the analysis of raw information and network pharmacology.To observe the effect of supplementing qi and nourishing yin on inflammatory indexes in SD rats with rheumatoid arthritis,and to explore the effects and mechanisms of IL7A/IL17RA/MMP9 pathway and Nrf2/Keap1/Ho1 pathway.Method:Bioinformatics analysis and network pharmacology: analyze the differentially expressed genes in Gene Expression Omnibus(GEO)database,and use the potential core target proteins identified by STRING platform and Cytoscape software to build a PPI network.GO enrichment analysis,Kyoto Encyclopedia of Genes and Genomes(KEGG)signal pathway analysis,gene set enrichment analysis(GSEA)were carried out for potential targets,and then disease target database was used to identify targets related to RA pathogenesis.Cross the relevant indicators with the action indicators of Yiqi Yangyin Tongluo Recipe,and obtain the potential targets considered by Yiqi Yangyin Tongluo Recipe in treating RA.Animal experiment: 50 SPF SD rats,male,fed under normal conditions,were divided into 5 groups based on different treatment methods.That is,the blank control group,model group,Yiqi Yangyin Tongluo group,Yiqi Yangyin Tongluo method in cooperation with methotrexate group,methotrexate group,were modeled by adjuvant arthritis method,and continued drug intervention for 4 weeks.The related experiments are divided into three parts.Part I: Effect of Yiqi Yangyin Tongluo Recipe on inflammatory expression in rheumatoid arthritis rats5.The swelling degree of ankle joint of experimental animals in each group was measured.6.The pathological changes of the synovium of the ankle joint were detected with light microscope.7.CRP contents in samples were detected by fluorescence immunoassay.8.Detection of IL-6,IL-17 and TNF in SD rats of different groups by ELISA-αHorizontal.Part II: Effect of Yiqi Yangyin Tongluo Recipe on IL7A/IL17RA/MMP9 signaling pathway in rheumatoid arthritis rats.Western blot assay was used to analyze the level of IL7A/IL17RA/MMP9 protein in rat synovium,and RT-PCR was used to detect the level of IL7A/IL17RA/MMP9 m RNA in rat synovium.Part III: Effect of Yiqi Yangyin Tongluo Method on Nrf2/Keap1/Ho1 Signal Pathway in Rheumatoid Arthritis Rats The content of Nrf2/Keap1/Ho1 protein in synovial tissue was detected based on WB method,and the expression level of Nrf2/Keap1/Ho1 m RNA in rat synovial tissue was detected by RT-PCR.Result:1.MMP9,CXCR4,CXCL10,CXCL13,TNFSF11,CCR5,ITGAX,TIMP1 and SPP1 were significantly up-regulated,and only ADIPOQ was down regulated in RA(Fig.3B,3C).These genes are expected to become potential targets for the treatment of RA.MMP9 was significantly enriched in three signaling pathways,including IL-17 signaling pathway,tumor necrosis factor(TNF)signaling pathway,and leukocyte trans endothelial migration,while CXCR4 was significantly enriched in chemokine signaling pathway,leukocyte trans endothelial migration,and cytokine receptor interaction.Yiqi Yangyin Tongluo Recipe may achieve its therapeutic purpose by targeting MMP9 and CXCR4 to regulate IL-17,TNF and chemokine signaling pathways.2.The swelling degree of ankle joint in model group was significantly higher than that in normal group(P<0.05);After 8 weeks of treatment,compared with the model group,the swelling degree of ankle joint in the Yiqi Yangyin Tongluo Formula group was significantly reduced,and the difference was statistically significant(P<0.05);Compared with the model group,the swelling degree of ankle joint in the methotrexate group and the methotrexate+Yiqi Yangyin Tongluo Formula group decreased significantly(P<0.05);There was no statistical difference in ankle swelling between the Yiqi Yangyin Tongluo Formula group and the methotrexate group(P>0.05);The ankle joint swelling in the combined group was significantly lower than that in the methotrexate group,and there was significant statistical difference in the results.3.In the model group,the cartilage tissue structure of rats was significantly damaged,and synovial cells were also observed to proliferate,deform,and