| Chinese herbal form ? la and processing are the are the two characteristics of clinical medicine in Traditional Chinese Medicine (TCM).Besides,they are also a significant difference between traditional Chinese medicine (TCM) clinical use and natural drugs.Spleen-kidney Yang deficiency diarrhea is prevalent in Chinese Medicine. Modern medical research proves that Spleen-kidney Yang deficiency diarrhea can cause function abate or recession, sluggish metabolism, insufficient thermogenesis. Patients with Spleen-kidney Yang deficiency diarrhea usually have symptoms such as chills, depressed. They are similar to those caused by modern medical theory of basal metabolic rate and energy metabolism level drops.Ershen Wan was established by Xu Shuwei, who was a famous physician lived in Song Dynasties. Ershen Wan comprises Buguzhi(Psoraleae Fructus) and Roudoukou (Myristicae Semen). Ershen Wan can warm the kidney and spleen and astringe the intestine to relieve diarrhea, which is a basal clinical treatment of Spleen-kidney Yang deficiency diarrhea. Buguzhi is the most important drug, tonify life-gate fire to warm the spleen. Roudoukou are assistant in the way of helping Buguzhi to promote the force of the warm the kidney and spleen, and astringe the intestine to relieve diarrhea. But the synergistic mechanism of salt-frying Buguzhi and whether Roudoukou of Ershen Wan need processing in clinical practice, which is necessary to be studied.OBJECTIVESBased on the previous studies, under the financial aid of the National Natural Science Fund Project "Research on Efficacy Increasing Mechanism of Processing for Chinese Medicine in the Form? la of Ershen Wan" (81274085),a Spleen-kidney Yang deficiency diarrhea model of rat will be established and a series of experiments will be carried out with the aim of studying the pharmacological effect of after and before processing of Buguzhi(Psoraleae Fructus) and Roudoukou(Myristicae Semen) which comprised Ershen Wan on rats with spleen-kidney yang deficiency diarrhea and analyze the metabolites in spleen-kidney yang deficiency diarrhea rat's serum and urine by metabolomics methods. The purpose of the above research is to investigate the effects of after and before processing of Buguzhi (Psoraleae Fructus) and Roudoukou(Myristicae Semen) which comprised Ershen Wan on Spleen-kidney Yang deficiency diarrhea rat's ATPase, lactate dehydrogenase (LDH) and succinate dehydrogenase (SDH) in the liver tissue, superoxide dismutase (SOD) and malonaldehyde (MDA) in serum, expression levels of uncoupling protein 2 (UCP2) mRNA in renal tissues and metabolites in serum and urine. Moreover, the meaning of the rationality and the synergistic mechanism of Ershen Wan, consisting of salt-frying Buguzhi and bran-roasting Roudoukou will be explored.METHODS1. The rats models for the Spleen-kidney Yang deficiency diarrhea were made through the perfusion of adenine, irreg? lar diet, and reception of ice senna extract. The models were treated with Ershen Wan, consisting of after and before processing of Buguzhi(Psoraleae Fructus) and Roudoukou(Myristicae Semen).2. Prepare the homogenate of liver tissue based on the Spleen-kidney Yang deficiency diarrhea rats treated with Ershen Wan, consisting of after and before processing of Buguzhi(Psoraleae Fructus) and Roudoukou(Myristicae Semen). Measure the activity of Na+-K+-ATPase?Ca2+-Mg2+-ATPase?lactate dehydrogenase (LDH) and succinate dehydrogenase (SDH) in the liver tissue with the kits. Investigate the effects of after and before processing of Buguzhi(Psoraleae Fructus) and Roudoukou(Myristicae Semen) which comprised Ershen Wan on the activity of enzymes related to the metabolics of the Spleen-kidney Yang deficiency diarrhea rats. Discussing the synergistic mechanism of alt-frying Buguzhi and bran-roasting Roudoukou from the viewpoint of energetic metabolism.3. Immunohistochemistry was used to measure the expression levels of UCP2 mRNA in renal tissues. Observe effects of Ershen Wan form? la and its processed product on expression levels of uncoupling protein 2 (UCP2) mRNA in renal tissues of rats with Spleen-kidney Yang Deficiency, to preliminarily explore the possible synergistic mechanism from the molec? lar level.4. Prepare the serum sample. Measure the activity of superoxide dismutase (SOD) and malonaldehyde (MDA) in serum with the SOD determination kit and that of MDA with its determination kit. Compare the effects of after and before processing of Buguzhi (Psoraleae Fructus) and Roudoukou (Myristicae Semen) which comprised Ershen Wan on the changes of SOD and MDA in serum of Spleen-kidney Yang deficiency diarrhea rats.5. Test Spleen-kidney Yang deficiency diarrhea rat's serum metabolic fingerprints in different stages by using gas chromatography-mass spectrometry (GC-MS) analyze by principal component analysis(PCA) and partial least squares discriminant analysis(PLS-DA)methods and screen out