| Backgroud:In Chinese medicine, TCM constitutions are closely related to the state of health, constitution types determine susceptibility to certain pathogenic factor and disease types of bias. Wet-heat constitution is now recognized as one type of nine biased constitutions, with hot and humid embodiment as the main characteristics. It often manifest as oily sticky facial skin, acne, dry or bitter mouth, heaviness of limbs, stool adhesion or dry knot, urinate short or yellow, male easy scrotal moisture, women with leukorrhea yellow or bad smell, red tongue, yellow greasy moss, rapid and slippery pulses, etc.. According to a national survey, the incidence of wet-heat constitution is 9.08%, the second of all the 8 kinds biased constitutions. Wet-heat constitution has been correlated with the digestive, respiratory, urinary system diseases etc.. Therefore, understanding the mechanism of wet-heat constitution, and how to change wet-heat constitution so as to prevent the occurrence of adverse health status becomes very meaningful.Causes of wet-heat constitution always comes from the traditional Chinese medicine dialectical analysis or cross-sectional epidemiological investigation, the results show that except by the hot and humid climate, there are much to do with heavy modern life and study pressure, adjustable eating habit, lack of exercise. These causes always lead to heart-yin deficiency, hyperactivity of ministerial fire, curb the spleen Yang, result in phlegm and heat. Intervention of wet-heat constitution mainly include the daily life, physical exercise, dietetic regulation, regulation of the mind and spirit and drugs, but are only stay in the traditional Chinese medicine syndrome differentiation and treatment, clinical trials have not been studied and all the intervention effect also lacks objective basis. Therefore, starting from the objective evaluation basis, carrying out randomized, controlled clinical study to research wet-heat constitution is very necessary.Ermiao pill is the common prescription by Zhu Danxi for treatment of wet-heat symptoms in the Yuan dynasty. The prescription can clear heat and dry dampness, jianyin bushen, jianpi shengyang, kidney water full will ease the internal heat fire and the lung will not be hot, since this can restrict liver wood, spleen will be protected from hurting. These are accord with the etiology and pathogenesis of the wet-heat constitution. Modern pharmacology study shows that ermiao pill main contain berberine, cortex phellodendri, magnoflorine, sesquiterpenoids and polyethylene ingredients acetylene, the whole prescription has antibacterial, antioxidant, fall blood sugar, adjusting immunity, regulating the central nervous system, cardiovascular system, digestive system, etc. It is very suitable to improve clinical manifestation for damp heat constitution.Metabonomics is processing by group index analysis, high-throughput detection and data. It investigated dynamic metabolic changes of the overall organisms or cells system, especially endogenous metabolism, genetic variation, environmental changes, as well as characteristics and influence of various substances into the metabolic system. By the method of metabonomics urine metabolites, our research found that compared with gentleness constitution, the fatigue model of sub-health population of wet-heat constitution have obvious different metabolites:creatinine, α-Oxoglutaric acid, coenzyme Q,3-phosphoglyceric acid, alanine et al.. These metabolites were significantly increased, and related with multiple energy krebs cycle, sugar, lipid, amino acids circulation, and other metabolic pathways.Therefore, this paper intends to carry out a randomized, controlled clinical trial, with ermiao pill to intervene wet-heat constitution healthy/sub-healthy students in Guangdong area, and take’H-NMR technique to investigate the metabolic mechanism of wet-heat constitution and its intervention metabolic mechanism, attempting to provide certain objective reference for wet-heat constitution clinical intervention.Object:Clarify the clinical efficacy of ermiao pill interventing wet-heat constitution, investigate the metabolic mechanism of wet-heat constitution’s formation and intervention.Method:Using "the standardized Constitution in Chinese Medicine Questionnaire" (CCMQ), the self-rated health questionnaire "sub-health measurement scale" by our research group and medical report for health diagnosis, we included healthy/sub-healthy people with gentleness constitution or pure wet-heat constitution in accord with the standards of the research. Wet-heat constitution individuals were randomly divided into control group and drug intervention group. Control group did not give any intervention drugs, except helath-promoting lifestyle guidelines, intervention group give ermiao pill intervention (6 g, po bid) for 28 days besides helath-promoting lifestyle guidelines. Experimental observation time points are before intervention (0 month),1 month after the intervention,6 months after intervention and 12 months after the intervention.