| BackgroundConstitution of Traditional Chinese Medicine(TCM)has relatively stable and inherent characteristics formed on the basis of innate endowment and acquired characteristics after birth,integrated with morphological structure,physiological function and psychological status in the process of human life.Constitution determines the kinds of disease,orientation,disease resistance,the course of the disease stage and pathological change trend.Yang-deficiency constitution is caused by lack of yang qi and is characterized by chills to the cold,hand and foot not warm,like hot food and depression.Yang-deficiency constitution is susceptible to phlegm retention,edema,bi syndrome,diarrhea,etc.In recent years,our previous studies suggested that the number of bias constitution in sub-health status and disease was larger than that in health status and yang-deficiency constitution contributed to a large proportion of bias constitution.Therefore,it is necessary to treat yang-deficiency constitution and explore molecular mechanism of TCM in the treatment of yang-deficiency constitution.The current researchesof constitution of traditional Chinese medicine mainlyfocus on the mechanism of gene polymorphism and differentially expressed genes.However,TCM constitution is made up of genetic factors and environmental factors.The difference of gene could not clarify the molecular mechanism of TCM constitution completely.Metabonomics is another important research field of systems biology following genomics and proteomics.It focuses on the overall dynamic metabolic changes,particularly on the characteristics of endogenous metabolism,genetic variation,environment changes,as well as a variety of substances transferred into metabolic systems.Metabolomics is a qualitative and quantitative analysis method which focus on low molecular metabolites(<1kD),reflecting changes in the organism after external stimuli or genetic modification.Compared with transcriptomics and proteomics,metabolomics has its own advantages.The metabolic is closest to the phenotype,which can reflect phenotypic changes.As a result,metabonomics has an important role in understanding the changes of nature.Small changes in gene and protein expression will be amplified in the metabolite.The number of metabolites is much less than the number of genes and proteins.Therefore,metabonomics is helpful for objective classification of different constitution types of TCM,which will lead to more comprehensive understanding of constitution phenomena.TCM constitution not only has stability but also has dynamic variability.Lifestyle interventions include promotion of health education,diet,living nursing,emotions and exercise.TCM plays an important.role in the treatment ofyang-deficiency constitution.Gui fu di huang wan derives from "Jin Kui Yao Lue",composed of cinnamon,monkshood,prepared rehmannia root,cornel,cortex moutan,yam,tuckahoe and alisma.Gui fu di huang wan has the function of warming kidney yang.Alisma and tuckahoe have the function of eliminating dampness and dieresis.Cortex moutan can clear liver-fire.Prepared rehmannia root,cornel and yam have the function of nourishing yin.Cinnamon and monkshood have the function of warming yang,Gui fu di huang wan has the function of warming kidney yang,which is commonly used to treat yang-deficiency constitution.However,the mechanism has not been fully understood.In our study,we included yang-deficiency constitution in healthy and sub-health population,which were randomly divided into control group and experimental group.The outcomes included scores of health promoting lifestyle questionnaire,TCM constitution questionnaire and self-rated health questionnaire.The plasma of yang-deficiency constitution and gentleness constitution were used by nuclear magnetic resonance(NMR)technology methods for metabonomics analysis.The plasma of control group and experimental group before and after the intervention were used by NMR technology methods for metabonomics analysis.ObjectiveTo assess the effect of gui fu di huang wan in the treatment of yang-deficiency constitution and explore the molecular mechanism of gui fu di huang wan in the treatment of yang-deficiency constitution.Methods1.We performed the clinical trials of gui fu di huang wan in the treatment of yang-deficiency constitution.The sub-health questionnaire and health-promoting lifestyles questionnaire were used to determine the health status.TCM constitution questionnaire was used to determine the type of constitution.Sixty two participants who were diagnosed with yang-deficiency constitution in healthy population were included and were randomly divided them into control group(n=31)and experimental group(n=31).The control group was given lifestyle intervention.The experimental group was given gui fu di huang wan combined withlifestyle interventionfor a month.The outcome was the scores of yang-deficiencyconstitution subscale,scores of other constitution subscale,sub-health questionnaire and health-promoting lifestyles questionnaire.2.We compared the metabolomics of yang-deficiency constitution and gentleness constitution.We selected 20 yang-deficiency constitution subjects and 10 gentleness constitution subjects.The fasting plasma was collected before the intervention and conserved at-80?.Metabolomic profiling was acquired by 'H-NMR.3.We compared the metabolomics of the control group before and after intervention.10 yang-deficiency constitution participants incontrol group were included.The fasting plasma was collected after the intervention and conserved at-80?.Metabolomic profiling was acquired by 'H-NMR.4.We compared the metabolomics of the experimental group before and after intervention.10 yang-deficiency