Constructing Evaluation Index Of Curative Effect Of Kidney-Yang Deficiency Syndrome Based On Three-dimensional Hybrid Method

Treatment based on syndrome differentiation is an important feature of Traditional Chinese Medicine.Syndrome differentiation is the core content of treatment,which is the basis of clinical diagnosis and treatment of TCM.In the clinical research of TCM,the objectivity,standardization and scientificity of the evaluation of the curative effect of Syndrome have always been the key issues affecting the clinical evaluation of TCM.Therefore,it is of great significance to perfect and establish the technique and method of Tcm Syndrome curative effect evaluation for constructing the evaluation system embodying TCM characteristics.The kidney-yang Deficiency Syndrome is one of the common clinical syndromes.This study takes the kidney-yang Deficiency Syndrome as an example to carry out the research on the therapeutic index of the kidney-yang Deficiency Syndrome.Therefore,based on the relevant research results of the "Diagnostic Criteria for Kidney-Yang Deficiency Syndrome" developed in the early stage,and in combination with the relevant requirements of syndrome evaluation in the "Guiding Principles for Clinical Research of Syndrome Type Chinese Drugs" promulgated by the National Medical Products Administration,our research group further studies the efficacy evaluation indicators of Kidney-Yang deficiency syndrome.This study refers to the development process and methods in the "Technical Specifications for the Development of Core Indexes for Clinical Trials of Traditional Chinese Medicine",and forms the efficacy evaluation index of Kidney-Yang deficiency syndrome through the mixed research methods of "combination of qualitative and quantitative research methods","combination of subjective and objective indicators" and "combination of doctor-patient evaluation".And we explore the effect and improvement of warming Kidney-Yang drugs on metabolomics related indexes,and to analyze the possible pathway and therapeutic effect index of warming Kidney-Yang drugs,so as to provide ideas and methods for formulating and applying the therapeutic effect evaluation standard of syndrome,which provides the basis for further research on the pathway and target of kidney-yang Deficiency Syndrome.This study consists of two parts.Part I:Adopting the research ideas and methods of "combination of qualitative and quantitative research methods","combination of subjective and objective indicators",and"combination of doctor-patient evaluation" to formulate the Core Outcome Set of kidney-yang deficiency syndrome to provide for the formulation of syndrome curative effect evaluation standards Methodological reference.Part Ⅱ:Exploring the influence and improvement of warming Kidney-Yang drugs on metabolomics related indicators,to analyze the possible action pathways and efficacy indicators of warming Kidney-Yang drugs from the metabonomics level,and to provide some ideas for the selection of objective indicators.(一)Study on the efficacy evaluation index of Kidney-Yang deficiency syndrome1 ObjectiveIn combination with the relevant requirements for syndrome evaluation in the "Technical Guidelines for the Clinical Research of Syndrome-type TCM New Drugs" promulgated by the National Medical Products Administration,referring to the methodological requirements of the"Technical Specifications for the Development of Core Outcome Set for Clinical Trials of Traditional Chinese Medicine",The three-dimensional mixed research method of" combination of qualitative and quantitative research methods ","combination of subjective and objective indicators",and"combination of doctor-patient evaluation" has been developed to develop the Core Outcome Set of Kidney-Yang deficiency syndrome to standardize the selection and application of curative effect evaluation indexes in clinical research of Kidney-Yang deficiency syndrome.It provides a basis for the research of " Kidney-Yang Deficiency Syndrome Efficacy Evaluation Criteria",and at the same time provides ideas and methods for the study of the core index set of syndromes.2 Methods2.1 Referring to the methodological requirements of the technical specification for the development of core index set of clinical trials of traditional Chinese medicine,the outcome index list of Kidney-Yang deficiency syndrome was preliminarily constructed through literature research,questionnaire survey and expert interview;Through Delphi method questionnaire survey to screen the core indicators and determine the Core Outcome Set of Kidney-Yang deficiency syndrome.Construct the core index set of Kidney-Yang deficiency syndrome of "combination of qualitative and quantitative research methods" and "combination of subjective and objective indicators".2.2 Referring to the model of MYMOP scale,investigate and summarize the clinical symptoms that patients focus on,and form the patient efficacy selfassessment scale,so as to construct the efficacy evaluation model of"combination of doctor-patient evaluation".2.3 Finally,based on the three-dimensional hybrid research method,a core index set of Kidney-Yang