Background:Graves’ disease(GD)is a systemic autoimmune disorder characterized by the infiltration of thyroid antigen-specific T cells into thyroidstimulating hormone receptor-expressing tissues.Stimulatory autoantibodies(Ab)in GD activate the TSH-R leading to thyroid hyperplasia and unregulated thyroid hormone production and secretion.The prevalence of GD in my country is about 0.53%,and the annual incidence is about 20/100,000 to 30/100,000.The disease has a long course,is easy to recur,and has obvious clinical symptoms and signs.It has a great impact on the life,work,and appearance of the patient.Traditional Chinese medicine has accumulated certain experience in the treatment of Graves’ disease,and has certain advantages in improving symptoms,anti-recurrence,and shortening the course of treatment.In our many years of clinical practice,we have summarized and formed a diagnosis and treatment plan for Graves’ disease combined with disease and syndrome.In the treatment,the combination of traditional Chinese and Western medicine is the main method.On the basis of the improvement indicators of Western medicine,the treatment is based on the syndrome differentiation of the disease,the person and the time,and a significant effect has been achieved.Preliminary research results show that the combination of disease and syndrome with sequential syndrome differentiation treatment can improve the long-term remission rate of Graves’ disease and reduce the recurrence rate.However,there is still a lack of high-quality clinical research evidence and the mechanism of action is not yet clear,so it cannot be widely used.Therefore,this study relies on the open project of the national clinical research base of traditional Chinese medicine(project batch number:JD2019SZXZD08),confirms the effect and advantages of disease syndrome combined with comprehensive therapy in the treatment of Graves’ disease,and discusses the law of traditional Chinese medicine and the relevant curative mechanism of core prescriptions,as well as the relevant biomarkers and their metabolic pathways.Chapter 1:Clinical observation on the treatment of Graves’ disease by combining disease and syndrome with sequential syndrome differentiationObjective:To evaluate the efficacy and safety of disease-syndrome combined with sequential syndrome differentiation in the treatment of Graves’ disease.Methods:A multicenter,randomized,open-label,case-control study design was used to randomly assign Graves’ disease patients who met the inclusion and exclusion criteria to the treatment group and the control group in a ratio of 3:1.All patients were given the guidance of diet and lifestyle.The patients in the treatment group were treated with a combination of disease and syndrome differentiation,while the control group was treated with conventional western medicine for 24 weeks.The baseline indicators,efficacy evaluation indicators and safety indicators of the two groups were observed.The baseline indicators included gender,age,height,weight,blood pressure,heart rate and other general conditions,thyroid volume,liver function,white blood cell count,etc.;efficacy evaluation indicators included Primary efficacy indicators:FT3,FT4,TSH,secondary efficacy indicators:aTPO,aTG,TRAb,ALT,AST,white blood cell count,exophthalmos grade,thyroid volume,hyperthyroidism diagnostic index score,TCM symptom score;safety indicators include general physical examination Project examination,renal function(BUN,Cr),urine routine,stool routine,electrocardiogram,adverse events,etc.Results:①Baseline:The two groups were comparable in age,gender,height,blood pressure,liver function,thyroid volume and other baseline data(P>0.05).②Clinical efficacy observation:The combination of disease and syndrome treatment with sequential syndrome differentiation can reduce the levels of FT3 and FT4(P<0.05),reduce the levels of TRAb,aTG and aTPO(P<0.05),and improve the negative rate of TRAb(P<0.05).It can reduce the thyroid volume in GD patients(P<0.05),improve exophthalmos(P<0.05),reduce TCM symptom score and hyperthyroidism diagnostic index score(P<0.05),and the treatment group can improve the above indicators,symptoms and signs.All aspects were better than the control group(P<0.05).The total effective rate of the treatment group was 94.5%,and that of the control group was 84.74%.The total effective rate of the treatment group was significantly higher than that of the control group(P<0.05).The recovery time in the treatment group was significantly shorter than that in the control group(P<0.05).