constitution participants inexperimental group were included.The fasting plasma was collected after the intervention and conserved at-80?.Metabolomic profiling was acquired by 1H-NMR.5.We analyzed the metabolomics data.MestReNova 5.3 software was used to preprocess the metabolic profiles.The values for each segment integral were normalized.The data was converted to excel format and imported into Simca-P 12.0.1.0(Umetrics AB,Sweden)for multivariate statistical analysis.Principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA)was used for analysis.Variable importance in projection(VIP)values was extracted from OPLS-DA model.We selected metabolites with VIP value>1.The normalization value(X±S)of the plasma metabolites with VIP value>1 was analyzed by two independent samples t test in the metabolomics of yang-deficiency constitution and gentleness constitution or paired t test in the metabolomics of control groupand experimental group before and after intervention.P<0.05 was considered as potential different metabolites.The metabolites were identified through human metabolite database(http://www.hmdb.ca)and references.Results1.Clinical trials of gui fu di huang wan in treating yang-deficiency constitution.There was 1 participant in control group dropout at 12 months after treatment.There was 1 participant in experimental group withdraw at 1 month after treatment.The general information in sex,age and BMI in thecontrol group and experimental group were comparable(P>0.05).1.1 Scores of health-promoting lifestyles questionnaireThere was no significant difference between control group and experimental group in each dimension score and total scores of health promoting lifestyle(P>0.05).The experimental group showed significant increase in scores of self-realization at 1 month and 12 monthsafter treatment compared with before(P<0.05).The control group showed significant increase in scores of exercise at 12 months after treatment compared with before treatment(P=0.028).The experimental group showed significant increase in scores of exercise at lmonth and 12 months after treatment compared with before treatment(P<0.05).The control group and experimental group showed significant increase in scores of nutrition at 1 month,6 months and 12 months after treatment compared with before treatment(P<0.05).The experimental group showed significant increase in scores of stress management at 1 month,6 months and 12 months after treatment compared with before treatment(P<0.05).There were significant differences of total scores of health promoting lifestyle between different time in control group and experimental group(F=5.595,P=0.002;F=8.605,P=0.000).The control group and experimental group showed significant increase in total scores of health promoting lifestyle at 1 month,6 months and 12 months after treatment compared with before treatment(P<0.05).1.2 The effect of gui fu di huang wan on scores of TCM constitution1.2.1 Conversion scores of yang-deficiency constitutionThere were significant differences ofconversion scores of yang-deficiency constitutionbetween different times in control group(F=5.207,P=0.002).The control group showed significant decrease in scores of conversion scores of yang-deficiency constitution at 12 months after treatment compared with before treatment(P=0.037).Compared with intervention before,conversion scores of yang-deficiency constitution in the experimental group decreased at 1 month,6 month and 12 month(P<0.05).There were no significant differences between control group and experimental group for conversion scores of yang-deficiency constitution before intervention(P>0.05).After intervention for one month,the experimental group showed significantly decrease in conversion scores of yang-deficiency constitution compared with the control group(F= 8.513,P=0.005).Six months and twelve months later,the experimental group also showed significantly decrease in conversion scores of yang-deficiency constitution compared with the control group(F= 4.163,P=0.046;F=4.776,P=0.033).1.2.2Each item of yang-deficiency constitution subscaleThe experimental group showed significant improvement for the hands and feet not warm and coldness symptom after intervention for one month compared with the control group(t=3.145,P=0.003;t=2.850,P=0.006).After six and twelve months,the experimental group showed significant improvement for stomach coldness and more coldness in winter symptom compared with the control group,respectively(t=2.060,P=0.044;t=2.309,P=0.024).1.2.3 Conversion scores of remaining constitutionThere were significant differences ofscores of gentleness constitutionbetween different times in experimental group(F=2.820,P=0.043).The experimental group showed significant increase in scores of gentleness constitution and decrease in scores of damp heat constitution at 6 month after treatment compared with before treatment(P=0.016,P=0.027).The experimental group showed significant decrease in scores of qi-deficiency constitution at 6 months after treatment compared with control group(F=4.290,P=0.043).1.3The effect of gui fu di huang wan on scoresof self-rated questionnaire1.3.1 The percentage of health and sub-healthBefore intervention,the control group included 16 subjects with health and 15subjects with sub-health;the experimental group included 14 subjects with health and 17subjects with sub-health.There were no significant differences between control group and experimental group in the percentage of health and sub-health before treatment(P>0.05).At 1 month,6 months and 12 months after treatment,there were no significant differences between control group and experimental group in the percentage of health and sub-health before