deficiency syndrome of "combination of qualitative and quantitative research methods","combination of subjective and objective indicators and combination of doctor-patient evaluation" is preliminarily formed,which will be used as the efficacy evaluation index of Kidney-Yang deficiency syndrome to form the efficacy evaluation scale of Kidney-Yang deficiency syndrome.3 Result3.1 Literature research3.1.1 Evaluation of curative effect of Kidney-Yang deficiency syndromeIn the early stage of this study,through literature retrieval and the process of term splitting and term standardization,through expert discussion and the combination of on-site Delphi and Brainstorming,the preliminary screening of items was completed.including 22 items of disease location and 15 items of disease nature.3.1.2 Construct the core index item pool of disease-related Kidney-Yang deficiency syndromeA total of 4425 literatures were obtained in the initial examination.According to the inclusion and exclusion criteria.215 RCTs were finally included.including 2 English literatures.A total of 314 outcome indicators were included in 215 RCTs.The extracted index names are standardized and unified,classified and differentiated to form the original item pool of core indicators of Kidney-Yang deficiency,with a total of 85 items,including 16 items with frequency≥5 and 69 items with frequency<5,so as to select indicators for questionnaire and interview research in the next stage.3.2 Questionnaire survey and Delphi method implementation3.2.1 Results of the first round of patient questionnaire surveyIn the first round of the questionnaire survey,the patients selected the symptoms of kidney-yang deficiency which were most expected to be improved.The disease position entrys are:Nocturnal Frequent micturition,Asthenia,Diarrhea.The pathological items are:Chills,Cold hands and feet.3.2.2 Results of the first round of expert questionnaire and interviewThe first round of expert questionnaire and interview research included 10 experts,and the positive coefficient of this round was 100%.3.2.2.1 Results of each item in the expert questionnaireScreening results of TCM symptom pool of Kidney-Yang deficiency syndrome:(disease position)the average ranking of the top three is:frequent nocturnal urination,waist pain and impotence,and the clinical significance ranking of the top four is:frequent nocturnal urination,impotence,clear urination and waist pain.Calculated coefficient of variation(CV):CV=0.13<0.25.The results show that experts have little differences on items.Coordination coefficient(W):W is 0.35,P=0.001<0.01.The results show that the expert coordination degree is good and the evaluation conclusion is credible.The top three on average(disease type)are:fear of cold,diarrhea on the fifth watch and Qingxi under the belt.The top three in clinical significance:fear of cold.Qingxi under the belt and diarrhea in the fifth shift.Calculated coefficient of variation(CV):CV=0.14<0.25.The results show that experts have little differences on items.Coordination coefficient(W):W is 0.27,P=0.004<0.01.The results show that the expert coordination degree is good and the evaluation conclusion is credible.Screening of original items of disease related indexes of Kidney-Yang deficiency syndrome:in the index pool with frequency≥5,experts selected the top three:sex hormone index,quality of life score and semen parameters;In the index pool with frequency less than 5,experts choose the top three:thyroid hormone,sexual living standard and sexual function.Other items:Syndrome Evaluation Method:Based on personal experience,supplemented by reference to the published syndrome efficacy evaluation standards(50%);Onset time of clinical medication:"2 weeks"(40%)and "4 weeks"(40%);Course of treatment(time for completely controlling the condition with drugs):"8 weeks"(70%);Efficacy evaluation method:"disappearance rate/normalization rate of multiple symptoms(core indicators)"(60%);Objective indexes may be related to the severity of syndrome(80%).3.2.3 The second round of patient questionnaire surveyCombined with the results of the first round of questionnaire and interview,the second round of questionnaire survey was conducted,including 100 patients.Among the TCM symptom indicators of KidneyYang deficiency syndrome,2-5 indicators that are most expected to be improved are selected.The top four frequency are:frequent urination at night,fatigue,low sexual desire,sore waist and knees.3.2.4 The second round of expert questionnaire surveyIn this round,100 experts with senior professional titles from all over the country were included for electronic questionnaire survey.The positive coefficient is 100%.Screening results of TCM symptom pool of Kidney-Yang deficiency syndrome:(disease location)the average ranking of the top three is:frequent urination at night,waist pain and clear and long urination,and the clinical significance ranking of the top three is:frequent urination at night.waist pain and clear and long urination.Calculated coefficient of variation(CV):CV=0.1 2<0.25.The results show that experts have little differences on items.Coordination coefficient(W):W