(1) Clinical trials on ermiao pill intervening wet-heat constitution:The main outcome measurements is wet-heat constitution transformation score, other constitution transformation points, self-test health status and wet-heat constitution entry scores for secondary efficacy index measurements. We carry out an index evaluation at 0 month,1 month,6 month and 12 month times respectively. Statistical analyses are used in order to make clear the clinical efficacy of Chinese traditional medicine recuperating the wet-heat constitution, attempting to provide a scientific basis for biased constitution rehabilitation by application of traditional Chinese medicine.(2) Metabolic mechanism of wet-heat constitution’s formation and intervention: Choose gentleness constitution participants and wet-heat constitution participants before the intervention (the control group and intervention group), wet-heat constitution control group before and after intervention, wet-heat constitution intervention group before and after intervention, get fasting blood sample early in the morning, extract plasma and-20 ℃ cryopreserved. Plasma thaw at room temperature, centrifugal (14000×g,10 min,4 ℃), take the supernatant 0.3 ml, add 0.1 ml of heavy water and 0.2 ml PBS buffer solution, blend and put into 5 mm nuclear magnetic pipe for metabonomics testing. Metabolomic finger printings of gentleness constitution, wet-heat constitution before and after the intervention were acquired by 1H-NMR. MestReNova 5.3 software is adopted to pretreatment the metabolic profiles, and normalize values for each subsection integral. The data output converted to excel format and imported to the software Simca-p 12.0.1.0 (Umetrics AB, Sweden) for multivariate statistical analysis.Through principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and orthogonal partial least-squares discriminant analysis (OPLS-DA) pattern recognition methods for data processing.We extract the VIP value in OPLS-DA model, combined with normalized integral value, we got different plasma metabolites between gentleness constitution and wet-heat constitution/wet-heat constitution group before and after intervention. And according to clinical symptoms, physical examination report, human metabolites database (database http://www.hmdb.ca) and literatures, metabolites were identificated, and the related metabolic pathways were analyzed. So as to reveal the metabolic mechanism of wet-heat constitution’s formation and intervention. Statistical analysis used SPSS 13.0, and the normalized integral value (X±S) compare used two independent sample t-test.Results:10 gentleness constitution cases and 88 wet-heat constitution cases were included in this study.88 wet-heat constitution cases were randomly divided into intervention group and the control group (n= 44).9 cases were fall off or got rid of, and a total of 79 cases complete the research, male 30, female 49, average age (19.51 ± 1.49) years old. There were 42 cases in wet-heat constitution intervention group, and 37 cases in the control group. There was no significant difference between two groups in age, sex and body mass index (P> 0.625).1. Clinical trials on ermiao pill intervening wet-heat constitution(1) The health-promoting lifestyle scores in the groups in different time pointsAccording to the results of repeated measurements analysis, between-group difference of HPLP-â…¡ scores is not significant (P=0.356). But the main effect of time points reach the significant level (P=0.000), there is no interaction effect between group and time (P=0.607). With the passage of time, both control and intervention group’s HPLP-â…¡ scores have a rise tendency, but the trend has no significant difference. Further analyze the scores between two groups in different time points, results show that there was no statistically significant difference between control group and intervention groupbefore or after intervention(p>0.158).Multiple comparision of different time points shows that HPLP-â…¡ scores in 6 month and 12 month after intervention rise significantly in intervention group (P<0.05), while in control group, 1monthã€6monthã€12 months after intervention rise significantly (P<0.05).(2)The transformation scores of wet-heat constitution in the groups in different time pointsAccording to the results of repeated measurements analysis, between-group difference of wet-heat constitution transformation scores is significant (P=0.015), The main effect of time points reaches the significant level too (P< 0.001), but there is no interaction effect between group and time (P=0.217). Conversion points has no statistical difference between two groups before the intervention(P=0.513), After intervention, the intervention group showed a trend of first decline and then rise, the control group showed a trend of first increase and then decrease, there was statistically significant between the two groups at lmonth/6 month time points(P=0.033,0.007), but the significance disappeared in 12 month time point. Multiple comparision of different time points shows that wet-heat constitution transformation scores in 6 month and 12 month after intervention rise significantly compared with 0 month and 1 month (P<0.05).