treatment(P>0.05).1.3.2 The scores of self-related health and sub-scaleThere was no significant differenceofscores of physical health sub-scale,psychological health sub-scale,social health sub-scaleand self-rated healthbetween control group and experimental group(P>0.05).There was nosignificant difference ofscores of physical health sub-scale,psychological health sub-scale,social health sub-scaleand self-rated healthbetween different times in control group and experimental group(P>0.05).1.3.3The scores of each dimension inphysical health sub-scaleThere was no significant difference of scores in each dimension between control group and experimental group(P>0.05).The experimental group showed significant increase in scores of organ function at 1 month after treatment compared with before treatment(P=0.031).The control group showed significant increase in scores of organ function at 12 months after treatment compared with before treatment(P=0.038).1.3.4 The scores of each dimension in psychological health sub-scaleThere was no significant difference of scores in each dimension between control group and experimental group(P>0.05).The control group showed significant increase in scores of cognitive function at 12 months after treatment compared with before treatment(P=0.006).1.3.5 The scores of each dimension in social health sub-scaleThe experimental group showed significant increase in scores of social adaption at 6 months after treatment compared with before treatment(P=0.027).The experimental group showed significant increase in scores of social support at 12months after treatment compared with before treatment(P=0.024).There were significant differences of scores of social support at 12months after treatment between control group and experimental group(P=0.036).1.4 Adverse eventsThere was no significant difference of blood routine,urine routine,liver and kidney function,blood lipids before and after treatment between control group and experimental group(P>0.05).There were no adverse events reported in the experimental group.2.The plasma metabolomic profiling of yang-deficiency constitution and gentleness constitution30 samples were analyzed by'H-NMR(10 in gentleness constitution and 20 in yang-deficiency constitution).PCA was used to make model and nine main components were calculated.The model parameters were RZX=0.966,QZ=0.743.The model analyzed 96.6%of the original data.The cumulative contribution rate was 74.3%.We then used OPLS-DA to make model and five main components were calculated.The model parameters were R2X=0.812,R2Y=0.714,Q2=0.452.We found 11 potential different metabolites,of which lactate,3-hydroxybutyrate,a-glucose,proline,alanine and glutamine decreased,trimetlylamine oxide,choline,glycine,myo-inositol,valine increased.They were mainly related to tricarboxylic acid cycle,fatty acid metabolism,amino acid metabolism,glucolysis,gluconeogenesis,alanine cycle,inositol metabolism,arginine and proline metabolism.3.The plasma metabolomic profiling of control group before and after intervention20 samples were analyzed by 1H-NMR(10 before intervention and 10 after intervention).PCA was used to make model and four main components were calculated.The model parameters were R2X=0.968,Q2=0.879.The model analyzed 96.8%of the original data.The cumulative contribution rate was 87.9%.We then used OPLS-DA to make model and two main components were calculated.The model parameters were R2X=0.783,R2Y=0.854,Q2=0.771.We found 9 potential different metabolites,includinglactate,valine,proline,choline,arginine,inositol,leucine,creatine and 3-hydroxybutyrate,which increased in the plasma mainly related to tricarboxylic acid cycle,fatty acid metabolism,amino acid metabolism,glucolysis,gluconeogenesis,inositol metabolism,arginine and proline metabolism.4.The plasma metabolomic profiling of experimental group before and after intervention20 samples were analyzed by1 H-NMR(10 before intervention and 10 after intervention).PCA was used to make model and four main components were calculated.The model parameters were R2X=0.896,Q2=0.646.The model analyzed 89.6%of the original data.The cumulative contribution rate was 64.6%.We then used OPLS-DA to make model and two main components were calculated.The model parameters were R2X=0.732,R2Y=0.705,Q2=0.550.We found 11 potential different metabolites,including valine,isoleucine,lactate,alanine,proline,arginine,betaine,a-glucose,glutamine,propanediol and 3-hydroxybutyrate,which increased in the plasma mainly related to tricarboxylic acid cycle,fatty acid metabolism,amino acid metabolism,glucolysis,gluconeogenesis,pyruvate metabolism alanine cycle,arginine and proline metabolism.Conclusions1.We found 11 potential different metabolites in yang-deficiency constitution,of which lactate,3-hydroxybutyrate,a-glucose,proline,alanine and glutamine decreased,trimetlylamine oxide,choline,glycine,myo-inositol,valine increased.Yang-deficiency constitution,characterized by decreased energy,has disorder of glucose and lipid metabolism,amino acid metabolism,tricarboxylic acid cycle and neurotransmitter.2.Lifestyle interventionpromotes the health promotion behavior and shows improvementinyang-deficiency constitution and health status,which maybe related to increasing the concentration oflactate,3-hydroxybutyrate,and proline.3.Gui fu di huang wan based on the lifestyle intervention can significantly improve the symptomsof yang-deficiency constitution and health status in the short term.Long-term follow-up also shows a significant effect.The mechanism maybe related to not only increasing the concentration oflactate,3-hydroxybutyrate,proline but also of alanine,glutamine and a-glucose. |
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