| Purpose:Through the retrospective analysis of the clinical data,TCM syndrome characteristics and comorbidities of patients with rheumatoid arthritis due to deficiency of both qi and yin,we explored the correlation between this syndrome type and the patients’ age,gender,course of disease,clinical manifestations,disease activity,laboratory indicators,combined diseases,etc.,and compared them with other syndrome types.It can provide certain evidence-based medicine evidence for TCM syndrome differentiation and treatment of rheumatoid arthritis due to deficiency of both qi and yin.The network pharmacology was used to analyze the mechanism of Yiqi Yangyin Tongluo Decoction in the treatment of rheumatoid arthritis.Animal experiments were conducted to evaluate the efficacy of Yiqi Yangyin Tongluo Decoction in the treatment of rheumatoid arthritis in rats,and the mechanism was explored based on TNF-α/MAPK/JNK signaling pathway.Materials and methods:1.Clinical observation: A total of 237 inpatients with rheumatoid arthritis from January 2019 to June 2021 in the Department of Rheumatology,Affiliated Hospital of Liaoning University of Chinese Medicine were included.The main observation items were as follows:(1)General situation: gender,age and course of disease;(2)Clinical manifestations: joint symptoms: the number of joint tenderness and joint swelling;Systemic symptoms: dry mouth and eyes,physical and mental fatigue,muscle soreness,soreness and weakness of waist and knees,shortness of breath,cough,weight loss,spontaneous sweating,night sweats,poor insomnia,and much nocturia.(3)Laboratory indicators and related examinations: inflammation indicators(ESR,CRP),immunological indicators(RF,CCP),and disease activity(DAS28 score);(4)Combined diseases: osteoporosis,osteoarthritis,pulmonary interstitial changes,Sjogren’s syndrome and type 2 diabetes mellitus.SPSS 22.0 statistical software was used to analyze and compare the results.2.Network pharmacology research: Through the TCM system pharmacology database(TCMSP)and the analysis platform and the Herbal Pharmacology database(HERB),and in combination with the literature,select the component targets,supplement the chemical components that have not been retrieved and find the corresponding action targets,and use the Uniport database to standardize the target information,use the Cytasape3.9.1 software to build the "drug-active component-target" relationship diagram,and screen the important active components of traditional Chinese medicine treatment according to the network topology parameters.Through Gene Cards,TTD,OMIM,Pharm Gkb and Drug Bank databases,the relevant target genes of rheumatoid arthritis diseases were obtained,the Wayne map was drawn,and the common targets of Yiqi Yangyin Tongluo Formula and rheumatoid arthritis were obtained.Through the String database,the protein interaction network analysis of the common targets obtained was carried out,and the core targets of Yiqi Yangyin Tongluo Formula in the treatment of rheumatoid arthritis were obtained according to the network topology parameters,Through the study of GO biological function and the enrichment analysis of KEGG signal pathway,the relevant signal pathway of Yiqi Yangyin Tongluo Formula in the treatment of rheumatoid arthritis was screened.3.Animal experiments: A CIA rat model of rheumatoid arthritis was established and 50 SPF SD rats were randomly divided into a normal control group(CON group),a model group(CIA group),a Yiqi Yangyin Tongluo decoction group(TCM group),a methotrexate group(MTX group),and a decoction of Yiqi Yangyin Tongluo decoction plus methotrexate group(TCM+MTX group),10 rats in each group.After continuous administration for 8 weeks,serum and joint synovial tissue were taken.The general state,joint swelling and arthritis index were observed,and the pathological sections of joints were prepared.Serum levels of interleukin-1β(IL-1β),interleukin-6(IL-6)and interleukin-17(IL-17)were measured to evaluate the inflammatory state of joints in rats.The expressions of TNF-a,MAPK,JNK,and P-JNK proteins as well as the gene expressions of TNF-a,MAPK,IL-1β and IL-6 in the synovial tissue of rats were detected by Western blot and QPCR,respectively.Result:1 clinical research results:(1)RA with deficiency of both qi and yin has no obvious relationship with gender and age,but the course of disease is positively correlated with deficiency of both qi and yin.Patients with deficiency of both qi and yin have a longer course of disease than those without deficiency of both qi and yin.(2)There is no obvious correlation between inflammation index(ESR,CRP)and immunological index(RF,CCP)and RA with deficiency of both qi and yin.(3)The relationship between RA with deficiency of both qi and yin is not obvious,but negatively related to the number of joint swelling and disease activity.The number of joint swelling and disease activity in RA patients with deficiency of both qi and yin are lower than those without deficiency of both qi and yin.(4)RA patients with deficiency of both qi and yin are more likely to be accompanied by osteoporosis,pulmonary interstitial changes and Sjogren’s syndrome than those without deficiency of both qi and yin,and have no obvious correlation with osteoarthritis and type 2 diabetes.