③Safety observation:the incidence of adverse reactions in the treatment group was 15.8%,and the incidence of adverse reactions in the control group was 32.2%,P<0.01.Conclusion:Combination of disease and syndrome with sequential syndrome differentiation in the treatment of Graves’ disease has significant curative effect and good safety.It can not only improve the level of thyroid function,but also regulate immunity,improve clinical symptoms and signs of patients,reduce the incidence of adverse reactions,and shorten the time for thyroid function to return to normal.Chapter 2:The medication rule of disease-syndrome combined with sequential syndrome differentiation based on data mining in the treatment of Graves’ diseaseObjection:To summarize the medication rules and core prescriptions for the treatment of Graves’ disease by combining disease and syndrome with sequential syndrome differentiation,and to provide data for network pharmacology research.Methods:The prescriptions of patients with Graves’ disease included in the first part of this study were collected by data mining method,and the extracted contents included general information(name,gender,age,etc.),clinical diagnosis and treatment information(western medicine diagnosis,traditional Chinese medicine diagnosis,prescription drug name and dosage,etc.)),establish a database for the extracted content and standardize the database,use Microsoft Office Excel to conduct descriptive statistical analysis on the frequency of medication,the four qi,five flavors,meridian return,and efficacy of the drug,and use IBM SPSS Statistics 22.0 software to make factor analysis of the drugs included in the prescription.Component diagram,dendrogram and cluster analysis,using IBM SPSS Modeler 18.0 software to make network diagram and association rule analysis of Apriori algorithm.Results:The core prescriptions were sorted out:the commonly used drugs for the treatment of Graves’ disease by combining disease and syndrome with sequential syndrome differentiation included licorice,Bupleurum,jujube,perilla,lily,Anemarrhena,white peony,Zhe Fritillaria,Scrophulariaceae,ebony,and tuckahoe,Huanglian,etc.The properties of the medicine are mostly warm,cold and flat;the taste of the medicine is mainly sweet,bitter and acrid;the main meridian is the liver meridian;the main effect of the medicine is the heat-clearing medicine and the tonic medicine.Classics,cold and temperature are unified,and the period is flat.High-frequency drug correlation analysis,factor analysis,cluster analysis,etc.show that the common prescriptions for treating Graves disease by combining disease and syndrome with sequential syndrome differentiation include Xiaochaihu Decoction,Baihe Zhimu Decoction,Banxia Houpu Decoction,Wuling Powder,Fangji Huangqi Decoction,etc.Commonly used drug pairs include Bupleurum-white peony,Lily-Anemarrhena,PoriaAlismatis,Shouwu Teng-Suanzaoren,Shanci Mushroom-Cat’s Claw,etc.The core prescriptions are Bupleurum,Perilla,Coptidis,Scrophularia,Jujube,White Peony,Black Plum,Lily,Anemarrhena,Papaya,Licorice,and Fritillaria.Conclusion:Combination of disease and syndrome with sequential syndrome differentiation in the treatment of Graves’ disease should combine the nature and flavor of ginseng,take into account the return of meridians,unify cold and temperature,and take a flat period.The selection of prescriptions and medication are flexible and changeable,focusing on syndrome differentiation and treatment due to disease,person,and time,and based on the research results,the core prescription for the treatment of Graves disease is obtained by combining disease and syndrome with sequential syndrome differentiation.Chapter 3:The mechanism of action of core prescriptions in the treatment of Graves’ disease based on network pharmacology researchObjection:To explore the potential pharmacological mechanism of the core prescription of disease-syndrome combined with sequential syndrome differentiation therapy in the treatment of Graves’ disease.Methods:Screen the main active ingredients of all drugs in the core prescription obtained in the second part of this study through TCMSP database,integrate and de duplicate the active ingredients of all drugs and relevant target information,and then conduct information standardization through UniProt protein database.The disease targets of Graves disease were obtained by searching the databases of Drugbank,TTD,OMIM and Genecards,and the above disease target information was integrated and de duplicated.Then the target name was standardized by using UniProt protein database to establish the target database of Graves’ disease.Take the intersection