potential bio-marks through VIP and t test. Through comparison after and before processing of Buguzhi(Psoraleae Fructus) and Roudoukou(Myristicae Semen) which comprised Ershen Wan of potential biomarkers in the serum of Spleen-kidney Yang Deficiency rats, explore the mechanism of Ershen Wan processing efficiency.6. Prepare the urine samples and profile the metabolites in the urine with the method of gas chromatography-mass spectrometry (GC-MS). Use softwares to analyze the data with the method of principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA).RESMLTS1. After Spleen-kidney Yang deficiency diarrhea modeling on the rat, drinking water intake decline, activity decrease, body quality reduce, body hair is scanty and lose luster, defecate pond drainage, urine volume increase, depression, hypothermia, chills,cluster, individual animals appear rectocele, and even death.We consider this method conforms to the symptoms of Spleen-kidney Yang deficiency diarrhea model.2. In comparison to the normal control group, processed Buguzhi and Roudoukou expressed a lower level of Na+-K+-ATPase(P< 0.01), Ca2+-Mg2+-ATPase(P<0.01)and SDH (P< 0.01), and a higher level of LDH(P< 0.01) in model group. Suggests that the rats in the aerobic oxidation power shortage, low energy metabolism, metabolic disorders, this accords with the state of model rats. Ershen Wan co? Id promote ATP decomposition, keep normal ion concentration and membrane potential, improve the energy metabolism and maintain normal metabolism of liver function to adjust the body energy metabolism by enhancing the level of Na+-K+-ATPase, Ca2+-Mg2+-ATPase and SDH activity and decreasing the level of LDH activity. Ershen Wan, consisting of salt-frying Buguzhi and bran-roasting Roudoukou, has best effect.3. Compared with that of normal group, the expression level of UCP2 mRNA in Spleen-kidney Yang deficiency diarrhea rat's renal tissues was significantly increased(P<0.01). Illustrate the Spleen-kidney Yang deficiency diarrhea rats in energy metabolism disorder status. Compared with that of model group, the expression level of UCP2 mRNA were recovered after receiving Ershen Wan (P<0.05), and the processing group (salt-frying Buguzhi and bran-roasting Roudoukou) is the best (P<0.05), which may be related to reg?lation of the body's basal metabolic rate.4. In comparison to the normal control group, it expressed a lower level of SOD (P< 0.01), expressed a higher level of MDA (P< 0.01) in model group. Illustrate the Spleen-kidney Yang deficiency diarrhea can lead to excessive oxygen free radicals accumulation and lipid peroxidation end product. Ershen Wan co? Id enhance the level of SOD and decrease the level of MDA, can improve the antioxidant ability of rats, reduce the lipid peroxide in the body. Res?lts show that Ershen Wan can improve the antioxidant ability and reduce the lipid peroxide. Ershen Wan, consisting of salt-frying Buguzhi and bran-roasting Roudoukou, has best effect.5. Identified 10 differentiated metabolites related to Spleen-kidney Yang deficiency diarrhea rats. Compared with the normal control group, the Alanine, Leucine, Valine, Glycine, Butanedioic acid, Pentanedioic acid, Proline, Glucose and Hexadecanoic acid were decreased significantly and Lactic acid increased(P<0.01). All the indicators were recovered after receiving Ershen Wan (P<0.05), and the processing group (salt-frying Buguzhi and bran-roasting Roudoukou) is the best.6. Twelve potential biomarkers were identified. But only eleven potential biomarkers including alanine, glycine, succinic acid, norvaline, L-proline, L-methionine, N-benzoylglycine,L-phenylalanine,hexadecanoic acid,tetradecanoic acid and tyrosine were definitely up-reg ? lated or down-reg?lated,and the intervention of Ershen Wan (salt-frying Buguzhi and bran-roasting Roudoukou) was the strongest.CONCLUSIONSThe activity of enzymes related to the metabolics of the Spleen-kidney Yang deficiency diarrhea rats went abnormal and metabolites in serum and urine were also abnormal, which indicates the basal metabolic rate and energy metabolism of Spleen-kidney Yang deficiency diarrhea rats. After treated with Ershen Wan, the markers of pharmcogical effect and metabolites in serum and urine were improved, which showed that Ershen Wan co?ld reg?late the basal metabolism and energy metabolism of Spleen-kidney Yang deficiency diarrhea rats. Ershen Wan, consisting of salt-frying Buguzhi and bran-roasting Roudoukou, has the best function, which showed the reasonability of the synergy in processing. Ershen Wan, consisting of salt-frying Buguzhi and bran-roasting Roudoukou, has the best function, which may be related to anti-lipid peroxidation, improve amino acid metabolism, lipid metabolism and energy metabolism, and reg? late the intestinal flora. It provides a scientific theory for guiding clinical rational drug use. |
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