The Clinical Observation On Treatment Of Yin-deficiency Constitution By Liu Wei Di Huang Wan And Its Metabonomic Characteristics

Backgroud:Yin-deficiency is a kind of Traditional Chinese Medicine (TCM) constitution which is influenced by innate endowment and acquired characteristics after birth. The characteristics of yin-deficiency include as follows:①patterns of physique:individual with yin-deficiency always thin; ②psychological trait:individual with yin-deficiency always vivacious, active, impatience; ③physical characteristics:tinnitus, amnesia, insomnia, dreaminess, or dry mouth; ④The tendency to disease:individual with yin-deficiency tend to suffer disease characteristic by dryness-heat;⑤The ability to adapt to the outside:can’t stand the heat.Many studies have shown that compared with the Gentleness constitution, people with Yin deficiency have metabolic disorder contributing to a significant rise of serum cortisol and adrenocorticotropic hormone. Our previous study found that there was a significant difference distribution between health and SHS. Yin-deficiency, Wet-heat, Blood stasis and Qi depression were respectively 1.618, 1.585,1.611,1.891 times more likely to develop SHS. So we speculate that yin-deficiency and health status has a closed connection. Adjusting an unbalanced TCMC has a significance. We designed a randomized controlled research to explore the effect of adjusting yin-deficiency by liu wei di huang wan and metabonomic characteristics of yin-deficiency constitution.Metabolomics is processing by group index analysis, high-throughput detection and data. It investigated the overall dynamic metabolic changes, particularly in studing the characteristics and effects of endogenous metabolic, genetic variation, environment changes, as well as a variety of substances transferred into metabolic systems. Is similar with the wholeness of TCM theory. The application of metabonomics maybe could find out the specific metabolites in the process of transformation of health status. It is conducive to understand the symptom of TCM theory. Metabonomics has became the bridge that connects the TCM and western medicine.Previous research has shown that there are significantly differences between the two samples after intervention. There are 11 specific metabolites, including betaine, proline, leucine, glutamine, serine, histidine, alanine, nicotinamide nucleotide, isoleucine, aspartic acid, inositol. It is related to fatty acid metabolism, Krebs cycle, ornithine cycle, amino acid metabolism, pyruvate metabolism, gluconeogenesis, alanine cycle, inositol metabolism, steroid synthesis, purine pyrimidine metabolism, ammonia recovery, glycine and serine metabolism multiple metabolic pathway. We speculate that the yin-deficiency also has a metabolic disorders. We applied the liu wei di huang wan to adjust the yin-deficiency, attempting to find out the metabolism characteristic of yin-deficiency.Object:1. Clarify the curative effect of bu liu di huang wan intervening yin-deficiency constitution. To provide the scientific basis for adjusting unbalanced TCMC.2.Explore the metabolism characteristic of yin-deficiency and relevant differences metabolites3.Explore the effect of liu wei di huang wan on metabolism of yin-deficiency and the changes of metabolism.Method:1. The research of the TCM intervening yin-deficiency constitution. The physical examination and self-rated health have been used to determine the health status. We selected yin-deficiency constitution in health status and randomly divided them into intervention group and control group. The intervention group was intervened by liu wei di huang wan for a month and TCM integrated regulation, the control group was only TCM integrated regulation. The main curative index is converted scores of yin-deficiency sub-scale and the subordination curative index is converted scores of CCMQ, SHMS V1.0 and HPLP-II. The health status has been observed to clarify the effect of TCM on health transformation.2. The research of the metabolomics of yin-deficiency constitution and gentleness constitution. We selected 18 yin-deficiency constitution participation and 10 gentleness constitution participation who enrolled in our clinical research in chapter two. The plasma was collected in empty stomach and cryopreserved at -20 ℃. And carry out bioinformatics analysis. Establish metabolic differences database.3. The research of the metabolomics of yin-deficiency intervention group before and after intervention. We selected 8 yin-deficiency constitution participation to