between the core prescription and the disease target,then import the common target of the core prescription Graves’ disease into the string database to obtain the PPI network,and then import the data of the drug’s active ingredients,intersection targets and disease targets into Cytoscape 3 8.2 construct the network diagram of "prescription active ingredient disease target",and finally conduct the enrichment analysis of go and KEGG pathways through Metascape database.Results:Core active ingredient:Based on the research of network pharmacology,it is found that the core effective components of the core prescription are quercetin、Kaempferol、luteolin、beta-sitosterol、naringenin、beta-carotene and stigmasterol can reduce the.level of thyroid hormone in serum and regulate immunity;The core targets include PTGS2,ESR1,PPARG,ESR2,ADRB2,KDR,TOP2A,CHRM2,etc.Go enrichment analysis found that the core prescription can affect a variety of biological processes such as the regulation of inflammatory response,the regulation of intercellular adhesion,the metabolic process of reactive oxygen species,the regulation of leukocyte cell adhesion and T cell activation.It can affect cell components such as secretory granule lumen,vesicle lumen,membrane raft,membrane micro region and membrane region,and can affect cytokine activity,cytokine receptor binding Receptor ligand activity,signal receptor activator activity,antioxidant activity and other molecular functions;KEGG pathway enrichment analysis found that the core prescription mainly acts on TNF signaling pathway,IL-17 signaling pathway and NF-κB signaling pathway to treat GD also involves AGE-RAGE signaling pathway,toll like receptor signaling pathway,T cell receptor signaling pathway,Th17 cell differentiation pathway,PI3K Akt signaling pathway and other signaling pathways.Conclusion:The core prescription can synergistically treat GD through multicomponent,multi-target,and multi-pathway,and its intervention mechanism mainly involves immune regulation,inflammatory response,oxidative stress and so on.Chapter 4:Study on the mechanism of disease-syndrome combined with sequential syndrome differentiation in the treatment of Graves disease based on non-targeted metabolomicsObjection:To explore the possible biomarkers and metabolic pathways for the treatment of Graves’ disease by combining disease and syndrome with sequential syndrome differentiation.Methods:Collect the serum samples of the disease syndrome combined with comprehensive therapy group before and after treatment,and detect the metabolites by ultra-high performance liquid chromatography-mass spectrometry.The differential metabolites were screened by univariate statistical analysis(t-test and multiple of variation analysis)and multivariate statistical analysis(unsupervised principal component analysis(PCA),supervised partial least squares analysis(PLS-DA)and orthogonal partial least squares analysis(OPLS-DA),Lipidmaps、METLIN database and self-built database were used for qualitative analysis,and finally the pathway enrichment analysis of differential metabolites was carried out through KEGG database.Results:Differential metabolite screening:in the non-targeted metabolomics study,103 significantly different metabolites in the disease syndrome combined with comprehensive therapy group before and after treatment were screened,with P≤0.05 as the threshold,and finally 12 related metabolic pathways were obtained.In this study,the metabolites that can be enriched into the pathway include phosphatidylcholine,phosphatidylethanolamine,lysophosphatidylethanolamine,sphingosine phosphate 1,sphingomyelin,thyroxine,etc;The enriched metabolic pathways include glycerol phospholipid metabolism,sphingolipid signaling pathway(sphingolipid metabolism),autoimmune thyroid diseases,thyroid hormone signaling and so on.Conclusion:Sphingosine phosphate 1 may be a potential biomarker for the treatment of Graves’ disease by combining disease and syndrome with sequential syndrome differentiation.The intervention mechanism mainly involves sphingolipid metabolism and glycerophospholipid metabolism.Through the above research,it is confirmed that the use of disease-syndrome combined with sequential syndrome differentiation in the treatment of Graves’ disease has significant curative effect and good safety,and fully utilizes the advantages of traditional Chinese medicine treatment;the traditional Chinese medicine drug law of this therapy and the related efficacy mechanism of core prescriptions are clarified;The therapy treats relevant biomarkers of Graves’ disease and its metabolic pathways. |