(5)Compared with non-deficiency of both qi and yin,RA with deficiency of both qi and yin is more prone to dry mouth and eyes,fatigue,muscle aches,soreness of waist and knees,shortness of breath,cough,emaciation,spontaneous sweating,night sweats,poor sleep and nocturia.2.Research results of network pharmacology:(1)The results of the "drug active ingredient target" in the treatment of RA with Yiqi Yangyin Tongluo Formula showed that quercetin,luteolin,kaempferol β-Components such as sitosterol and skullcap are the main active components of the prescription for supplementing qi,nourishing yin and dredging collaterals in the treatment of RA.(2)The target interaction network regulated by the active ingredients of Yiqi Yangyin Tongluo Recipe in the treatment of RA shows that PTGS2,PTGS1,TNF,JUN,IL-6,MAPK1,IL-1β,AKT1,etc.are the key targets of this network,which may play a major role in the treatment of RA by Yiqi Yangyin Tongluo Recipe.(3)Enrichment of GO function of the key target of Yiqi Yangyin Tongluo Recipe in treating RA shows that Yiqi Yangyin Tongluo Recipe may treat RA through biological processes such as signal transduction,positive regulation of cell proliferation,positive regulation of gene expression,negative regulation of apoptosis,inflammatory reaction,reaction to lipopolysaccharide,etc.Most of its targets are in plasma membrane,cell fluid,cytoplasm,extracellular space,cell surface,membrane raft,etc.,with enzyme binding and protein serine/threonine/lipopolysaccharide.KEGG results show that Yiqi Yangyin Tongluo recipe can treat RA by interfering with cancer pathways,lipid and atherosclerosis,AGE-RAGE signaling pathway,PI3K-Akt signaling pathway,IL-17 signaling pathway,apoptosis,TNF signaling pathway and other signaling pathways in diabetic complications.3.Results of animal experiments(1)Yiqi Yangyin Tongluo recipe can significantly improve the joint swelling degree of RA rats,and HE staining can be used to observe that inflammatory cell infiltration,synovial tissue proliferation,pannus formation and cartilage tissue destruction can be effectively alleviated in the joints of rats.Compared with methotrexate alone,Yiqi Yangyin Tongluo Recipe combined with methotrexate has a better effect.(2)According to the results of ELISA,Yiqi Yangyin Tongluo Recipe can reduce the expression of IL-1β,IL-6 and IL-17 in the serum of RA rats,regulate the level of inflammatory factors,and achieve the effect of controlling inflammation.Compared with methotrexate alone,Yiqi Yangyin Tongluo Recipe combined with methotrexate has a better effect.(3)According to the experimental results of Western blot and Q-PCR,the model animal in this experiment is RA rat model,and a large number of MAPK-related proteins are found in the relevant results of target protein analysis,which proves that the activation of TNF-α/MAPK/JNK signaling pathway causes inflammatory reaction and forms rheumatoid arthritis.Yiqiyintongluo recipe can reduce the expression levels of TNF-a,MAPK,JNK,P-JNK protein and TNF-a,MAPK,IL-1β,IL-6 m RNA in synovial tissue of RA rats,and compared with methotrexate alone,Yiqiyintongluo recipe combined with methotrexate has a better effect.It is proved that Yiqi Yangyin Tongluo recipe can play a role in treating RA through TNF-α/MAPK/JNK signal pathway.Conclusions:1.RA with deficiency of both qi and yin has no obvious relationship with gender,age,inflammatory index(ESR,CRP),immunological index(RF,CCP)and joint tenderness,but it is positively correlated with the course of disease,and negatively correlated with joint swelling and disease activity.Compared with non-deficiency of both qi and yin,RA patients with deficiency of both qi and yin are more likely to be accompanied by osteoporosis,pulmonary interstitial changes and Sjogren’s syndrome,which is related to osteoarthritis and type 2 diabetes.In terms of clinical symptoms,RA with deficiency of both qi and yin is more prone to dry mouth and eyes,fatigue,muscle aches,soreness of waist and knees,shortness of breath,cough,emaciation,spontaneous sweating,night sweats,poor sleep and nocturia.2.Using the method and technology of network pharmacology,it is predicted that the active ingredients of Yiqi Yangyin Tongluo recipe may be quercetin,luteolin,kaempferol,β-sitosterol,wogonin,etc.,and its target points may be PTGS2,PTGS1,TNF,JUN,IL-6,MAPK1,IL-1,AKT1,etc.,through GO enrichment analysis.Its action pathway may be through regulating cancer pathway,lipid and atherosclerosis,AGE-RAGE signaling pathway in diabetic complications,PI3K-Akt signaling pathway,IL-17 signaling pathway,apoptosis,TNF signaling pathway,etc.Therefore,the treatment of RA with Yiqiyangyin Tongluo Recipe is realized through multiple components,multiple targets and multiple pathways.3.Combined with the results of network pharmacology,CIA rat model was made,and the mechanism of RA treatment was finally verified by animal experiment.Through the analysis of tissue,protein and gene expression,it is shown that Yiqi Yangyin Tongluo Recipe can reduce the activity of TNF-α/MAPK/JNK signal pathway by acting on it,so as to reduce inflammatory factors,improve inflammatory response and finally achieve the purpose of treating RA.Moreover,Yiqi Yangyin Tongluo Recipe combined with Methylamine Dieyin has a better therapeutic effect. |
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