The Clinical Observation On Treatment Of Qi-deficiency Constitution By Bu Zhong Yi Qi Wan And Its Metabonomic Characteristics

Backgroud:Traditional Chinese Medicine (TCM) constitution is the relatively stable, 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. The qi-deficiency is characteristic as low voice, shortness of breath, lassitude, easy to sweat, weak pulse, reddish tongue with tooth print. So far, the study on qi-deficiency is focus on epidemiological investigation and poor in genes, proteins and metabolic mechanism. Our previous studies suggested that the number of bias constitution in sub-health status is more than that in health. As a result, we inferred that the qi-deficiency constitution is associated with health status. In this study, a randomized, controlled study was conducted to study the bu zhong yi qi wan intervening qi-deficiency constitution to test its mechanism.The study on qi-deficiency still aimed at epidemiological investigation and poor in gene chip, proteomics, metabonomic. Our previous result show:(Dthe number of bias constitution in health status is in minority (18.7%), and the gentleness constitution in health status is in majority (81.3%). The number of bias constitution in sub-health status is more than that in health (64.0%), most of whom are qi-deficiency constitution (41%).②As the logistic result shows, the gentleness constitution is positive related to health status and negative related to sub-health status. The qi-deficiency constitution is positive related to sub-health status.③The gentleness constitution is hard to transforme into sub-health status and disease, while the bias constitution is much more easier in sub-health status and disease. As a result, we inferred that the bias constitution is associated with health status. The qi-deficiency constitution volunteers in health status cannot be interferenced by disease. Therefore, we studied the metabonomic characteristics of qi-deficiency constitution volunteers in health status, and intervented them by bu zhong yi qi wan to study its mechanism.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, of. Metabolomics is qualitative and quantitative analysis low molecular metabolites (<1kD) to reflect changes in the organism after external stimuli or genetic modification. Compared with transcriptomics and proteomics, metabolomics has its own advantages. Firstly, the metabolic is the closest to the phenotype, which can reflect phenotypic changes. The changes in gene caused the changes of protein expression, and ultimately occured changes in metabolic processes. Therefore, the study of the metabolic group 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. As a result, we used the metabolitic to study the metabolic disorders of qi-deficiency constitution. Our group found that the urine of qi-deficiency constitution contained creatinine, histamine, glucose, alanine, guanosine triphosphate glycerol acid, creatine phosphate, hippuric acid, histidine, glycerol acid levels. These metabolites, which were related with multiple energy krebs cycle, glycolysis, amino acid metabolism, fatty acid metabolism and other metabolic pathway, were significantly increased. The results from the preliminary study shows that long-term heavy work and mental stress causes the body and blood problems, the energy consumption rate increased, gives rise to a sense of fatigue, and at last in sub-health status. The qi-deficiency constitution participates have yin and yang imbalance, more susceptible to the external environment and other factors lead to sub-health or disease. Therefore, we hypothesized that the presence of energy metabolism in qi-deficiency constitution, may contribute to the health sub-health or disease. The intervention of qi-deficiency constitution can be adjusted to prevent the occurrence of sub-health and disease.Bu zhong yi qi wan is the representative prescription for Li Dongheng, who is one of the four famous doctors in Jinyuan period. The medicine can treat fever, diarrhea, bleeding and others. Modern pharmacology studies found that the element of bu zhong yi qi wan is complexity, which contains saponins, polysaccharides, flavonoids, alkaloids, amino acids and volatile oil. It is useful to regulate gastrointestinal motility, secretion of digestive juice, promote intestinal absorption, anti-tumor, regulate immune function, promoting protein synthesis, anoxia, and combined effects of antibacterial anti-inflammatory. The qi-deficiency is characteristic as low voice, shortness of breath, lassitude, easy to sweat, weak pulse, reddish tongue with tooth print and usually have some digest problems. The bu zhong yi qi wan is suitable for regulate qi-deficiency.Object:Clarify the curative effect of bu zhong yi qi wan intervening qi-deficiency constitution. Clarify the metabolic disorders of bu zhong yi qi wan intervening qi-deficiency constitutionMethod:Firstly, we study the TCM intervening qi-deficiency constitution. The sub-health questionnaire and physical examination have been used to determine the health status. We selected qi-deficiency constitution in health status and randomly divided them into control group and intervention group. The intervention group was intervened by Bu zhong yi qi wan for a month and the control group was not intervened. The main curative index is converted scores of qi-deficency subscale and the subordination curative index is