| Backgroud and ObjectiveSyndrome of spleen deficiency is prevalent in Chinese Medicine. Patients with spleen deficiency usually have symptoms such as low functions of digestion and absorption, malnutrition, fatigue and magersucht. Modern medical research proves that spleen deficiency is relevant to the human body’s function of digestion, absorption and metabolish.Buzhongyiqi Decoction was established by Li Dongyuan, who was a famous physician lived in Jin and Yuan Dynasties. Buzhongyiqi Dectoction is a representative prescription for tonifying Qi and raising Yang, which is most characterized by the compatibility of drugs strengthening Qi plus cimicifuga and bupleurum. In this decoction, astragalus is the most important drug, while cimicifuga and bupleurum are assistant in the way of helping astragalus to promote Yang to uprise. According to ancient literature, dosage of cimicifuga and bupleurum should be light. But in clinical practice nowadays, the proportion of astragalus, cimicifuga and bupleurum is different, which is necessary to be studied.In recent years, the development of science and technology helps to provide new ways for modern research in Chinese Medicine. Metabolomics, one of the new methods, can be used to analyze the metabolites in organisms and to find the correlation between these metabolites and physiological and pathological changes. To some degree, Metabolomics run in the same way with the whole concept in Chinese Medicine. Therefore, its application may help to study and analyze the theory of Chinese Medicine.Based on the previous studies, a spleen deficiency model of rat will be established and a series of experiments will be carried out with the aim of studying the pharmacological effect of Buzhongyiqi Decoction and its altered prescription on spleen deficiency rat and analyze the metabolites in spleen rat’s urine by metabolomics methods. The purpose of the above research is to investigate the effects of Buzhongyiqi Decoction and its altered prescription on spleen rat’s body weight, organ index, amylase in serum, gastric H+-K+-ATP enzyme, sodium-dependent glucose transporter (SGLT1), glucose transporter2(GLUT2), a peptide transporter1(PepT1) in intestine, glucokinase (GK) in liver, lactate dehydrogenase (LDH), succinate dehydrogenase (SDH) and isocitric dehydrogenase (IDH) in serum and metabolites in urine. Moreover, the meaning of the compatibility of tonifying Qi and raising Yang in Buzhongyiqi Decoction will be explored.Methods1、Establish the spleen deficiency rat by the method of "food preferences+food control+exhaustive swimming". Observe and record the rats’changes of weight, food intake, stool, spirit condition and behavior, et al. Grade according to "quantitive table of spleen deficiency syndrome symptoms in animal models". Evaluate whether the spleen model established successfully by comparing the weight and total scores of spleen syndrome before and after the model is established.2、Establish the spleen deficiency rat by the method of "food preferences+food control+exhaustive swimming" and treat the rats with Buzhongyiqi Decoction and its altered prescriptions-Buzhongyiqi Decoction without cimicifuga and bupleurum (hereinafter called "no cimicifuga and bupleurum") and Buzhongyiqi Decoction with doubled cimicifuga and bupleurum (hereinafter called "doubled cimicifuga and bupleurum"). Investigate the effects of Buzhongyiqi Decoction and its altered prescriptions on the changes of weight and organ index of the spleen deficiency rats.3、Prepare the serum sample and homogenate of stomach mucosa based on the spleen deficiency rats treated with Buzhongyiqi Decoction and its altered prescriptions. Measure the activity of amylase in serum with the amylase determination kit and that of H+-K+-ATPase with its determination kit. Investigate the effects of Buzhongyiqi Decoction and its altered prescriptions on the changes of amylase in serum and H+-K+-ATPase in stomach mucosa of the spleen deficiency rats.4、Prepare the homogenate of intestine mucosa and determine the protein expression level of sodium-dependent glucose transporter1(SGLT1), glucose transporter2(GLUT2) and peptide transporter1(PepTl). Investigate the effects of Buzhongyiqi Decoction and its altered prescriptions on the protein expression level of glucose transporter and peptide transporter of the spleen deficiency rats.5、Prepare the homogenate of liver and determine the activity of glucokinase (GK) in liver with the method of Elisa. Prepare the serum samples and determine the activity of lactate dehydrogenase (LDH), succinate dehydrogenase (SDH) and socitric dehydrogenase (IDH) in serum with the method of Elisa. Investigate the effects of Buzhongyiqi Decoction and its altered prescriptions on the activity of enzymes related to the metabolics of the spleen deficiency rats.6、Prepare the urine samples and profile the metabolites in the urine with the method of1H-NMR. Ascribe the peaks of metabolites according to the chemical shift values, peak splitting case, the coupling constants and references. Use softwares to analyze the data with the method of principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA).Results1、Establishment of spleen deficiency model:Weight of the model rats by the