(3)The transformation scores of other constitutions in the groups in different time pointsAccording to the results of repeated measurements analysis, none between-group differences of constitution transformation scores are significant (P>0.061), But the main effect of time points all reach the significant level (p<0.045), there is no interaction effect between group and time except for yin-deficiency constitution (P=0.012). With the passage of time, both control and intervention group’s gentleness constitution transformation scores have a rise tendency, while the other seven kinds of biased constitution transformation scores have a downward trend, although with no statistical difference.Multiple comparison of different time points shows that all the constitution transformation scores except for gentleness and qi-depression constitution after intervention in 6 month and 12 months compared with 0 month have significant difference(p< 0.05).(4)Item scores of wet-heat constitution in the groups in different time pointsAccording to the results of repeated measurements analysis, none between-group differences of different items scores are significant in addition to urinate fever or color thick. But the main effect of time points all reach the significant level except for easy scrotal moisture (P< 0.001), there is no interaction effect between group and time (P>0.062). With the passage of time, both control and intervention group’s items scores have a reduced tendency. The.tendency mainly manifested in oily sticky facial skin and acne in control group, and also manifested in dry or bitter mouth, leukorrhea yellow or bad smell, stool adhesion or dry knot and urinate short or yellow in intervention group. Multiple comparison of different time points shows that after intervention in 1 monthã€6 month and 12 month, sitems scores of nose greasy or facial glossy and easy to acne or furuncle have significant difference compared with 0 month (P< 0.05), other items scores in 6 month and 12 month after intervention also had a significant reduce(P< 0.05).(5)The transformation scores of self-rated health status in the groups in different time pointsAccording to the results of repeated measurements analysis, between-group difference of self-rated health transformation scores is not significant (P=0.125). But the main effect of time points reach the significant level (P<0.001), there is no interaction effect between group and time (P=0.518). With the passage of time, both intervention and control group’s self-rated health transformation scores have a rise tendency but the change trend has no significant difference between the two groups. Multiple comparison of different time points shows that self-rated health transformation scores in 1 months 6 month and 12 months after intervention all rise significant in intervention group (P<0.05), while in control group, only 6 month and 12 months after intervention rise significant (p<0.05).With the passage of time, both group’s physiological, psychological and social health transformation scores have a rise tendency. For physiological health transformation scores, there was significant between-group difference (P< 0.001) and main effect of time points also reach the significant level (P<0.001), there is no interaction effect between group and time (P=0.785), organ function scores in 6 month after intervention rise significant in intervention group (P<0.05), while in control group, both 6 month and 12 months after intervention rise significant (P<0.05). For psychological health transformation scores, there was no significant between-group difference (P=0.941) and main effect of time points (P=0.083), there is no interaction effect between group and time (P=0.568), transformation scores in 6 month after intervention rise significant in intervention group (P<0.05). For social health transformation scores, there was no significant between-group difference (P=0.941), but main effect of time points also reach the significant level (P=0.019), there is no interaction effect between group and time (F=0.612), transformation scores in 6 month after intervention rise significant in intervention group (P<0.05).2. Metabonomic characteristics of wet-heat constitution’s formation and intervention(1)The plasma’H-NMR profiling of wet-heat constitution and gentleness constitutionThe experiment took gentleness constitution cases (10) and wet-heat constitution cases before intervention (20) as the research object for modeling. After data processing and pattern recognition, the PCA and PLS-DA model can divided the gentleness constitution group and wet-heat constitution group. And the OPLS-DA model can divided better. Then we found 28 significant metabolites by the load scatter plot, S-plot, VIP value and normalized integral value (P=0.000-0.049). The results showed that compared with gentleness constitution group, isoleucine, leucine, valine, threonine, lactic acid, a-Hydroxyisobutyrate, proline, choline, histidine, trimethylamine oxide (TMAO), betaine, a and β glucose, taurine, propylene glycol, cholesterol, glycine, glycerol, citrulline, propionylglycine, glutamine, alanine, 3-Phosphoglyceric acid, aspartic acid, creatine,3-hydroxybutyric acid, aldosterone, unsaturated lipid.