is 0.25.P<0.01.The results show that the expert coordination degree is good and the evaluation conclusion is credible.The top three on average(disease type)are:chills.diarrhea on the fifth watch and fatigue.The top three in clinical significance:chills,diarrhea in the fifth shift and fatigue.Calculated coefficient of variation(CV):CV=0.12<0.25.The results show that experts have little differences on items.Coordination coefficient(W):W is 0.1 9.P<0.01.The results show that the expert coordination degree is good and the evaluation conclusion is credible.Screening of disease-related indicators of Kidney-Yang deficiency syndrome:results there were four items with more than 50%,namely:quality of life score(scale form)(84%),sexual life standard(sexual life satisfaction,sexual life log.etc.)(79%);Sex hormone index(60%);International erectile dysfunction index(IIEF-5)scale score(52%).Other items:mainly referring to the published syndrome efficacy evaluation criteria,supplemented by personal experience(50%);Onset time of clinical medication:"2 weeks"(50%);Course of treatment(time for completely controlling the disease with drugs):"8 weeks"(46%);Evaluation method:"clinical control rate(disease remission and/or partial remission and/or stable condition for at least 4 weeks)"(44%);Objective indexes may be related to the severity of Kidney-Yang deficiency(56%);When selecting the above objective indexes and scales as the curative effect judgment of Kidney-Yang deficiency syndrome,it must be combined with the disease(53%);The correlation between objective indexes and KidneyYang deficiency syndrome was greater(59%).According to the results of two rounds of questionnaires,after the demonstration meeting of the research group,sort out the core indicators of Kidney-Yang deficiency syndrome,and select 15 items with frequency>25 to enter the next round of expert consensus meeting for selection.3.3 Consensus meeting of experts15 senior professional experts of clinical and methodology from 7 hospitals in Beijing,Shanghai,Jiangsu,Sichuan and other places carried out online consensus meeting.The consensus meeting adopted the nominal group method,in which the conference secretary led a reading of the previous literature and Delphi’s findings,and presented the consensus entries.Experts are asked to vote on each item in turn based on the results.Considering the principle of "The simplest feasible,the most important,the standardized and the common understanding" of the core index set and the clinical practicability of the evaluation method,more than 70%of the votes for 4-5 points(very important)items into the core indicator set of kidneyyang Deficiency Syndrome.3.3.1 Outcome of the consensus meeting of expertsKidney-yang Deficiency Syndrome of TCM symptoms core indicators:Frequent nocturnal urination.waist ache,low libido,chills,fatigue,Enterorrhea before Dawn,listlessness.Disease-related kidney-yang Deficiency Syndrome core indicators:Quality of life score,sexual life level.At the same time,the expert opinion holds that the kidney-yang Deficiency Syndrome related to the disease should be defined as the diagnosis of the disease in western medicine,but not as the main content of the curative effect evaluation of the kidney-yang Deficiency Syndrome.4 Conclusion4.1 By using the methods of "Combination of qualitative and quantitative research methods" and "Combination of subjective and objective indexes",and referring to the technical methods and procedures for the development of the Core Outcome Set of clinical trials of TCM,through literature research and questionnaire investigation,at last,the COS of kidney-yang Deficiency Syndrome was formed by consensus meeting of experts.including:Frequent nocturnal urination.waist ache,low libido,chills,weakness,Enterorrhea before Dawn,listlessness;Disease-related kidneyyang Deficiency Syndrome core indicators:Quality of life score,sexual life level.The core index set of kidney-yang Deficiency Syndrome takes the core index of TCM symptoms of kidney-yang Deficiency Syndrome as the main evaluation index The index of kidney-yang Deficiency Syndrome related to the disease should be defined as the recommended content,not as the main content of the curative effect evaluation of the Syndrome.4.2 Referring to the model of MYMOP scale,investigate and summarize the clinical symptoms that patients focus on,and form the efficacy selfassessment scale based on patient evaluation,so as to construct the efficacy evaluation model of "combination of doctor-patient co evaluation",which includes 26 items aim to construct a curative effect evaluation model of"combined doctor-patient evaluation".4.3 Finally,based on the three-dimensional hybrid research method,the therapeutic effect evaluation index of kidney-yang Deficiency Syndrome was initially formed,which was "The combination of qualitative and quantitative research method,the combination of subjective and objective indexes,and the combination of doctor-patient evaluation." It provides the foundation of the early work for the forming of the therapeutic effect evaluation scale of kidney-yang Deficiency Syndrome.