method of "food preferences+food control+exhaustive swimming" significantly decreased (P<0.05). Score of syndrome of the model group was significantly higher than the control group (P<0.05), which was more than4point. Results indicated that the spleen deficiency model was established successfully.2、Weight and organ index:Weight of the spleen deficiency rats increased slowly, and that of rats treated with Buzhongyiqi Decoction and with "no cimicifuga and bupleurum", rats’weight increased significantly, which was similar to that of the control group. However, the effect of "doubled cimicifuga and bupleurum" treatment was not significant. Spleen index and thymus index of the spleen deficiency rats decreased significantly (P<0.05). After treated with Buzhongyiqi Decoction, spleen index of rats with "no cimicifuga and bupleurum" and "doubled cimicifuga and bupleurum" rats’restored to the level of that of the control group (P<0.05). Buzhongyiqi Decoction and "except cimicifuga and bupleurum" treatment had effect of enhancing thymus index of spleen deficiency rats while the effect of "doubled cimicifuga and bupleurum" was not significant (P>0.05).3、Amylase in serum and H+-K+-ATPase in stomach mucosa:Activity of amylase in serum of the spleen deficiency rats decreased significantly (P<0.05). Activity of amylase in serum of rats increased significantly (P<0.05) after treated with Buzhongyiqi Decoction,"no cimicifuga and bupleurum" and "doubled cimicifuga and bupleurum" treatments. Among the three treatments, the effect of Buzhongyiqi Decoction’s effect was the best. Activity of H+-K+-ATPase in stomach mucosa of the spleen deficiency rats increased significantly (P<0.05) than the control group. Activity of H+-K+-ATPase of rats after treated with Buzhongyiqi Decoction,"no cimicifuga and bupleurum" and "doubled cimicifuga and bupleurum", activity of H+-K+-ATPase was restored to the normal level (P<0.05).4、Protein expression level of glucose and peptide transporters:Protein expression level of sodium-dependent glucose transporter1(SGLT1), glucose transporter2(GLUT2) and peptide transporter1(PepT1) significantly decreased compared with the control group (P<0.05). After treated with Buzhongyiqi Decoction, protein expression level of the three transporters was significantly higher than that of the model group of spleen deficiency rats (P<0.05). After treated with "no cimicifuga and bupleurum" treatment, expression level of GLUT2enhanced (P<0.05) but that of SGLT1and PepTl didn’t increase significantly (P>0.05). After treated with "doubled cimicifuga and bupleurum" treament, expression level of SGLT1and GLUT2 enhanced (P<0.05) but that of PepTl didn’t increase significantly (P>0.05), when compared with that of model group.5、Activity of LDH in serum and GK in liver:Activity of succinate dehydrogenase (SDH) and socitric dehydrogenase (IDH) in serum of different groups didn’t differ significantly (P>0.05). Activity of lactate dehydrogenase (LDH) of spleen deficiency rats decreased significantly compared with the control group (P<0.05). Being treated with Buzhongyiqi Decoction could enhance the activity of LDH in serum of spleen deficiency rats (P<0.05). However,"no cimicifuga and bupleurum" and "doubled cimicifuga and bupleurum" treatments could not enhance the activity of LDH in serum. Activity of GK in liver of spleen deficiency rats was significantly higher than that of control group (P<0.05). Buzhongyiqi Decoction had an effect of reversing the enhanced level of GK activity (P<0.05) and restored it to normality. But "no cimicifuga and bupleurum" and "doubled cimicifuga and bupleurum" treatments didn’t show significant effect to restore the activity of GK.6、Analysis of urine by metabolomics:Determine eight metabolites which might be related to spleen deficiency syndrome by metabolomics. In urine of spleen deficiency rats, content of the eight metabolites all decreased compared with the control group (P<0.05). Buzhongyiqi Decoction tends to increase six of the eight metabolies."No cimicifuga and bupleurum" treatment tends to increase all of the eight metabolies, in which effect on dimethylglycine increase is significant (P<0.05)."Doubled cimicifuga and bupleurum" treatment tends to to increase only three of the eight metabolies.ConclusionsWeight and organ index of spleen deficiency rats decreased; Digestion, absorption and metabolism function of spleen deficiency rats went abnormal and metabolites in urine were also abnormal, which indicates the metabolic mal-function of substance and energy of spleen deficiency rats. After treated with Buzhongyiqi Decoction, the markers of pharmcogical effect and metabolites in urine were improved, which showed that Buzhongyiqi Decoction could restore the function of digestion, absorption and metabolism of spleen deficiency rats. Its effect was better than its altered prescriptions ("no cimicifuga and bupleurum" and "doubled cimicifuga and bupleurum"), which showed the reasonability of the compatibility of tonifying Qi and raising Yang in Buzhongyiqi Decoction and demonstrated that the dosage of cimicifuga and bupleurum should be light in the traditional theory based on scientific experimental data. |
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