(2)The plasma’H-NMR profiling of wet-heat constitution before and after health-promoting lifestyle interventionThe experiment took wet-heat constitution control group cases (10) as the research object for modeling. After data processing and pattern recognition, the PCA and PLS-DA model can divide wet-heat constitution before and after lifestyle intervention. And the OPLS-DA model can divided better. Then we found 26 significant metabolites by the load scatter plot, S-plot, VIP value and normalized integral value (P=0.000-0.035), all decreased after intervention. They are aldosterone, cholesterol, isoleucine, glutamic acid, glutamine, oxoglutarate, pyruvic acid, choline, arginine, histidine, trimethylamine oxide (TMAO), a and β glucose, taurine, propylene glycol, threonine, glycine, glycerol, valine, leucine, citrulline, propionylglycine, alanine,3-Phosphoglyceric acid, aspartic acid, unsaturated lipid.Compared with the gentleness constitution,22 kinds of metabolites such as aldosterone, cholesterol, leucine, isoleucine, valine, glutamine, choline, histidine, trimethylamine oxide (TMAO), a and β glucose, taurine, propylene glycol, threonine, glycine, glycerol, citrulline, propionylglycine, alanine,3-Phosphoglyceric acid, aspartic acid and unsaturated lipid were increased in wet-heat constitution, but after health-promoting lifestyle intervention, the expression of these 22 metabolites significantly decreased.(3)The plasma 1H-NMR profiling of wet-heat constitution before and after ermiao pill interventionThe experiment took wet-heat constitution intervention group cases (10) as the research object for modeling. After data processing and pattern recognition, the PCA and PLS-DA model can divide wet-heat constitution before and after lifestyle and ermiao pill intervention. And the OPLS-DA model can divided better. Then we found 26 significant metabolites by the load scatter plot, S-plot, VIP value and normalized integral value (P=0.000-0.037). Of which, lactate acid, a-Hydroxyisobutyrate and 3-hydroxybutyrate increased after intervention, while all others decreased, such as aldosterone, cholesterol, glutamic acid, glutamine, choline, arginine, trimethylamine oxide (TMAO), proline, methanol, α and β glucose, taurine, propylene glycol, threonine, glycine, glycerol, valine, leucine, propionylglycine, alanine,3-Phosphoglyceric acid, aspartic acid, unsaturated lipid.Compared with the gentleness constitution,20 kinds of metabolites such as aldosterone, cholesterol, leucine, valine, proline, glutamine, choline, trimethylamine oxide (TMAO), α and β glucose, taurine, propylene glycol, threonine, glycine, glycerol, propionylglycine, alanine,3-Phosphoglyceric acid, aspartic acid and unsaturated lipid were increased in wet-heat constitution, but after ermiao pill intervention, the expression of these 20 metabolites significantly decreased.Conclusion:1.The results of clinical trials found that, lifestyle and lifestyle with ermiao pill intervention both have significant effects on the improvement of the situation of wet-heat constitution, have the trend to lead the whole constitution change to gentleness constitution. Lifestyle intervention worked slowly, need longer time to work; and with the addition of drug intervention can work fast, have more obvious improving, and long time efficacy is obvious too.2. On the symptoms improved, compared with lifestyle intervention, ermiao pill intervention besides can improve not only symptoms of oily sticky facial skin, easy to acne, bad breath, but also dry or bitter mouth, urinate short or yellow, leukorrhea yellow or bad smell, stool adhesion or dry knot.3. Lifestyle and ermiao pill intervention wet-heat constitution can improve the self-rated health status, mainly reflected in terms of physical health, lifestyle improving gastrointestinal discomfort, eye discomfort and other symptoms of organ function iseffective, also to problems of properly handling the unpleasant things, relationship satisfaction, self satisfaction, etc. But on tension, depression, insecurity, fear, loneliness and suspicious etc. psychological symptoms, combined with ermiao pill intervention would be effective.4. The formation of wet-heat constitution may be related to tricarboxylic acid cycle, fatty acid metabolism, degradation metabolism of leucine/isoleucine/valine, sugar dysplasia, pyruvate metabolism, urea cycle, aspartic acid metabolism, bile acid synthesis, purine or pyrimidine metabolism, arginine and proline metabolism, glycine and serine metabolism, betaine metabolism, ammonia recovery, amino sugar metabolism, and other associated metabolic pathways.The main involved metabolic disorders include three major energy metabolic pathways, enhanced inflammatory/immune response, the central nervous system inhibition, muscle fatigue, cell permeability enhancement, and gastrointestinal disorders. Those mechanisms of lifestyle and ermiao pill regulate wet-heat constitution probably be adjusting these metabolic disorders. |
PDF Full Text Request