analyze the metabolic differences of self-contrast before and after the treatment and establish metabolic differences database. The plasma was collected in empty stomach and cryopreserved at -20℃.4. The research of the metabolomics of yin-deficiency control group before and after intervention. We selected 10 yin-deficiency constitution participation to analyze the metabolic differences of self-contrast before and after the treatment and establish metabolic differences database. The plasma was collected in empty stomach and cryopreserved at -20℃.5.Analyze the metabolomics data. The plasma was thawed at room temperature and centrifuged (14000×g, 10min,4℃).0.3 ml supernatant buffer solution,0.2 ml PBS,0.1 ml heavy water was added together, and injected in 5mm NMR tubes to test metabolic genomics. The plasma of intervention group, control group and gentleness group had been collected before and after intervention for the metabolomics analysis. MestReNova 5.3 software has been used to pretreatment the metabolic profiles, and normalized the values for each segment integral. The data output will be converted to Excel format and imported into Simca-P 12.0.1.0 (Umetrics AB, Sweden) for multivariate statistical analysis. Principal component analysis and partial least squares discriminant analysis have been used to analyze. In order to strengthen inter-group differences, the orthogonal partial least squares discriminant analysis (PLS-DA) has been used as well. The results were expressed in scores plot. Statistical analysis was performed using SPSS 13.0. The normalization value of the plasma metabolites has analysis by two independent samples t test. VIP values have been extracted from OPLS-DA model. We selected metabolites VIP value>1, P<0.05 as difference variables. Combination of clinical symptoms, medical reports and questionnaires content, VIP value and normalized results were identified to find represent metabolites. The component identification was identified through human metabolite database (http://www.hmdb.ca) and literature. We found related metabolic markers of yin-deficiency constitution to explore mechanisms of energy metabolism disorder.Results:1. Clinical observation of TCM intervening yin-deficiency constitution(1) Main indicators evaluation:Comparison between intervention group and control groups Before the intervention, there are no significantly different between control group and intervention group(F=0.1, P=0.753). After a month later, the scores of yin-deficiency constitution in intervention group have decreased significantly (F=0.021, P=0.044). There was also significant difference between intervention group and control group (F=4.559, P=0.044; F=5.076, P=0.028) in six month and twelfth month. Comparison in the group. In the intervention group, there were significant difference between before the intervention and one month, one month and sixth month(P=0.020, P=0.036). After a year later the scores of yin-deficiency constitution has decreased significantly(44.60±11.75,29.36±15.39, P=0.000). While in the control group only first month and sixth month comparison has difference(46.78± 14.85, 40.05±12.96, P=0.005). And there was a effects of interaction between intervention group and control groups (F=3.084, P=0.029). The comparison of difference of scores of yin-deficiency constitution. There were significant difference between intervention group and control groups(F=5.906, P=0.018; F=5.56, P=0.021; F=4.559, P=0.037). Compared with control group, the symptoms of body fever, trick heart generate heat, feel thirsty, xerocheilia has changed a lot(F=4.700, P=0.034; F=7.135, P=0.010; F=5.289, P=0.025; F=8.526, P=0.005). (2) Secondary endpoints: ①The scores of other TCMC. As time goes on. There was a decrease of scores of gentleness (F=2.969, P=0.036) and a increase of yang deficiency in control group(F=3.653, P=0.022). There was a decrease of scores of blood stasis and special diathesis(F=3.032, P=0.033; F=4.599, P=0.036). ②After intervention, the scores of HPLP-II in intervention group have increased significantly(F=3.983, P=0.024), but there was no significant difference in control group. ③After intervention, the scores of Self-rated health score in intervention group have increased significantly(F=4.546, P=0.013). But but there was no significant difference between intervention group and control group.2. The plasma 1H NMR profiling of yin-deficiency constitution and gentleness constitutionWe selected 28 volenteers (10 gentleness constitution and 13 yin-deficency constitution) to study its