fibrosis,in which there was significant inflammatory infiltration;In the model group,the surface of synovial tissue was thickened,inflammatory infiltration was obvious,synovial cells were disordered,and pannus could be observed;In the two treatment groups,the cartilage surface was smooth,no structural damage was found,the synovial hyperplasia was not obvious,and the cell arrangement was not disordered;The cartilage tissue of the combined treatment group was smooth without damage,and the synovial structure was basically normal,with regular arrangement and normal shape.4.The comparison results showed that the C-reactive protein index of the model group was significantly higher than that of the normal group(P<0.05);The CRP indexes of the Yiqi Yangyin Tongluo Formula group were significantly lower than those of the model group,with statistical differences(P<0.05);The CRP level in the methotrexate group and the combined treatment group was significantly lower than that in the model group(P<0.05);The CRP level in the methotrexate group was lower than that in the Yiqi Yangyin Tongluo Formula group,with no significant difference(P<0.05);Compared with the methotrexate group,the level of C-reactive protein in the Yiqi Yangyin Tongluo Fang+methotrexate group was significantly lower than that in the methotrexate group,and there was a statistical difference(P<0.05).5.Model group IL-6,IL-17,TNF-αThe level was significantly higher than that of the normal group(P<0.05);Compared with the model group,these indexes of the Yiqi Yangyin Tongluo Formula group were significantly reduced(P<0.05);Compared with the model group,the levels of IL-6,IL-17 and TNF in the methotrexate group,methotrexate+Yiqi Yangyin Tongluo Formula group-αThere was a significant difference between the two groups(P<0.05);Compared with the methotrexate group,the IL-6,IL-17,TNF in the Yiqi Yangyin Tongluo Formula group and the methotrexate group-αThe level was similar,and the difference was not statistically significant(P>0.05);Compared with methotrexate group,Yiqi Yangyin Tongluo Fang+methotrexate group IL-6,IL-17,TNF-αThe level was significantly lower than that of methotrexate group(P<0.05).6.The protein levels of IL7 A,IL17RA and MMP9 in the model group were significantly higher than those in the normal group(P<0.05);Compared with the model group,the protein levels of IL7 A,IL17RA and MMP9 in the Yiqi Yangyin Tongluo Formula group were significantly lower(P<0.05);Compared with the model group,the protein levels of IL7 A,IL17RA and MMP9 in the methotrexate group,methotrexate+Yiqi Yangyin Tongluo Formula were significantly lower(P<0.05);The protein levels of IL7 A,IL17RA and MMP9 in the Yiqi Yangyin Tongluo Formula group and the methotrexate group were similar,and there was no significant difference(P>0.05);Compared with the methotrexate group,the levels of IL-7A,IL-17 RA and MMP9 in the Yiqi Yangyin Tongluo Fang+methotrexate group were significantly lower than those in the methotrexate group(P<0.05).7.Compared with the normal group,the m RNA levels of IL7 A,IL17RA and MMP9 in the model group were significantly higher(P<0.05);Compared with the model group,the m RNA levels of IL7 A,IL17RA and MMP9 in the Yiqi Yangyin Tongluo Formula group were significantly lower(P<0.05);Compared with the model group,the m RNA levels of IL7 A,IL17RA and MMP9 in the methotrexate group and the methotrexate+Yiqi Yangyin Tongluo Formula group were significantly lower(P<0.05);Compared with the methotrexate group,the m RNA levels of IL7 A,IL17RA and MMP9 in the Yiqi Yangyin Tongluo Recipe group and the methotrexate group were similar,and the difference was not statistically significant(P>0.05);Compared with the methotrexate group,the m RNA levels of IL7 A,IL17RA and MMP9 in the Yiqi Yangyin Tongluo Fang+methotrexate group were significantly lower than those in the methotrexate group(P<0.05).8.After successful modeling,the expression of Keap1 protein in the model group was significantly higher than that in the normal group,while Nrf2/Ho1 was on the contrary,with a statistically