converted scores of CCMQ, SHMS V1.0and HPLP-II. The health status has been observed to clarify the effect of TCM on health transformation.Secondly, we study the metabolomics of qi-deficiency constitution and gentleness constitution. We selected19qi-deficiency constitution people and15gentleness constitution. The plasma was collected in empty stomach and cryopreserved at-20℃.Thirdly, we study the metabolomics of qi-deficency intervention group before and after intervention. We selected20qi-deficiency constitution people (10in control group and10in intervention group). The plasma was collected in empty stomach and cryopreserved at-20℃.Fourthly, analysed the metabolomics data. The plasma was thawed at room temperature and centrifuged (14000×g,10min,4＞).0.3ml supernatant buffer solution,0.2ml PBS,0.1ml heavy water was added together, and injected in5mm 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. MestReNova5.3software 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-P12.0.1.0(Umetrics AB, Sweden) for multivariate statistical analysis. Principal component analysis and partial least squares discriminant analysis have been used to analyse. In order to strengthen inter-group differences, the orthogonal partial least squares discriminant analysis (OPLS-DA) has been used as well. The results were expressed in scores plot. Statistical analysis was performed using SPSS13.0. The normalization value (X±S) of the plasma metabolites has analysis by two independent samples t test. VIP values have been extracted from PLS-DA model. We selected metabolites VIP value>1, P<0.05as 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 qi-deficiency constitution to explore mechanisms of energy metabolism disorder.Results:(1) Clinical observation of TCM intervening qi-deficiency constitutionBefore the intervention, there are no significantly different between control group and intervention group. After a month later, the scores of qi-deficiency constitution in intervention group have decreased significantly (t=4.355, P=0.000), while the control group was increased, but there was no significant difference (t=-1.145, P=0.26). The control group and intervention group had significant difference after the intervention (t=2.053, P=0.044). Bu zhong yi qi wan can improve the symptoms significantly. In the secondary evaluation indicators, scores of other constitutions don’t change after a month intervention in intervention group (P>0.05), while the scores of yang-deficiency constitution, yin-deficiency constitution, blood stasis constitution changed in the control group, the difference was statistically significant (P<0.05). After a month later, the scores of HPLP-II in intervention group have decreased significantly (t=-2.879, P=0.007), but there was no significant difference in control group(t=-1.366, P=0.181). There are no significant difference in control group and intervention group in HPLP-II after1month (t=-0.811, P=0.421). The scores of some symptom increased in control group after1month, but there was no significant difference, while in intervention group, the scores have declined. The scores of palpitation, dizziness, lazy improved significantly (P<0.05).(2) The plasma1H NMR profiling of qi-deficiency constitution and gentleness constitutionWe selected35volenteers (15in gentleness constitution and19in qi-deficency constitution) to study its metabolism characteristics. PCA method was used to build model and six main components was calculated. The model parameters R2X=0.856, Q2=0.504, the model analyzed85.6%of the original data. Based on the data to establish the OPLS-DA model and two main components was calculated. The model parameters R2X=0.531, R2Y=0.388, Q2=0.0787. There are significantly differences between the two samples after intervention. There are11specific 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.(3) The plasma1H NMR profiling of intervention group before and after interventionWe selected20volenteers (10in control group and10in intervention group) to study its metabolism characteristics. PCA method was used to build model and five main components was calculated. The model parameters R2X=0.725, Q2X=0.382, the model analyzed72.5%of the original data. Based on the data to establish the PLS-DA model and three main components was calculated. The model parameters R2X=0.502, R2Y=0.967, Q2X=0.721. There are significantly differences in intervention group before and after intervention. There are35specific metabolites, including renal steroids, coenzyme A, sitosterol, aldosterone, valine, cholesterol sulfate, ethanol, prostaglandin E1, isoleucine, alanine, gamma amino acid, glutamine, methionine, betaine, benzene alanine, phenylpropanolamine, alanine, glycerol acid, aspartic acid, serine, histidine, a-linolenic acid, prostaglandin D2,2-ethyl acrylate, different dimension A acid, sphingosine, urea, tyrosine, epinephrine, acetylamino, methyl hippuric acid, vanillic acid, hippuric acid, N-acetyl-tyrosine, p-hydroxybenzoic acid. 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:Firstly, it indirectly shows that if the constitution can not be improved, it may convert to other constitutions.Secondly, Bu zhong yi qi wan can significantly improve the qi-deficiency constitution.Thirdly, the different metabolites of intervention group before and after intervention related to the citric acid cycle and bu zhong yi qi wan maybe regulate the disorder.