metabolism characteristics. PCA method was used to build model and 7 main components was calculated. The model parameters R2X=0.641, Q2X=0.156, the model analyzed 64.1% of the original data. Based on the data to establish the PLS-DA model and 4 main components was calculated. The model parameters R2X=0.436, R2Y=0.99, Q2X=0.775. Based on the data to establish the OPLS-DA model and 5 main components was calculated. The model parameters R2X=0.906, R2Y=0.874, Q2X=0.349. There are significantly differences between the two groups. There are 11 specific metabolites, including Cholesterol, L-Isoleucine, L-Leucine, L-Lactic acid, L-Alanine, Glycoprotein, L-Proline, L-Valine, 3-hydroxybutyrate, Creatine, Threonine, D-Aspartic acid, Unsaturated lipid. It is related to fatty acid metabolism, Krebs cycle, ornithine cycle, amino acid metabolism, pyruvate metabolism, gluconeogenesis, alanine cycle, inositol metabolism, steroid synthesis, purine pyrimidine metabolism, ammonia recovery metabolism multiple metabolic pathway.3. The plasma 1H NMR profiling of intervention group before and after interventionWe selected 8 volenteers in intervention group to study its metabolism characteristics. PCA method was used to build model and 5 main components was calculated. The model parameters R2X=0.938, Q2X=0.751, the model analyzed 93.8% of the original data. Based on the data to establish the PLS-DA model and 4 main components was calculated. The model parameters R2X=0.524, R2Y=0.996, Q2X=0.832. Based on the data to establish the OPLS-DA model and 4 main components was calculated. The model parameters R2X=0.744, R2Y=0.917, Q2X=0.296. There are significantly differences in intervention group before and after intervention. There are 24 specific metabolites, including mianliy VLDL, Lipid. Fucose, Threonine, L-Isoleucine, Lysine, L-Alanine,2-hydroxy isovaleric acid, Glycoprotein, L-Proline, Glutamic acid, L-Glutamine, Acetoacetic acid, Gamma-Aminobutyric acid,2-oxoglutarate, Citrate, Myoinositol, Threonine, Tyrosine, D-Serine, Lactate, β-glucose, Unsaturated lipid,1-Methylhistidine It is related to fatty acid metabolism, the TCA cycle, ornithine cycle, catecholamine synthesis, amino acid metabolism, pyruvate metabolism, gluconeogenesis, synthesis of steroids, purine pyrimidine metabolism, metabolism of sphingolipids metabolism, retinol, ammonia recovery, synthetic phospholipids metabolism, glycine and serine metabolism, mitochondrial electron transport chain and multiple metabolic pathways.4.The plasma 1H NMR profiling of control group before and after interventionWe selected 10 volenteers in intervention group to study its metabolism characteristics. PCA method was used to build model and 8 main components was calculated. The model parameters R2X=0.927, Q2X=0.705, the model analyzed 92.7% of the original data. Based on the data to establish the PLS-DA model and 4 main components was calculated. The model parameters R2X=0.921, R2Y=0.866, Q2X=0.472. Based on the data to establish the OPLS-DA model and 4 main components was calculated. The model parameters R2X=0.921, R2Y=0.866, Q2X=0.637. There are significantly differences in intervention group before and after intervention. There are 22 specific metabolites, including mianliy VLDL, Lipid. Fucose, Threonine, L-Isoleucine, Lysine, L-Alanine,2-hydroxy isovaleric acid, Glycoprotein, L-Proline, Glutamic acid, L-Glutamine, Acetoacetic acid, Gamma-Aminobutyric acid,2-oxoglutarate, Citrate, Myoinositol, Threonine, Tyrosine, D-Serine, Lactate, β-glucose, Unsaturated lipid,1-Methylhistidine It is related to fatty acid metabolism, the TCA cycle, ornithine cycle, catecholamine synthesis, amino acid metabolism, pyruvate metabolism, gluconeogenesis, synthesis of steroids, purine pyrimidine metabolism, metabolism of sphingolipids metabolism, retinol, ammonia recovery, synthetic phospholipids metabolism, glycine and serine metabolism, mitochondrial electron transport chain and multiple metabolic pathways.Conclusion:1. Liu wei di huang wan and lifestyle regulate can significantly improve the yin-deficiency constitution.2. There was energy and fats metabolism disorders, after the intervention of liu wei di huang wan these disorders have improved, disturbance in the energy metabolism maybe the mechanism of symptoms.3. After the intervention of liu wei di huang wan there was a decreased of amino acid connect with neurotransmission, maybe the excitatory and inhibitory signal pathways of CNS was in a unbalanced status. The character traits of yin-deficiency maybe the consequence of this unbalanced.