significant difference(P<0.01).Compared with the model group,the level of Keap1 protein in the Yiqi Yangyin Tongluo Formula group was significantly lower,and the level of Nrf2/Ho1 protein was significantly higher,with a statistically significant difference(P<0.05);Compared with the model group,the levels of Keap1 protein in the methotrexate group and the methotrexate+Yiqi Yangyin Tongluo Formula group were significantly lower,and the levels of Nrf2/Ho1 protein were significantly higher(P<0.05);Compared with the methotrexate group,the Nrf2-Keap1-Ho1 protein level in the Yiqi Yangyin Tongluo Formula group and the methotrexate group was equivalent,and there was no significant difference(P>0.05);Compared with the methotrexate group,Keap1 in the Yiqi Yangyin Tongluo Fang+methotrexate group was significantly lower than that in the methotrexate group,and Nrf2/Ho1 was significantly higher than that in the methotrexate group,with a statistically significant difference(P<0.05).9.The level of Keap1 m RNA in the model group was significantly higher than that in the normal group after modeling,and the level of Nrf2/Ho1 m RNA was significantly lower than that in the model group(P<0.01).Compared with the model group,the level of Keap1 m RNA in the Yiqi Yangyin Tongluo Formula group was significantly lower,while the level of Nrf2/Ho1 m RNA was on the contrary,and the results were statistically different(P<0.05);Compared with the model group,the level of Keap1 m RNA in the methotrexate group and the combination treatment group decreased to a certain extent,while the level of Nrf2/Ho1 m RNA was on the contrary,with a significant difference(P<0.05);The Nrf2-Keap1-Ho1 m RNA level of Yiqi Yangyin Tongluo Formula group and methotrexate group was similar,and there was no statistical difference(P>0.05);Compared with the methotrexate group,the Keap1 in the combined treatment group was significantly lower,and Nrf2/Ho1 was significantly higher,both of which were statistically significant(P<0.05).Conclusion:1.The Yiqi Yangyin Tongluo Formula can improve inflammation in rats with rheumatoid arthritis,and the Yiqi Yangyin method is an effective method for treating rheumatoid arthritis.2.During the process of rheumatoid arthritis,the expression of MMP9,CXCR4,CXCL10,CXCL13,TNFSF11,CCR5,ITGAX,TIMP1,and SPP1 genes is significantly upregulated,while ADIPOQ is downregulated.The combination of five traditional Chinese medicines in the Yiqi Yangyin Tongluo formula acts on the top 10 central genes of RA,among which MMP9 and CXCR4 play a key role in the core targets of the five traditional Chinese medicine diseases and are recommended as key genes.MMP9 was significantly enriched in three signaling pathways,including IL-17 signaling pathway,tumor necrosis factor(TNF)signaling pathway and leukocyte trans endothelial migration,while CXCR4 was significantly enriched in the interaction of viral protein with cytokines and cytokine receptors,chemokine signaling pathway,human immunodeficiency virus 1 infection,leukocyte trans endothelial migration,and cytokine cytokine receptor interaction.This indicates that the Yiqi Yangyin Tongluo Formula may treat rheumatoid arthritis by targeting MMP9 and CXCR4 to regulate the IL-17 signaling pathway,TNF signaling pathway,and chemokine signaling pathway.3.The therapeutic effect of Yiqi Yangyin Tongluo Formula on rheumatoid arthritis is roughly the same as that of Western medicine methotrexate.4.The combination of Yiqi Yangyin Tongluo Formula and Methotrexate has a slightly better therapeutic effect on rheumatoid arthritis than the single use of Western medicine Methotrexate.5.Yiqi Yangyin Tongluo Formula can inhibit IL-17,IL-6,TNF-α Factor levels can improve inflammation in rheumatoid arthritis rats.6.The Yiqi Yangyin Tongluo Formula may be one of the anti-inflammatory mechanisms of rheumatoid arthritis by inhibiting the IL7A/IL17RA/MMP9 signaling pathway.7.The inhibition of Nrf2/Keap1/Ho1 signaling pathway by the Yiqi Yangyin Tongluo Formula may be one of the anti-inflammatory mechanisms of rheumatoid arthritis.