(二)Exploratory Metabonomics Study of Warming Kidney-Yang drug intervention in human body1 ObjectiveUsing metabolomics research methods,to explore the metabolic process of warming Kidney-Yang drugs before and after treatment in healthy subjects,the impact and improvement of metabolomics related indicators,and to explore the possible action pathway and efficacy indicators of warming Kidney-Yang drugs from the perspective of metabolomics,to provide an idea and method for the selection of objective indicators.2 MethodsBlood and urine samples of 6 healthy subjects before and after taking Fuqi Guben ointment(Composition:Aconite.Cinnamon,Medlar)were collected from the phase Ⅰ clinical trial ward of Xiyuan Hospital,Chinese Academy of Traditional Chinese Medicine.UPLC/Q-TOF-MS detection method and non targeted metabolic profile analysis technology were used.Principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA)were performed by Simca 14.1,and thermogram analysis was performed by R 4.02,Biomarkers of inter group differences were identified and analyzed using MetaboAnalyst 5.0 metabolic pathway.3 Results3.1 Blood sample analysis results3.1.1 PCA analysis resultsPCA model parameter R2 is good and the representative model interpretation rate is good.The three time points before,30 min and 24 h after positive ion administration were well distinguished.3.1.2 OPLS-DA analysis resultsOPLS-DA analysis found that positive and negative ions were well distinguished before and 24 hours after administration.The model parameter data is good and the model is stable and reliable.3.1.3 Identification of differential metabolitesA total of 16 differential metabolites were identified,including 11 lysophosphatidylcholine,Iphenylalanine,L-tyrosine,OLEIC ACID,stearicacid and stearoylcarnitine.3.1.4 Metabolic pathway analysisMainly focuses on phenylalanine tyrosine and tryptophan biosynthesis,phenylalanine metabolic pathway,unsaturated fatty acid biosynthesis,ami noacyl tRNA biosynthesis,ubiquinone and other terpenoid quinone biosynthesis.Phenylalanine and tyrosine are in the biosynthesis of phenylalanine tyrosine and tryptophan,phenylalanine metabolism pathway and aminoacyl tRNA biosynthesis pathway.Oleic acid and stearic acid are in the biosynthesis pathway of unsaturated fatty acids,and tyrosine is in the biosynthesis pathway of ubiquinone and other terpenoid quinones.3.2 Urine sample analysis results3.2.1 PCA analysis resultsPCA model parameter R2 is good and the representative model interpretation rate is good.The time points before and 48 hours after administration of positive and negative ions were well distinguished.3.2.2 OPLS-DA analysis resultsOPLS-DA analysis was conducted on the samples before and 48 hours after administration,and the positive and negative ions were well distinguished.The model parameter data is good and the model is stable and reliable.3.2.3 Identification of differential metabolitesDifferential metabolite identification a total of 19 differential metabolites were identified,including 4-hydroxy proline,L-proline,citrulline,ornithine and L-leucine.3.2.4 Metabolic pathway analysisMetabolic pathway analysis mainly focuses on arginine and proline metabolism,arginine biosynthesis and tryptophan metabolism pathway.Hydroxyproline,proline and ornithine are at the center of arginine and proline metabolic pathway;Citrulline and ornithine are in arginine biosynthesis pathway;Uracil and 5-hydroxyindole acetaldehyde are in the tryptophan metabolic pathway.4 ConclusionThe results of metabolomics showed that 35 biomarkers and 8 pathways were changed,involving amino acid metabolism,fat metabolism and energy metabolism.The above pathways and markers are mainly related to oxidative stress,apoptosis and dysfunction of hypothalamus pituitary target gland axis,involving cardiovascular diseases,neurodegenerative diseases and infertility of Kidney-Yang deficiency type.(三)CONCLUSION"Part Ⅰ" uses three-dimensional hybrid research methods to initiallyform the Core Outcome Set of Kidney-Yang deficiency syndrome in the three dimensions of "combination of qualitative and quantitative research methods","combination of subjective and objective indicators",and"combination of doctor-patient evaluation",which serve as the efficacy evaluation index of Kidney-Yang deficiency syndrome." Part Ⅱ" carried out a metabonomics study of human intervention of warming Kidney-Yang drugs.From the analysis of differential metabolites,the effect of this warming Kidney-Yang drug is related to the level of sex hormones,sexual life level,semen parameters which are closely related to the Disease-related indicators in Part Ⅰ",and indirectly provide a scientific basis and microsubstance supplement for Core Outcome Set of Kidney-Yang deficiency syndrome,and can reflect the clinical application value of objective indicators of Kidney-Yang deficiency syndrome to a certain extent.However,metabonomics in this study is only Exploratory research,which reflects the clinical value of objective index of kidney-yang Deficiency Syndrome to a certain extent,in order to provide reference for further research on the pathway and target of kidney-yang Deficiency Syndrome.